heat shock and uv-c abiotic stress treatments as alternative tools to

HEAT SHOCK AND UV-C ABIOTIC STRESS TREATMENTS AS ALTERNATIVE

TOOLS TO PROMOTE FRESH-CUT CARROT QUALITY AND SHELF-LIFE

TESE APRESENTADA PARA OBTENÇÃO DO GRAU DE DOUTOR EM

ENGENHARIA ALIMENTAR

Carla Sofia Marques de Alegria

ORIENTADORA: Doutora Margarida Gomes Moldão-Martins

COORIENTADORES: Doutora Marta Maria Moniz Nogueira de Abreu

Doutor Luis Cisneros-Zevallos, professor associado da Texas A&M

University

JÚRI:

Presidente: Reitor da Universidade de Lisboa

Vogais: Doutor António Augusto Martins de Oliveira Soares Vicente, professor associado

com agregação da Escola de Engenharia da Universidade do Minho

Doutora Cristina Luísa Miranda Silva, professora associada da Escola Superior de

Biotecnologia da Universidade Católica Portuguesa

Doutora Margarida Moldão Martins, professora auxiliar com agregação do Instituto

Superior de Agronomia

Doutor Victor Manuel Delgado Alves, professor auxiliar do Instituto Superior de

Agronomia

Doutora Susana Maria Gomes Caldas da Fonseca, professora auxiliar convidada

da Faculdade de Ciências da Universidade do Porto

Doutora Marta Maria Moniz Nogueira de Abreu, investigadora auxiliar do Instituto

Nacional de Investigação Agrária e Veterinária, I.P.

LISBOA

2015

Este trabalho foi financiado pela Fundação para a Ciência e Tecnologia pela atribuição de uma bolsa individual de doutoramento com referência SFRH/BD/62211/2009.

i

ABSTRACT

Abiotic stress treatments, heat shock (HS_100 C/45 s) and UV-C (0.1-5 kJ.m-2

), and two passive

modified atmosphere packaging conditions were evaluated under the hurdle concept as alternative

approaches to the standard processing of fresh-cut carrot (FCC). The significant phenolic

accumulation, via phenylalanine-ammonia lyase activation, showed to be independent on key

factors contributing to raw material bioactivity, cultivar and crop season, but dependent on

treatments intensity (HS_[100 C/45 s], UV-C_[2.5 kJ.m-2

]) and on oxygen availability (>2%). The

low decontamination efficiency of UV-C_[2.5 kJ.m-2

] as single hurdle was similar (p>0.05) to that of

sodium hypochlorite (<1 Log10 cycle in aerobic mesophilic microflora) and was responsible for FCC

early sensorial rejection (<7 days), regardless packaging conditions. The simultaneous effects on

microbiological control and reduced metabolic rates yielded by HS_[100 C/45 s] significantly

contributed to the preservation of FCC sensorial quality allowing the full recovery (100%) of raw

materials’ phenolic levels (≥5 days, 5 C), with no synergic effects in the combination with UV-C,

resulting in FCC shelf-life extension by two-fold (14 days, 5 C) when using a bi-oriented

polypropylene micro-perforated film.

KEY WORDS: · Fresh-cut carrot · Heat shock · UV-C · Wounding · Phenolic synthesis ·

iii

Stresses abióticos de choque térmico e radiação UV-C para a promoção da

qualidade e período de vida útil de cenoura minimamente processada

RESUMO

Foram avaliados tratamentos de stress abiótico, segundo o conceito “hurdle” - choque térmico

(CT_100 C/45 s), UV-C (0.1-5 kJ.m-2

) e duas condições de embalagem em atmosfera modificada

passiva, em alternativa ao processamento convencional de cenoura minimamente processada

(CMP). O incremento (p<0.05) da composição fenólica, via activação da fenilalanina-amónio-liase,

mostrou ser independente de factores determinantes na bioactividade da matéria-prima, cultivar e

época de colheita, e dependente da intensidade dos tratamentos (CT_[100 C/45 s], UV-

C_[2.5 kJ.m-2

]) e dos níveis de oxigénio disponíveis (>2%). A baixa eficiência de descontaminação

do UV-C_[2.5 kJ.m-2

] enquanto tratamento singular foi semelhante (p>0.05) ao tratamento com

hipoclorito de sódio (<1 ciclo Log10 microrganismos aeróbios a 30 C) sendo responsável pela

rejeição sensorial precoce de CMP (<7 dias), independentemente das condições de embalagem.

Os efeitos concomitantes do CT_[100 C/45 s] no controlo microbiológico (redução 3 ciclos Log10

microrganismos aeróbios a 30 C) e na redução da actividade metabólica preveniram a

deterioração da qualidade sensorial de CMP (p<0.05), possibilitando a recuperação (100%) dos

níveis fenólicos da matéria-prima (≥5 dias, 5 C), sem acréscimos sinérgicos na combinação com

o UV-C, e com duplicação do período de vida da CMP (14 dias, 5 C) quando se utilizaram filmes

de polipropileno biorientado micro-perfurado.

PALAVRAS-CHAVE: · Cenoura minimamente processada · Choque térmico · UV-C · Corte ·

Síntese fenólica ·

v

ACKNOWLEDGMENTS

Despite the fact that my name is printed on the cover of this thesis, “I” is something that it is not

included within its chapters. This page is dedicated to the countless contributions of my supervisors

and to the support of my family, friends and colleagues.

To my supervisors, Doctor Margarida Moldão-Martins, Doctor Marta Abreu and Doctor Luis

Cisneros-Zevallos, I would like to thank you for providing me the opportunity to pursue my PhD, for

the research environment you created for me and for your support and guidance through the

learning curve required to develop this thesis. A word for each is necessary…

Margarida, from the very first day your constant encouragement, teachings, generosity and

guiding nature gave me the necessary confidence to pursuit this endeavor!

Luis, you are without doubt an oasis of ideas and your passion for science is inspiring and

enriches every student. I was no exception!

Marta… Your dedication, encouragement, hard work, the use of your own experiences to

motivate me, your support through thick and thin… Be sure that you constantly inspired

and challenged me to better myself and aim higher. I am at a loss for words and I am

indebted to you more than you know.

These studies were financially supported via a Doctoral fellowship (SFRH/BD/62211/2009) from

Fundação para a Ciência e Tecnologia, I.P., which I acknowledge and thank. Through this

scholarship I could support myself and pursue the PhD.

I would like to thank Doctor Carlos Santos (Chair of Unidade Estratégica de Investigação e

Serviços de Tecnologia e Segurança Alimentar, INIAV, I.P.-Polo do Lumiar) for always allowing me

to pursue my academic goals in his Department.

To Doctor Elsa M. Gonçalves, besides her friendship, has much taught me through her critical view

and also for her great attitude, constant motivation and support in several ways during all these

years.

My wholehearted thanks to Joaquina Pinheiro and Ana Gomes, who are also experiencing the feat

of developing their PhD thesis, thank you for the continuous support and friendship… At stormy

times you provided me the necessary anchor and even when your own lives were overwhelming,

you remained present! I do not know how I would have got through some stages without you!

To all members of the labs, both in Lisbon and in College Station, Texas, for your technical

assistance, friendship and smiling faces, thank you! Maria do Carmo Paula e Ana Magalhães,

thank you for your collaboration and support regarding the microbiological determinations. Paula

Castillo and Freddy Ibañez, thank you for your hospitality, you guys made my stay at Texas more

enjoyable and sociable.

ACKNOWLEDGMENTS

vi

To all members of the sensorial panel, thank you for your input and patience. Your constant

feedback made my work more challenging.

Unrestrained comments from anonymous reviewers provided equal parts of useful feedback and

the determination to keep pushing forward.

I wish also to thank CAMPOTEC, in the person of Eng. Tânia Caracol, for providing me the

packaging films used in the shelf-life studies.

To all that took time away from their own over-extended lives to provide feedback on this thesis, I

am extremely grateful. I falter to include such a list, since surely I would omit some who deserves

mention.

Eduardo, MEU LOBO, thank you for believing in me and for all the sacrifices, support (including

countless hours peeling carrot…) and love that you always have given me! Thank you for even

learning how to pronounce phenylalanine ammonia-lyase! You gave me the strength, the passion

and the will to keep on going, even when things were looking darker… Thank you for your smile

and calm… FOREVER ALWAYS!

To my parents, Olga and Fernando, thank you for giving me the freedom and opportunity to chase

after my own dreams, even when they appeared to be beyond your understanding or somewhat

risky. You’ve always been a true source of inspiration to me and you always will! Thank you!

I also thank my grandmother, ‘Vó Amélia, a she-warrior, for teaching me that there is nothing like

honest hard work and that you always should run after your dreams.

All errors and limitations remaining in this thesis are mine alone.

Thank you!

vii

AGRADECIMENTOS

Apesar de estar editado o meu nome na capa desta tese, “Eu” é algo que é ofuscado nos seus

capítulos. Este espaço é dedicado às inúmeras contribuições dos meus orientadores e ao apoio da

minha família, amigos e colegas.

Aos meus orientadores, Doutora Margarida Moldão-Martins, Doutora Marta Abreu e Doutor Luis

Cisneros-Zevallos, gostaria de agradecer a oportunidade que me deram para a realização deste

doutoramento, pelo ambiente de trabalho que me providenciaram e por todo o apoio e orientação

através da curva de aprendizagem necessária ao desenvolvimento desta tese. Torna-se

necessária uma palavra de agradecimento a cada um de vós…

Margarida, desde o primeiro dia, o seu constante incentivo, ensinamentos, generosidade e

orientação deram-me a confiança necessária para a prossecução deste projecto!

Luis, sem dúvida que és um oásis de ideias e a tua paixão pela ciência é inspiradora e

enriquecedora para qualquer aluno. Eu não fui excepção!

Marta… A tua dedicação, incentivo, trabalho árduo, o uso das tuas próprias experiências

para me motivar, o teu apoio nos bons e maus momentos… Tem a certeza que sempre

me inspiraste e desafiaste a me melhorar e apontar mais alto. Faltam-me as palavras e

estarei sempre em dívida para contigo.

A realização deste doutoramento só foi possível através do financiamento de uma bolsa de

doutoramento (SFRH/BD/62211/2009) pela Fundação para a Ciência e Tecnologia, I.P., que

reconheço e agradeço. Através da atribuição desta bolsa foi-me possível sustiver durante estes

anos de desenvolvimento do doutoramento.

Gostaria de agradecer ao Doutor Carlos Santos, responsável pela Unidade Estratégica de

Investigação e Serviços de Tecnologia e Segurança Alimentar (INIAV, I.P.-Polo do Lumiar), por

sempre me apoiar na prossecução dos meus objectivos académicos no seu departamento.

À Doutora Elsa M. Gonçalves que, para além da sua amizade, pelo seu espírito crítico muito me

ensinou e também pela sua boa disposição, apoio e encorajamento a quaisquer níveis durante

todos estes anos de convívio.

Os meus mais sinceros agradecimentos à Joaquina Pinheiro e à Ana Gomes, que estão também a

viver a façanha de desenvolver as respectivas teses de doutoramento, pela sua amizade e

contínuo apoio… Em muitas situações foram uma âncora durante a tempestade! Mesmo quando

as suas vidas pregaram rasteiras, continuaram sempre presentes! Não sei como teria superado

algumas fases sem vocês!

A todos os membros dos laboratórios, quer em Lisboa como em College Station, Texas, os meus

agradecimentos pela assistência técnica, amizade e sorrisos! Às técnicas Maria do Carmo Paula e

Ana Magalhães, muito obrigada por todo o apoio prestado na realização das análises

AGRADECIMENTOS

viii

microbiológicas. Paula Castillo e Freddy Ibañez, muito obrigada pela vossa hospitalidade, vocês

fizeram a minha estadia no Texas muito mais sociável e agradável.

A todos os membros do painel sensorial, obrigada pelo vosso contributo e paciência. Os vossos

comentários foram desafiantes para a continuidade destes estudos.

As mais variadas críticas de revisores anónimos providenciaram igualmente contributos

inestimáveis para o trabalho e a determinação para continuar em frente.

Gostaria também de agradecer à empresa CAMPOTEC, na pessoa da Eng. Tânia Caracol, por me

fornecerem os filmes de embalagem utilizados neste trabalho.

A todos os que me ofereceram tempo das suas próprias vidas para a concretização desta tese, o

meu muito obrigada! Retenho-me de os nomear na medida em que receio omitir alguém meritório

de tal agradecimento.

Eduardo, MEU LOBO, obrigada por acreditares em mim e por todos os sacrifícios, apoio (até

mesmo as inúmeras horas a descascar cenoura…) e amor que sempre me dedicaste. Obrigada

até por teres aprendido a pronunciar fenilalanina amónia-liase! Deste-me a força, a paixão e a

vontade para seguir em frente mesmo quando as coisas pareciam menos boas… Obrigada pelo

teu sorriso e calma… SEMPRE SEMPRE!

Aos meus pais, Olga e Fernando, agradeço por sempre me terem dado asas para perseguir os

meus sonhos e por sempre ajudarem o ar a sustentar o voo, mesmo quando isso poderia parecer

ser algo inatingível. Sempre foram uma fonte de inspiração para mim e sempre o serão! Obrigada!

Agradeço também à minha ‘Vó Amélia, a minha guerreira, que sempre me ensinou que não existe

nada melhor do que aquilo que é atingido através do nosso esforço e que devemos ir sempre atrás

dos nossos sonhos!

Todos os erros e limitações inclusos nesta tese são apenas meus.

Muito obrigada!

ix

TABLE OF CONTENTS

Abstract ............................................................................................................................................... i

Resumo .............................................................................................................................................. iii

Acknowledgments ............................................................................................................................. v

Agradecimentos ............................................................................................................................... vii

Table of contents .............................................................................................................................. ix

Abbreviation list ............................................................................................................................... xv

THESIS OUTLINE ...........................................................................................................................XVII

PART I – INTRODUCTION

1 Scope & Aims ..................................................................................................................... 3

2 Research design ................................................................................................................. 6

PART II - LITERATURE REVIEW

1 Why fresh-cut carrot is a healthy food? ........................................................................... 9

1.1 Fresh-cut carrot features ...................................................................................................... 9

1.2 Nutritional and bioactive composition ................................................................................. 11

1.2.1 Carotenoids ........................................................................................................................ 12

1.2.2 Phenolic compounds .......................................................................................................... 14

1.2.3 Dietary fibers ...................................................................................................................... 18

1.3 Botanical and agronomical aspects and postharvest behavior ........................................... 20

2 Why minimal processing affects fresh-cut carrot quality and which are the standard preservation treatments? ................................................................................................ 27

2.1 Minimal processing unit operations .................................................................................... 27

2.2 Quality decay ...................................................................................................................... 29

2.2.1 Physiological and biochemical changes ............................................................................. 31

2.2.2 Microbiological changes ..................................................................................................... 34

2.2.3 Nutritional and bioactive changes ....................................................................................... 36

2.3 Preservation treatments by default ..................................................................................... 38

TABLE OF CONTENTS

x

2.3.1 Decontamination ................................................................................................................. 38

2.3.2 Modified atmosphere packaging ......................................................................................... 40

3 How abiotic stresses can improve fresh-cut products quality? ................................... 43

3.1 Abiotic stresses features..................................................................................................... 43

3.2 Wounding ........................................................................................................................... 44

3.3 Heat shock .......................................................................................................................... 47

3.4 UV-C radiation .................................................................................................................... 50

PART III – MATERIAL & GENERAL METHODS

1 Plant material .................................................................................................................... 57

2 Sample preparation .......................................................................................................... 57

2.1 Wounding intensities .......................................................................................................... 57

2.2 Induced phenolic synthesis studies .................................................................................... 58

2.3 Shelf-life studies ................................................................................................................. 58

3 Heat shock and UV-C stress treatments procedures .................................................... 59

3.1 Single application ............................................................................................................... 59

3.1.1 Heat shock .......................................................................................................................... 59

3.1.2 UV-C ................................................................................................................................... 59

3.2 Combined application ......................................................................................................... 61

4 Analytical procedures ...................................................................................................... 62

4.1 Headspace gas analysis ..................................................................................................... 62

4.2 Total phenolic content ........................................................................................................ 62

4.3 Phenolic profile and chlorogenic acid quantification ........................................................... 63

4.4 Total carotenoid content ..................................................................................................... 63

4.5 -carotene content ............................................................................................................. 64

4.6 Antioxidant capacity ............................................................................................................ 64

4.7 Phenylalanine-Ammonia Lyase (PAL) Activity .................................................................... 65

4.8 Polyphenol Oxidase (PPO) Activity .................................................................................... 65

4.9 Peroxidase (POD) Activity .................................................................................................. 66

4.10 Dietary fiber content ........................................................................................................... 66

4.11 pH ....................................................................................................................................... 66

TABLE OF CONTENTS

xi

4.12 Soluble solids content ......................................................................................................... 66

4.13 Color ................................................................................................................................... 67

4.14 Sensorial analysis............................................................................................................... 67

4.15 Microbiological responses .................................................................................................. 68

5 Statistical analysis ........................................................................................................... 68

5.1 Analysis of variance ............................................................................................................ 68

5.2 Response surface methodology and model fitting .............................................................. 68

5.3 Hierarchical cluster and principal component analysis (PCA) ............................................. 70

5.4 Modeling microbial changes ............................................................................................... 70

5.5 Modeling phenolic content changes ................................................................................... 71

PART IV - DEVELOPED RESEARCH STUDIES

1 Fresh-cut carrot: raw material bioactive quality and decay profile .............................. 75

1.1 Morphological distribution of bioactive composition along cv. Nantes carrot tissues from two crop seasons................................................................................................................ 75

1.1.1 Results and discussion ....................................................................................................... 76

1.1.1.1 Bioactive markers & related enzymes .............................................................................. 76

1.1.1.2 Antioxidant capacity.......................................................................................................... 79

1.2 Characterization of the total phenolic content in two carrot cultivars (Nantes vs. Navajo) and in two cv. Nantes crop seasons (spring vs. fall) ........................................................... 81

1.2.1 Results & Discussion .......................................................................................................... 81

1.2.1.1 Total phenolic content ...................................................................................................... 81

1.3 Phenolic synthesis as affected by tissue type and wounding intensity ............................... 83



1.3.1.2 PAL activity ....................................................................................................................... 85

1.3.1.3 PPO activity ...................................................................................................................... 87

1.3.1.4 Color ................................................................................................................................. 87

1.4 Phenolic synthesis as affected by peel removal and shredding .......................................... 89


1.4.1.1 Total phenolic content and chlorogenic acid quantification ............................................... 90

1.4.1.2 PAL activity ....................................................................................................................... 95


1.5 Standard minimal processing and storage effects on fresh-cut carrot quality ..................... 98


1.5.1.1 Headspace gas analysis ................................................................................................... 98

TABLE OF CONTENTS

xii


1.5.1.3 Total carotenoid content ................................................................................................... 101

1.5.1.4 Antioxidant capacity .......................................................................................................... 102

1.5.1.5 Dietary fiber content ......................................................................................................... 103

1.5.1.6 pH ..................................................................................................................................... 104

1.5.1.7 Soluble solids content ....................................................................................................... 105

1.5.1.8 Sensorial analysis ............................................................................................................. 106

1.5.1.9 Microbiological responses ................................................................................................ 108

2 Heat shock and UV-C stress effects on carrot quality: single and combined application ....................................................................................................................... 113

2.1 Heat shock and UV-C stress effects on fresh-cut carrot quality ........................................ 113

2.1.1 Results & Discussion ........................................................................................................ 115


2.1.1.2 Total phenolic and carotenoid contents ............................................................................ 115

2.1.1.3 POD activity ...................................................................................................................... 117

2.1.1.4 Color ................................................................................................................................. 118

2.1.1.5 Sensorial analysis ............................................................................................................. 119


2.2 UV-C single stress effects on the wound-induced dynamics ............................................ 122

2.2.1 RSM modeling of UV-C treatment .................................................................................... 123

2.2.1.1 Results & Discussion ........................................................................................................ 124

Total phenolic content ........................................................................................................ 124 Total carotenoid content ..................................................................................................... 128 Color ................................................................................................................................... 129

2.2.2 UV-C dose validation ........................................................................................................ 129

2.2.2.1 Results & Discussion ........................................................................................................ 130

Total phenolic content ........................................................................................................ 130 PAL activity ......................................................................................................................... 131

2.3 Heat shock [100 c/45 s] single stress effects on the wound-induced dynamics .............. 132

2.3.1 Phenolic synthesis as affected by heat shock application before or after peel removal .... 132

2.3.1.1 Results & Discussion ........................................................................................................ 134 Total phenolic content ........................................................................................................ 134

2.3.2 Heat sock effects on wound-induced phenolic synthesis dynamic ................................... 135

2.3.2.1 Results & Discussion ........................................................................................................ 136 Total phenolic content & PAL activity.................................................................................. 136 Total carotenoid content ..................................................................................................... 139

POD activity ........................................................................................................................ 139

2.4 Heat shock and UV-C combined stress effects on the wound-induced dynamics ............ 142



TABLE OF CONTENTS

xiii

2.4.1.2 Total carotenoid content ................................................................................................... 146


2.4.1.4 PAL activity ....................................................................................................................... 147

2.4.1.5 POD activity ...................................................................................................................... 149

2.4.1.6 pH, SSC, Dry matter and CIELab color ............................................................................ 150

2.5 Closing remarks................................................................................................................ 152

3 Technological proposal: hurdle concept into action .................................................. 155

3.1 Selected heat shock and UV-C stress treatments and MAP effects on fresh-cut carrot quality ............................................................................................................................... 155




3.1.1.3 Total carotenoid and -carotene content .......................................................................... 162


3.1.1.5 PAL activity ....................................................................................................................... 165

3.1.1.6 POD activity ...................................................................................................................... 168

3.1.1.7 Phenolic synthesis dynamic ............................................................................................. 169

3.1.1.8 pH and soluble solids content (SSC) ................................................................................ 171

3.1.1.9 Color ................................................................................................................................. 172

3.1.1.10 Sensorial analysis............................................................................................................. 173


3.1.1.12 Hierarchical cluster and principal component analyses .................................................... 180

3.2 Proposed technological alternative: effects on fresh-cut carrot quality and shelf-life ........ 184




3.2.1.3 Total carotenoid and -carotene content .......................................................................... 188


3.2.1.5 PAL activity ....................................................................................................................... 191

3.2.1.6 POD activity ...................................................................................................................... 193

3.2.1.7 pH and soluble solids content (SSC) ................................................................................ 194

3.2.1.8 Color ................................................................................................................................. 195

3.2.1.9 Sensorial analysis............................................................................................................. 196


3.2.1.11 Product shelf-life estimation ............................................................................................. 202

4 Final Remarks ................................................................................................................. 207

4.1 General conclusions ......................................................................................................... 207

4.2 Future prospects............................................................................................................... 211

TABLE OF CONTENTS

xiv

References ...................................................................................................................................... 213

Appendixes ..................................................................................................................................... 241

Appendix 1 ...................................................................................................................................... 243

Appendix 2 ...................................................................................................................................... 248

Appendix 3 ...................................................................................................................................... 255

xv

ABBREVIATION LIST

AOx Antioxidant capacity

AOxH Hydrophilic antioxidant capacity AOxL Lipophilic antioxidant capacity AOxT Total antioxidant capacity

CA Chlorogenic acid

CAE Chlorogenic acid equivalents

CCRD Central composite rotatable design

cfu Colony forming units

CRD Completely randomized design

CTR Carbon dioxide transmission rate

F&V Fruits and vegetables

FC Fresh-cut

GAE Gallic acid equivalents

HIPO Sodium hypochlorite solutions (chlorinated water)

HS Heat shock

HSP(s) Heat shock protein(s)

LAB Lactic acid bacteria

LTS Low temperature storage

MAP Modified atmosphere packaging

MeJa Methyl jasmonate

OPP Oriented polypropylene film

OTR Oxygen transmission rate

PAL Phenylalanine ammonia-lyase

PCA Principal component analysis

PME Pectin methylesterase

PM-MAP Perforation-mediated modified atmosphere packaging

POD Peroxidase

PPO Polyphenol oxidase

RH Relative humidity

RM Raw material

ROS Reactive oxygen species

RSM Response surface methodology

SSC Soluble solids content

TAPC Total mesophilic aerobic count

TCC Total carotenoid content

TE Trolox equivalents

TPC Total phenolic content

UV-C Short wavelength ultraviolet radiation

WI Whiteness index

Y&M Yeasts and molds

xvii

THESIS OUTLINE

This thesis is organized in 4 parts as shown in the schematic below. The first part introduces the

background information concerning the research topic, aims and research design. The second part

is dedicated to the review of the relevant literature on which the research is based. The third part

includes the common methods, technological and analytical procedures and statistical tools used in

the experimental research. Finally, the fourth part contains the set research studies in compliance

with the thought-out strategies where the specific methodology of each study is detailed. Closing

this part are the general conclusions together with some proposals for future works.

PART I – INTRODUCTION

3

1 SCOPE & AIMS

Fresh-cut (FC) vegetables mean raw fresh vegetables that have been peeled, cut, decontaminated

and packed products which are maintained under refrigerated conditions at all stages, from cutting

through distribution. These 100% edible products are intended to be consumed in its raw form and

have enjoyed an increasing presence in the marketplace since the 1980's to meet consumer

expectations in terms of freshness and nutrition value, variety, packaging and product appeal.

However the most important driving force behind fresh-cut product purchases is convenience.

Nonetheless, the rapid growth rates of fresh-cut products, sold via both retail and foodservice

channels, have begun to stabilize with growth trends leveling off as we enter the twenty-first

century. In fact, the establishment of the fresh-cut industry has experienced some difficulties mostly

because of the high perishability of the product.

It is well recognized that minimal processing and wounding have profound physiological effects on

plant tissues, and most consequences of cutting are physiologically deleterious. The higher nutrient

availability of cut vegetables and the presence of different microbial populations typical of the

individual ingredients as well as the caused damage promote biochemical and microbial instability.

Thus, the minimal processing renders fresh-cut fruits vegetables (F&V) highly perishable and the

loss of sensorial, nutritional and functional properties takes place quickly during products storage.

In terms of processing technologies a key strategy to control fresh-cut deterioration is to combine a

multiplicity of effective non-damaging treatments (regardless its nature), since interesting

synergistic effects might be developed (hurdle technology) to maintain product safety and help

preserve the food quality over a longer period of time. Quality control in the supply chain (from raw

materials, production and distribution) is crucial in terms of food safety, quality and environmental

impact. The globalization of market opportunities has shown that production systems need a new

approach that should focus on safety, quality and health rather than quantity and has shown that a

fully integrated and complex supply chain must be able to fulfill the consumers’ needs.

Actually, the demand for highly convenient fresh-cuts still increases, especially in light of renewed

consumer health-conscious attitudes brought on by awareness and appreciation of natural

phytochemicals and antioxidants in fruits and vegetables. This consumer trend is the result of

national and international health bodies increasing effort on defining and promoting healthy diets,

most commonly accepted that a diet rich in fresh and fresh-cut fruit and vegetables (F&V) can

reduce the risk of most cancers and heart disease. However, credible promotion of fresh-cut F&V

consumption on the basis of its specific nutritional value and health-related benefits requires clear

definition concerning processing and storage/marketing chain conditions.

Carrot (Daucus carota L.) composition contains high amounts of carotenoids, phenolic compounds,

vitamins and dietary fiber which have been specifically highlighted regarding its potential to prevent

chronic and oncological diseases. In fact, carrot stands out as the main vegetable source of -

carotene (about 60-80% of the total carotenoid content) and, apart from its input to the vegetable

antioxidant capacity, significantly contributes to fulfill vitamin A daily requirements (FDA/USDA,

SCOPE & AIMS

4

2011). Moreover, its phenolic composition, mainly composed by hydroxycinnamic acids, also

actively contributes to carrots antioxidant capacity.

The rapid quality deterioration and reduced shelf-life of fresh-cut carrot (sliced, chopped or

shredded) is in part due to microbial proliferation during storage, especially lactic acid bacteria,

quickly surpassing the well-defined microbiological thresholds (7 days). Nonetheless, it was been

identified that changes in fresh-cut carrot sensory quality such as losses in orange intensity and

fresh-like flavor caused by minimal processing (Seljåsen et al., 2001; Barry-Ryan et al., 2000;

Sant’Ana et al., 1998) could occur sooner than critical microbial development and impair the

products’ acceptance (4-5 days). These important components of fresh-cut carrot quality are

difficult to commercially analyze and usually it is simply assumed that “if it looks good, it tastes

good”. Consequently, the shelf life of the product will, very likely, be overestimated and the repeat

buying by consumers could be affected.

As a result, development of integrated alternative technologies and product shelf life estimation

based on all aspects of quality of fresh-cut carrot, such as heat shock and UV-C radiation in

concert with packaging are considered. These treatments, known as abiotic stresses by nature, act

simultaneously over the main deterioration mechanisms, microbial and physiological, provide a

chemical-free product as well with potential to improve products quality, safety, bioactivity,

sensorial attributes and shelf-life.

Heat treatments are generally effective in reducing microbial levels and, under suitable

time/temperature conditions, it proves to be an effective methodology to control microbial growth in

several fresh-cut F&V (Aguayo et al., 2008; Li et al., 2001 a). Several biochemical pathways are

likely to be involved in the stress responses imposed by heat, namely synthesis of heat shock

proteins, interruption of normal cellular protein synthesis, including quality-related enzymes

(polyphenol oxidase, peroxidase and phenylalanine ammonia-lyase) and also changes in the

kinetic behavior of already synthesized enzymes (Loaiza-Velarde et al., 2003; Saltveit, 2000).

Reports on the effectiveness of UV-C treatments in reducing microbial loads on the surface of

fresh-cut F&V and particularly over pathogens are found (Schenk et al., 2007; Allende et al.,

2006 b; Fonseca and Rushing, (2006). Moreover, UV-C is currently considered among the abiotic

stress treatments as the one of highest potential to enhance bioactivity content of fresh-cut F&V.

This treatment acts as an elicitor of resistance mechanisms in fruit and vegetables, and thus leads

to a rapid increase of stress-responses such as biosynthesis of phenolic compounds (via

phenylalanine ammonia-lyase activation) (Schreiner and Huyskens-Keil, 2006; Cisneros-Zevallos,

2003). At last, many researchers found that other physiological effects induced by UV-C benefit the

sensorial quality of fresh-cut F&V (Erkan et al., 2008; Lamikanra et al., 2005).

The aim of this thesis was to evaluate the effects of heat shock and UV-C radiation treatments on

fresh-cut shredded carrot quality as a multi-target technological approach. The pursued strategy

involved the study of the underlying biochemical effects triggered by the application of both

treatments as single or in combination (hurdle concept perspective) and focused on the use of the

controlled abiotic stress treatments towards the maintenance of fresh-cut carrot bioactive

SCOPE & AIMS

5

composition and microbial control, without impairment of products fresh-like quality during low

temperature storage (5 C). The above aim will be accomplished by fulfilling the following research

objectives:

- Evaluation of the main factors influencing fresh-cut carrot bioactive quality:

o Cultivar, crop season and tissues types;

o Tissues ability to accumulate phenolic compounds as a stress response to

wounding;

o Minimal processing and storage effects on quality changes;

- Selection and optimization of the abiotic stress treatments (heat shock and UV-C radiation)

conditions to improve products quality just after processing and during low temperature

storage;

- Validation of the selected abiotic stress treatments and evaluation of different MAP solutions

as to assure a high-quality product while estimating achieved shelf-life in regard to the

standard (industrial) practice.

6

2 RESEARCH DESIGN

The diagram below (Figure 1) is a schematic of the experimental setup showing the three research

topics main objectives.

Figure 1 Research design.

PART II - LITERATURE REVIEW

9

1 WHY FRESH-CUT CARROT IS A HEALTHY FOOD?

1.1 FRESH-CUT CARROT FEATURES

Current life standards bring forward some issues regarding taking on a healthy diet, where

convenience plays a key role in consumer food choices. Beside the convenience requirement,

acceptance of a food product depends on the fulfillment of nutritional and sensorial requirements

and also on the products functional claims. The last requirement is related to the presence and

amount of bioactive compounds with known effects on human health, namely those that contribute

to reduce risk factors of several non-transmissible chronic diseases (e.g., cardiovascular disease,

diabetes type 2 and certain types of cancer).

This consumer trend forced the food industry to react and provide to consumers practical and

healthy food choices. In that sense, the development of fresh-cut or minimally processed fruits and

vegetables was undertaken. These products fulfill the above mentioned requirements seeing as

they are healthy, convenient, ready-to-eat and commonly preserved using combined low intensity

preservation methods that assure fresh-like quality maintenance during the respective shelf-life

(Gómez et al., 2011, Artés et al., 2009, Allende et al., 2006a, Ahvenainen, 1996). Also, fresh-cut

products are processed as to provide a 100% edible product not requiring further processing prior

to its consumption.

Several definitions concerning either minimal processing or fresh-cut minimally processed products

are found in the literature (Gómez et al., 2011, Oms-Oliu et al., 2010, Rico et al., 2007, Allende et

al., 2006a, Watada and Qi, 1999, Ahvenainen, 1996, Tapia de Daza et al., 1996, Wiley, 1994,

Huxsoll and Bolin, 1989). Nonetheless, all report to the simultaneity of convenience – ready-to-eat

products, and fresh-like characteristics. Maintenance of the fresh-like characteristic implies that the

product consists of living tissues with an active respiratory metabolism, which is to say that fresh-

cut products are physiologically active.

In this work, the adopted definition for fresh-cut (FC) products is given by Watada and Qi (1999)

which define it as convenient, ready-to-eat, refrigerated, fresh, cut, decontaminated and packed

products, with minimal nutritional and sensorial losses during the respective shelf-life. In Table 1,

products that fall under this definition are identified and it can be observed that fresh-cut products

share in common a short shelf-life.

The quality decay profile of a fresh-cut product is extremely dependent on raw material types and

cultivar as it determines the factors that soon compromise products acceptance by consumers’,

such as susceptibility to browning, softening and microbial spoilage. Minimal processing

technology, designed specifically for each product and above used preservation methods

(conventional and/or emerging), requires a priori selection of specific nutritional and bioactive

markers and the detailed knowledge of the time-decaying sequential pattern that lead to fresh-like

quality losses. In this sense, cautious selection of preservation treatments that assure the prime

WHY FRESH-CUT CARROT IS A HEALTHY FOOD?

10

objective of freshness implies a comprehensive knowledge of the raw material and respective

quality-determinant factors, namely cultivar features, agronomical practices, harvest conditions and

postharvest behavior (Watada and Qi, 1999; Ahvenainen, 1996; Kim et al., 1993).

Table 1 Requirements for the commercial manufacture of fresh-cut fruits and vegetables (pre-peeled and/or sliced, grated or shredded) and expectable product shelf-life (Adapted from Ahvenainen,

1996)

Target consumer

Working principle

Processing demands Examples of suitable fruits and

vegetables

Shelf-life

(days at 5 C)

Catering industry; Restaurants; Schools; Industry

Preparation today, consumption tomorrow

- Standard kitchen hygiene and tools;

- No heavy washing for peeled and shredded produce (except potato);

- Packages can be returnable container.

Most fruits and vegetables 1 - 2

Preparation today, consumption within 3-4 days

- Disinfection (washing of peeled and shredded produce at least with water);

- Permeable packages (except potato).

Carrot, cabbages, iceberg lettuce, potato, beetroot, acid fruits and

berries 3 - 5

Retail stores and above mentioned consumers

Retail market

- Good disinfection (chlorine or acid washing for peeled and shredded produce);

- Additives; - Modified atmosphere

packaging.

Carrot, Chinese cabbage, red cabbage, potato, beetroot, acid

fruits and berries 5 - 7

Over the last century, carrots have largely been used as a popular cooking vegetable, salad item,

snack food, and raw vegetable and, as time-pressed consumers quickly demanded the

convenience factor, carrots market was swiftly broadened, offering consumers carrots in a more

portable and convenient way without losing its fresh-like characteristics. As a result, fresh-cut carrot

products, such as shredded, grated, pie-cut, sliced, sticks and baby carrots, emerged and are one

of the most consumed ready-to-eat vegetables worldwide (Ragaert et al., 2004). According to

Lucier and Lin (2007), just in the United States, about 80% of fresh-cut carrot products are

consumed at home. Carrots predominance in fresh-cut vegetable production, independently of its

cut format, characterizes it as an important component in this market owing success to carrots

freshness, pleasant flavor, nutritional and health benefits as well as to respective versatility of use

(Alasalvar et al., 2001).


11

1.2 NUTRITIONAL AND BIOACTIVE COMPOSITION

Carrot chemical composition varies according to the respective cultivar, agricultural handling,

harvest period and root development stage. An average chemical composition is shown in Table 2.

Carrots moisture content is in the narrow range of 86-90% and is considered as a reliable source of

carbohydrates and minerals, especially Ca, P, Fe and Mg.

Table 2 Average carrot chemical composition (values based on 100 g of edible portion). USDA, 2012.

Centesimal composition Vitamins Minerals Fatty acids

Water (g) 88.29 Vitamin C (total ascorbic acid, mg)

5.9 Ca (mg) 33 Total saturated (g) 0.037

Energy (kcal) 41 Thiamin (mg) 0.066 Fe (mg) 0.30 Total monounsaturated (g) 0.014

Protein (g) 0.93 Riboflavin (mg) 0.058 Mg (mg) 12 Total polyunsaturated (g) 0.117

Total lipid (g) 0.24 Niacin (mg) 0.983 P (mg) 35 Others

Carbohydrate, by difference (g)

9.58 Vitamin B-6 (mg) 0.138 K (mg) 320 -carotene (g) 3477

Fiber, total dietary (g)

2.8 Folate, DFE (µg) 19 Na (mg) 69 -carotene (g) 8285

Sugars, total (g) 4.74 Vitamin A, RAE(µg)/ IU

835/16706 Zn (mg) 0.24 -cryptoxanthin (g) 125

Vitamin E (-tocopherol, mg)

0.66 Lutein + zeaxanthin (g) 256

Vitamin K (phylloquinone, µg)

13.2 Lycopene (g) 1

Carrots supply a minimal amount of calories (Table 2) and, as most vegetables, holds low protein

and lipid content. Its richness in sugars mainly composed of sucrose, glucose, xylose and fructose

(Carlin et al., 1990) is highly correlated to the sweet taste perception which, in turn, is very

appreciated by consumers (Seljåsen et al., 2001).

Furthermore, carrots phytochemical composition is diverse and includes carotenoids (Block, 1994),

phenolics (Babic et al., 1993), polyacetylenes (Koidis et al., 2012), vitamins (especially does from

the B complex) and dietary fibers (Kochar and Sharma, 1992). The resulting balance between

lipophilic and hydrophilic antioxidant compounds allows it to be considered as a functional food with

significant health promoting properties (Arscott and Tanumihardjo, 2010; Hager and Howard,

2006). Besides, for health promotion and disease prevention, it is considered as important to know

the total antioxidant capacity (AOxT) since it represents the cumulative capacity of the dietary

components to scavenge free radicals (Pellegrini et al., 2003), allowing to quench free radicals in

both aqueous and lipid phases, reflecting an integrated and/or synergistic effects of all the

antioxidants present (Wu et al., 2004), which in carrots are mainly attributed to the presence of

phenolics and carotenoids, respectively, considered as carrots bioactive markers.


12

1.2.1 Carotenoids

Carotenoids are lipophilic compounds that are associated with the lipidic fractions. The respective

biosynthesis is well known (Clotault et al., 2008), where two C20 intermediate molecules

(geranylgeranyl diphosphate) are converted into phytoene, precursor molecule for all carotenoids

(Figure 2).

Chemically, carotenoids are polyisoprenoid compounds which can be divided into two major groups

(Figure 3): carotenes – strictly composed by carbon and hydrogen atoms, and xanthophylls –

oxygenated hydrocarbon derivatives with at least one oxygen function (e.g., hydroxy, keto, epoxy,

methoxy or carboxylic acid groups). The characteristic structural feature of these compounds is a

conjugated double bound system that acts on the respective chemical, biochemical and physical

properties (Quirós and Costa, 2006). These natural pigments are responsible for the foods

perceived colors, from red to yellow, which is an attribute of significant importance, as it can be

considered as a quality criterion susceptible to change by food processing (Chen et al., 1995).

a)

b)

Figure 2 Carotenoid biosynthetic pathway. Boxed compounds indicate main carotenoids found in carrot (Clotault et al., 2008).

Figure 3 Chemical structure of carotenoids. a) Carotenes. b) Xanthophylls. (Adapted from

Landrier et al., 2012).


13

In carrot roots these compounds are present in high amounts and are confined in chromoplasts. No

photosynthetic function is recognized in the carrot root, and the high carotenoid concentration

found may be due to a mutation on its evolutionary history (Simon et al., 2008; Gross, 1991) since

carotenoid biosynthesis genes may have been targeted by artificial selection for the development

of orange colored roots (Clotault et al., 2012). Also, it has been shown that developmental and

environmental signals that are involved in plastid differentiation are related to carotenoid

accumulation in carrot roots (Fuentes et al., 2012).

Increasing interest in dietary carotenoids is related to respective biological functions. Carotenoids,

beside holding pro-vitamin A activity, are involved in gene expression regulation and act as

inhibitors of monocyte adhesion and in platelet activation and these effects are attributed to the

antioxidant properties of carotenoids, namely due to the ability to deactivate free radicals and

physically quench singlet oxygen (Rahman, 2007). These properties label carotenoids as

biologically active molecules with health-promoting properties such as anticancer activity,

prevention of development of cardiovascular pathologies, and antioxidant activity (Stahl and Sies,

2005; Alasalvar et al., 2001; Sant’Ana et al., 1998; Desorby et al., 1998; Wright and Kader, 1997;

Chen et al., 1995). The abundance of antioxidant carotenoids in carrots, especially β-carotene, is

one reason for carrot to be considered as a functional food (Figure 4).

Figure 4 Health promoting properties attributed to carotenoids (adapted from Sharma et al., 2012).

Carotenoids are precursors of vitamin A (retinol) which is the primary physiological function of

these compounds. Vitamin A acts on different levels, being involved in the maintenance of healthy

epithelial cell differentiation, normal reproductive performance, bone development and visual

functions (Gerster, 1997; Hinds et al., 1997; Futoryan and Gilchrest, 1994; Ross and Gardner,

1994). In carrot, the amount of carotenoids ranges between 6000 to 54800 µg.100 g-1

from which

40 to 80% is -carotene, entrusting carrot as the main vegetable source of -carotene (Simon and


14

Wolff, 1987 cit in Desorby et al., 1998). Related to its activity as precursor of vitamin A, theoretically

one molecule of -carotene yields two molecules of retinol with a conversion ratio of 100%,

whereas two molecules of -carotene yields one with a 50% conversion ratio (Sharma et al., 2012;

Heinonen, 1990 cit in Desorby et al., 1998; Simpson, 1983; Bushway and Wilson, 1982). On the

other hand, it has been identified that dietary -carotene is less easily absorbed by the body than

retinol (vitamin A) since there is first the need to convert -carotene to retinal and retinol, justifying

the found 12:1 retinol activity equivalent ratio (RAE), meaning that it takes about 12 µg of dietary -

carotene to provide the body with 1 µg of retinol. As suggested by the FDA (USDA, 2011), the

recommended daily allowance (RDA) in adults for vitamin A is around 2800 IU1 (averaged between

male and female RDA) and 100 g of carrot provide about 100% of the suggested daily value

(835 µg RAE equivalent to 2780 IU). In addition, -carotene has been reported to hold antioxidant,

antimutagenic, chemopreventive, photoprotective and immunoenhancement properties (Sharma et

al., 2012; van Helden et al., 2009; Rahman, 2007; Kopsell and Kopsell, 2006).

Besides -, and -carotene, four more carotene species are found in carrot, - and -carotene,

lycopene, and -zeacarotene. The usual respective percentage is 33:60:1:4:1:1, but it is known to

vary significantly when one considers genotype, location and year (Desorby et al., 1998). Usually,

90% of the carotenes found in carrots are in the trans form and the occurrence of cis forms in raw

carrots can be attributed to isomerization during the extraction procedures (Chen et al., 1995),

especially when organic solvents are used.

1.2.2 Phenolic compounds

Other phytochemicals that raised the scientific community interest due to the respective

antioxidant, antimutagenic and antitumor activities are the phenolic compounds (Tsao, 2010; Kris-

Etherton et al., 2002; Rice-Evans et al., 1997). Phenolic compounds are strong antioxidants that

potentiate the activity of antioxidant vitamins and enzymes serving as a defense system against

oxidative stress, namely by the accumulation of reactive oxygen species (ROS) (Tsao, 2010).

Phenolic compounds are plants ubiquitous secondary metabolites which are biosynthesized via

phenylpropanoid pathway (Dixon and Paiva, 1995), where phenylalanine ammonia-lyase (PAL, EC

4.3.1.5) plays a key role. PAL catalyzes the non-oxidative deamination of L-phenylalanine resulting

in trans-cinnamic acid and a free ammonium ion which is first step in the phenolic biosynthesis

(Figure 5).

1 1 IU is equivalent to 0.3 µg of retinol.


15

Figure 5 “Biosynthetic relationships among stress-induced phenylpropanoids”. (Dixon and Paiva,

1995).


16

More than 8000 phenolic compounds have been found, ranging from simple molecules (phenolic

acids) to highly polymerized compounds such as tannins (Bravo, 1998). The basic chemical

structure is the phenol molecule and rarely sugar residues are directly linked to an aromatic carbon

atom. The conjugated forms (sugar residues linked to hydroxyl groups present in the basic

structure) are more abundant and range from monosaccharides to oligosaccharides. The main

sugar residue found is glucose but galactose, rhamnose, xylose, and arabinose are also found.

Links to carboxylic and organic acids, amines, lipids and even other phenolics can also be found

demonstrating the diversity of known phenolic structures, allowing phenolics to be categorized in

different ways. Phenolic compounds have been classified according to their respective source,

biological activity and chemical structure. In the present work, the adopted classification concerns

the respective aglycone chemical structure (nonsugar component of the glycoside molecule) which

considers 4 major classes: phenolic acids, flavonoids, phenolic amides and other non-flavonoid

compounds (e.g., resveratrol). Included in the phenolic acids are the benzoic and cinnamic acids

derivatives based on a C1-C6 and C3-C6 backbones (Figure 6) (Tsao, 2010).

Figure 6 Phenolic acids (Tsao, 2010).

The great variability of phenolic compounds and respective occurrence as a complex mixture in

plant matrices adds difficulties to study the respective bioavailability (primarily determined by their

chemical structure) and physiologic and functional effects. Nonetheless, phenolic compounds have

what is considered the ideal chemical structure for free radical-scavenging activity since they

gather the necessary properties to define an antioxidant (Figure 7), such as reactivity as a

hydrogen/electron-donating agent and the transition metal-chelating potential.


17

Figure 7 Phenolic compound structural properties contributing to the respective antioxidant activity.

In carrot phenolic compounds are present through the entire root, but higher concentrations are

found in the epidermis (Mercier et al., 1994). Phenolic acids are the main phenolics found,

particularly hydroxycinnamic acids and derivatives, but p-hydroxybenzoic acids have also been

identified (Surjadinata and Cisneros-Zevallos, 2012; Talcott and Howard, 1999; Babic et al., 1993).

This specific phenolic composition provides carrot a high antioxidant potential since in vitro studies

have shown that phenolic acids such as caffeic, chlorogenic, ferulic and p-coumaric have higher

antioxidant capacity than that of vitamins E and C on a molar basis (Rice-Evans et al., 1997). A

typical orange carrot phenolic profile can be seen in Figure 8.

Figure 8 Typical chromatograph of phenolic compounds in orange carrot varieties. (Source: Alasalvar

et al., 2001; Peak identification: #1: 3’-caffeoylquinic acid; #3: 5’-caffeoylquinic acid; #5: 3’-p- coumaroylquinic acid; #6: 3’- feruloyquinic acid; #7: 3’,4’-dicaffeoylquinic acid; #8: 5’-feruloyquinic acid; #10: 5’-p-coumaroylquinic acid; #12: 4’-feruloyquinic acid; #13: 3’,5’-dicaffeoylquinic acid; #14: 3’,4’- diferuloylquinic acid; #16: 3’,5’- diferuloylquinic acid).


18

Chlorogenic acid has been identified as the major hydroxycinnamic acid in carrot, representing 42-

62% of the total detected phenolic compounds in carrots tissues (Zhang and Hamauzu, 2004). This

compound has also been identified as the major phenolic accumulated as a stress response to

wounding, increasing from an initial concentration of 16.2 mg.kg-1

up to 920 mg.kg-1

(Jacobo-

Velazquez and Cisneros-Zevallos, 2012).

Clinical trials have demonstrated that chlorogenic acid can be used to decrease glucose absorption

and consequently reduce the body mass of overweight and obese people (Thom, 2007). Other

biological activities associated with chlorogenic acid are anti-hepatitis B virus activity and inhibitory

effects on brain-tumor progression (Wang et al., 2009; Belkaid et al., 2006).

It has been found that the chlorogenic acid derivatives 4,5-dicaffeoylquinic acid and 3,5-

dicaffeoylquinic acid (isochlorogenic acids), also found in carrot tissues, are highly bioavailable in

humans. Known biological activities of these compounds include the respective use in anti-HIV

drugs (Zhu et al., 1999; Robison et al., 1996), treatment of hepatic failure (Choi et al., 2005) and

prevention of neurodegenerative diseases related to oxidative stress (Kim et al., 2005).

Phenolic compounds, apart from the biological activities, play an important role in fruit and

vegetables quality. These compounds have a preponderant effect in terms of the visual quality,

seeing as phenolics such as anthocyanins are natural pigments responsible for blue, purple and

red colors. Moreover, hydroxycinnamic acids, as an example, are substrates to polyphenol

oxidases which are responsible for the production of brown polymers that impair fruit and

vegetables visual quality (Tomás-Barberán and Espín, 2001). Taste is another sensory attribute

that phenolic compounds are (in part) responsible for since they interfere with the bitter (e.g.,

flavanone neohesperidosides in citrus fruits, 6-methoxymellein in carrot), sweet (e.g.,

dihydrochalcones) and pungent (e.g., capsaicin in paprika) taste perception. Also, the characteristic

astringency of some foods is attributed to the presence of tannins. The aroma of a given fruit and

vegetable is either influenced by the presence and respective amount of volatile phenolics.

1.2.3 Dietary fibers

Carrot is also considered as a privileged source of dietary fibers. Dietary fiber is mostly found in

plants structural components and is composed of complex, nonstarch carbohydrates and lignin

which are indigestible to humans since they do not produce enzymes capable for the respective

hydrolyzation into constituent monomers (Sharma et al., 2012; Turner and Lupton, 2011). As a

result of this inability, dietary fiber is considered to hold no caloric value. However, the associated

health benefits are considerable and include regulation of blood sugar, constipation prevention,

protection against heart diseases and also prevention of some forms of cancer (Turner and Lupton,

2011).

The definition of “fiber” and “dietary fiber” is under constant update and, as a result, several

definitions are found in the literature. The first concept was perhaps introduced by Hippocrates

(400 BC) stating “To the human body it makes a great difference whether the bread be made of

fine flour or coarse, whether of wheat with the bran or without the bran” but it was Hipsley (1953, cit


19

in Brownlee, 2011) who introduced the term “dietary fiber” when he observed that populations with

high fiber-rich food diets tended to also have lower rates of pregnancy toxaemia. In Table 3, a

collection of proposed definitions is shown, where fiber categories are also included. Despite the

differences found in some definitions, all have in common that fibers are not digestible by humans

and hold beneficial effects over the well-being.

Table 3 Proposed definitions for fiber and/or dietary fiber.

Author(s) Definition

Trowell (1972) Fiber is the food proportion derived from plants cellular wall that is poorly digested in human beings.

Trowell (1974) “Dietary fiber consists of remnants of the plant cells resistant to hydrolysis (digestion) by the alimentary

enzymes of man”.

Asp and Johansson

(1984); Selvendran

and Robertson

(1994)

Fiber is the group of non-starch polysaccharides and lignin, which includes several indigestible

polysaccharides in addition to the main components of the cell wall.

American

Association of

Cereal Chemists

(AACC, 2001)

Dietary fiber is defined as ‘‘the edible parts of plants or analogous carbohydrates that are resistant to digestion

and absorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary

fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances. Dietary fibers

promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood

glucose attenuation”.

U.S. Institute of

Medicine (2002)

Divides “fiber” into three categories: (1) dietary fiber, (2) added fiber, and (3) total fiber, and defines them as:

(1) Intrinsic, intact and nondigestible carbohydrates and lignin found in plants; (2) Isolated nondigestible carbohydrates with beneficial effects in humans; (3) Sum of dietary and added fiber.

Tungland and

Meyer (2002);

Meyer (2004)

Fibers are an integral part of daily consumed foodstuffs, namely vegetables, cereal grains, fruits, legumes,

leguminous plants, among others. Dietary fiber is classified according to the respective “simulated intestinal

solubility” into insoluble or soluble fiber. Insoluble fibers include lignin, cellulose, and hemicelluloses, while

soluble fibers include pectins, beta-glucans, galactomanan gums, and a large range of nondigestible

oligosaccharides including inulin.

European

Commission

Directive

2008/100/EC

(2008)

Defines “fiber” as carbohydrate polymers with three or more monomeric units that are neither digested nor

absorbed in the human small intestine, and further divides it into the following categories:

(1) Edible carbohydrate polymers naturally occurring in the food as consumed; (2) Edible carbohydrate polymers which have been obtained from food raw material by physical,

enzymatic or chemical means and which have a beneficial physiological effect demonstrated by generally accepted scientific evidence;

(3) Edible synthetic carbohydrate polymers which have a beneficial physiological effect demonstrated by generally accepted scientific evidence.

WHO/FAO;

Codex Alimentarius

Commission

(2008a,b)

Defines “dietary fiber” as carbohydrate polymers with ten or more monomeric units that are not hydrolyzed by

the endogenous enzymes in the small intestine of humans and belong to the following categories:

(1) Edible carbohydrate polymers naturally occurring in the food as consumed; (2) Carbohydrate polymers, which have been obtained from food raw material by physical, enzymatic

or chemical means and which have been shown to have a physiological effect of benefit to health as demonstrated by generally accepted scientific evidence to competent authorities;

(3) Synthetic carbohydrate polymers which have been shown to have a physiological effect of benefit to health as demonstrated by generally accepted scientific evidence to competent authorities.

Dietary fiber can be further classified into insoluble and soluble fractions. Tungland and Meyer

(2002) established this difference based on the respective fiber solubility in a pH-controlled enzyme

solution (representative of human small intestine enzymes), while Elleuch et al. (2011) distinguish


20

these two fractions based on the respective solubility in water. Soluble dietary fibers include

oligosaccharides, pectins, -glucans, and galactomanan gums, whereas included in the insoluble

fraction are mainly cell wall components such as cellulose, hemicellulose and lignin (Yoon et al.

2005; Davidson and McDonald, 1998; Roehrig, 1988; Schneeman, 1987).

Soluble and insoluble dietary fibers hold distinct physiological functions and nutritional effects

(Tosh and Yada, 2010). Soluble fiber aids to reduce blood cholesterol and to regulate blood

glucose levels (Abdul-Hamid and Luan, 2000; Roehrig, 1988; Olson et al., 1987; Schneeman,

1987) due to the respective ability to increase its viscosity. On the other hand, insoluble fibers, due

to the respective porosity and low density, promote an increase of fecal bulk, reducing intestinal

transit (Elleuch et al., 2011), and are mostly fermented in the large intestine favoring the growth of

intestinal microflora, namely probiotic species (Tosh and Yada, 2010).

It has been reported that carrot cell wall is composed of pectin (7.41%; galacturonans,

rhamnogalacturonans, arabinans, galactans and arabinogalactans-1), cellulose (71.7 - 80.94%; β-

4, D-glucan), lignin (2.48 - 15.2%; trans-coniferyl alcohol, trans-sinapyl alcohol and trans-p-

coumaryl alcohol) and hemi-cellulose (9.14 - 13.0%; xylans, glucuronoxylans β-D-glucans and

xyloglucans) (Nawirska and Kwasniewska, 2005; Lineback, 1999; Kochar and Sharma, 1992). The

dietary fiber composition is known to be fairly stable and during fresh carrot postharvest storage

(six months; 8±2 C; RH of 85±1%) since no significant changes in this content are described

(Augšpole et al., 2013). In fact, due to carrot richness in dietary fiber, considerations about the

feasibility of using carrot peels (industry by-product) as a food ingredient has been raised

(Chantarro et al., 2008).

1.3 BOTANICAL AND AGRONOMICAL ASPECTS AND POSTHARVEST BEHAVIOR

Worldwide, carrot (Daucus carota L.) is one of the most cultivated crops and can be divided into

two groups of domesticated varieties: the Eastern or Asian (var. atrorubens), comprising mainly

purple and yellow roots, and the Western (var. sativus) mainly of orange coloration (Almeida, 2006;

Rubatzky et al., 1999). Historically, the first evidences of carrot cultivation/domestication dates

back 5000 years, in the now known region of Afghanistan. The wild carrot cultivars were of a

whitish to purple coloration from which the wide range of phenotypic and molecular diversity found

on the todays domesticated cultivars originated (Vivek and Simon, 1998; Heywood, 1983). The

domestication process of carrot is mainly attributed to Mediterranean societies, where the Greeks

had a significant part to the recognition of carrots health benefits and, consequently, its introduction

into the human diet (“Let your food be thy medicine and your medicine be thy food” – Hippocrates).

After the domestication and respective widespread trough Europe and Asia, the major change that

occurred on its historic evolution was the color change to orange in late XVI early XVII centuries.

Also, the works by Vilmorin-Andrieux (XIX century) led to the development of other varieties, with

different colors and without a hard core, which contributed to the acceptance and increased

consumption of this vegetable (Mazza, 1989).


21

The genus Daucus belongs to the Apiaceae (sin. Umbelliferea) family and it comprises about 25

species (Almeida, 2006). The domesticated carrot belonging to the sub-species Daucus carota

sativus is a biennial herbaceous plant, with short vegetative cycle, slow initial growth but steady

until harvest. Carrot plants morphologically consist of a stem, leaves, roots, flowers and fruits. The

plants’ stem is barely above ground during its vegetative state and has a somewhat convex apical

meristem from which the leaves develop establishing, at the base, a rosette-like shape (Rubatzky

et al., 1999). Carrot flowers are small, white to greenish-white or light yellow in color, with five

petals, five stamens and an entire calyx (Rubatzky et al., 1999; Ripado, 1981). The fruits are

characterized for being a small and light seed, aromatic and of a greenish-gray color (Ripado,

1981).

Three different anatomical tissues are

visually recognized in the carrot root

(Figure 9): peel or epidermis, cortical

parenchyma (also known as cortex) and

the central stele. The epidermis is the

most exterior layer that protects the root,

while the cortical parenchyma acts as

the roots storage unit where sugars and

pigments such as carotenoids are

contained and also facilitates water

movement from the exterior to the

xylem. The endodermis is lined with

casparian strips (bands consisting of

suberin) that act as a water regulation system since it prevents water and minerals from passively

seeping between the cells forcing water into endodermal cells as to reach the stele. The phloem

and xylem vessels (vascular tissues) are included in the central stele and are responsible for the

transport of photosynthetic metabolites from leaves to root and water and nutrients from the root to

the upper plant, respectively.

Carrots chemical composition, particularly its bioactive composition, varies according to the

respective cultivar, agricultural handling, harvest period and root development stage (Gajewski and

Dąbrowska, 2007; Brunsgaard et al., 2006; Kidmose et al., 2004; Seljåsen et al., 2001; Warman

and Havard, 1997; Sandhu et al. 1988). As examples, Seljåsen et al. (2013) reported that carrot

phenolic content is dependent on the genetic background and temperature during root

development plays a key role to carotenoid synthesis as temperature outside the range 16 C to

25 C compromises root color (Almeida, 2006).

The color of carrot roots is one of the most visible traits that can be used to distinguish carrot

cultivars (Figure 10 a) but, usually, carrots are classified according to the respective root shape

(conical, spherical or cylindrical; Figure 10 b) and length (Figure 10 c).

Figure 9 Schematic internal structure of carrot roots. (image retrieved from: http://cnx.org/content/m43142/latest/?collection=col11410/latest; June 2013)


22

a)

b)

c)

Figure 10 Classification of carrot cultivars according to root (a) color, (b) shape (Rubatzky et al., 1999) and (c) length (www.carrotmuseum.co.uk).

The main grown carrot cultivars worldwide are Nantes variants. Nantes carrots belong to the semi-

late group of roots with high production yields, harvested 70 to 200 days after seedling,

characterized by an intense orange color, cylindrical uniform shape, without lateral or secondary

roots and with a relatively small and soft core (Almeida, 2006, Ripado, 1981). These characteristics

alone make Nantes carrot an ideal raw material for minimal processing.

Harvest is usually mechanical and carrots are harvested in an immature or partially mature state,

when the root reaches the desired diameter. Carrot is an intensive outdoor crop where higher

production yields are obtained when cultivation takes place in deep-neutral soils and temperate

climates (Anuário Hortofrutícola, 2000). Yields of about 50 t/ha and 80-100 t/ha are harvested in

the fall and the spring crops, respectively, although higher yields can be achieved in late harvests

by keeping mature roots in the fields (Almeida, 2006).

Even though root shape is primarily determined by the genotype, climate conditions play a major

role during root development, influencing both shape and size. For instance, soil temperature

between 15.5 C and 21.1 C leads to the development of good quality roots, while temperatures

below 15 C lead to the development of long, conical and thin roots (Joubert et al., 1994). Carrot

can be considered as a cool season crop, but it is suitable for regions with temperatures from 5 C

to 35 C, with 18 C being considered as the optimal temperature (Almeida, 2006; Romer, 1999).

Its cultivation is world spread and is marketed all year. According to the latest data from FAOSTAT

(2013), in 2011 carrot and turnip ranked 12th among the most cultivated vegetables worldwide,

showing the consumer demand for this vegetable. According to the same source and in reference


23

to the same year, China, USA and the Russian Federation led the world’s carrot and turnip

production, while Portugal figured as the 38th contributor (Table 4), with a 144 262 t production

corresponding to a productive area of 4 638 ha.

Table 4 World main contributors to carrot and turnip production and respective harvested area in 2011 (FAOSTAT, 2013).

Region/Country Production (t) Area harvested (ha)

World 35 658 466 1 184 284

Europe 8 629 810 278 469

EU (27) 5 224 941 113 638

Russian Federation 1 735 030 74 300

China 16 233 213 466 562

USA 1 305 600 34 070

Japan 602 003 18 740

Oceania 279 910 6 047

In Portugal the main carrot production areas are located in the “Ribatejo e Oeste”, “Póvoa de

Varzim-Esposende” and “Aveiro” regions, where the predominant cultivar is the Nantes, although

others, such as Anglia, Puma, Tancar, Nerac, Bangor and Bolero, can also be found (Anuário

Vegetal, 2006). Portuguese carrot has a reasonable quality where the main identified issue is the

existence of heterogeneous lots in terms of root caliber. This problem causes some constraints

regarding its acceptance in detriment to imported roots. Even though Portugal has excellent climate

and soil conditions for carrot production, some carrot productive areas are experiencing

abandonment due to the high production cost, labor shortage, market prices or even due to Spain,

Germany and France product imposition in the national market. The national production is

therefore insufficient to comply to its needs and, in 2009, the major suppliers were Spain and

France with a joint share of 97% of the 28 179 tons of imported carrot (Anuário Agrícola, 2011).

Concerns over appropriate cultivar selection and respective storage conditions should be taken in

order to produce high quality fresh-cut vegetables. Some studies have demonstrated that not all

cultivars are adequate for minimal processing and, in fact, this is a determinant feature to products

respective commercial shelf-life (Cabezas-Serrano et al., 2009; Gorny et al., 1999; Kim et al.,

1993). Choosing cultivars appropriate for minimal processing could rely in the taste and aroma

characteristics of the raw material and also those which hold lesser probability for color and

firmness deterioration during processing and storage (Gorny et al., 2000). Also, raw material

physiological maturity should be adequate for processing since it affects not only the FC sensorial

quality (e.g. color, firmness, sweetness) but also the respiratory and ethylene production rates

(Oms-Oliu et al., 2007; Soliva-Fortuny et al., 2004; Soliva-Fortuny et al., 2002). Therefore,

knowledge of the postharvest behavior of a commodity is of extreme importance as to define


24

respective storage conditions as well as to minimize the evolution of the ripening process in order

to maximize its shelf-life.

During the postharvest period, fruits and vegetables (F&V) undergo physical, chemical and

enzymatic changes namely in texture, loss in astringency, biosynthesis of volatile compounds and

conversion of starch into sugars (Beattie and Wade, 1996; Wills et al., 1989). The continuity of the

biochemical reactions such as respiration, transpiration and ethylene production during the

postharvest period, induce changes in color, aroma, taste and texture (Kader, 1992; Wills et al.,

1989) culminating in the F&V senescence.

The respiratory metabolism has major implications in what concerns postharvest storability of F&V.

The respiratory rate is taken as an indicator of F&V physiological activity and is directly proportional

to the produce perishability (Watada et al., 1990). Carrot is a vegetable that has a moderate

respiratory rate, of about 10-20 mg CO2.kg-1

.h-1

at 5 C (Saltveit, 2004a), however susceptible to

changes according to the respective storage temperature (Table 5).

Table 5 Carrot respiration rate according to storage temperature (adapted from Luo et al., 2004).

Respiration rate (mg CO2.kg-1.h-1)

Temperature (C) Topped (top trimmed) Bunched (presence of leaves)

0 10-20 18-35

5 13-26 25-51

10 20-42 32-62

15 26-54 55-106

20 46-95 87-121

As evidenced on Table 5, the respiration rate dependence on temperature shows the importance of

maintaining carrot at low temperatures in order to keep low respiratory levels and delaying decay.

Carrots optimal storage temperature is of 0-1 C with a 98-100% relative humidity (RH) and, when

stored in these conditions, carrots have a storage-life of 7 to 9 months. However, these conditions

are rarely achieved during commercial storage and distribution, where carrots are usually kept 5 to

6 months at 0-5 C with a 90-95% RH (Luo et al., 2004). Even though the use of controlled

atmospheres, particularly the decrease of O2 levels to 1-3%, is beneficial to significantly lower

produce respiration rates (Saltveit, 2004a), in carrot the use of controlled atmospheres does not

further increase the respective storage-life in comparison to air with high RH (Luo et al., 2004). In

fact, lowering O2 levels to about 1%, despite the inhibitory effect over sprouting, promotes carrot

decay with increased bacterial rot and off-flavors / odors development (Leshuk and Saltveit, 1990

cit in Luo et al., 2004). Also, carrot is susceptible to CO2 injury, where levels above 5% induce

spoilage due to increased microbial proliferation (Suslow et al., 2002).


25

As respiration, the produce transpiration also induces changes during the postharvest period,

leading to water losses which compromise texture and color. The use of adequate storage

temperature and RH minimizes the transpiration process and, concerning carrot, 3-4 C with a 98-

100% RH are suitable conditions for a 3 to 5 months postharvest storage (Suslow et al., 2002).

Ethylene (C2H4) is a naturally occurring hormone which is physiologically active at very low

concentrations – in the order of 0.1 ppm (Saltveit, 2004b; Kader, 1980). This colorless gas is

synthetized in small amounts by plants and is related to the respective growth and development.

Ethylene is readily diffused and the respective synthesis is continuously maintained in order to

sustain biologically active tissues (Saltveit, 2004b). Carrots produce very low ethylene

( < 0.1 μL.kg-1

.h-1

at 20 C), but are ethylene-sensitive where exposure to as little as 0.5 ppm

induce isocoumarin formation leading to the development of a bitter flavor (Luo et al. 2004; Suslow

et al., 2002). As a result, carrots should not be mixed with ethylene-producing commodities and

ethylene absorbers in the refrigerated storage chambers should also be used (Moldão and Empis,

2000).

27

2 WHY MINIMAL PROCESSING AFFECTS FRESH-CUT CARROT QUALITY AND

WHICH ARE THE STANDARD PRESERVATION TREATMENTS?

2.1 MINIMAL PROCESSING UNIT OPERATIONS

The production of fresh-cut fruits and vegetables involves preparation and preservation operations.

The preparation operations leading to a ready-to-use final product are mainly physical processes of

separation and downsizing while preservation operations, designed to extend products shelf-life,

include a multiplicity of methods, including the packaging operation. Critical to the production,

distribution and marketing of FC fruits and vegetables is the continuous maintenance of low

temperature as to preserve product quality. Temperature should be kept at levels adequate to keep

cells alive and to minimize the adverse effects of minimal processing, namely by reducing

enzymatic processes, respiration rates and ethylene production slowing even microbial

development (Oliveira Silva et al., 2012; Watada and Qi, 1999).

Prior to minimal processing, raw materials should be sampled and tested for incidence of defects

(e.g. physical damage) and insect infestation, and quickly moved into an appropriate temperature

storage room. Many quality problems can be avoided by rejecting inferior quality raw material on

the reception dock and not allowing it into cold storage or the processing room.

A description of the general manufacturing unit operations flow chart for the production of fresh-cut

shredded carrot is shown in Figure 11 along with the respective recommendation/aim for each

processing step.

Physical segregation of laboring areas should be well defined within the minimal processing

technological flow chart (Figure 11). All preparation operations, including carrots (raw material)

preliminary wash, are usually confined to a work area designated by “low care” seeing as the

hygienic standards are less demanding than those regarding the subsequent operations. After

these operations, striker hygienic standards are imposed in the new working area designated as

“high care”. This physical separation of working areas is fundamental in minimal processing since,

once vegetable tissues are cut and become exposed, the respective susceptibility to degradation

and/or contamination increases drastically. As a result, controlling higher hygienic measures should

therefore avoid possible cross contamination from the raw material and from the equipment to the

cut product.

From the unit operations included in the technological processing of fresh-cut shredded carrot,

wounding operations such as peeling and shredding are of extreme importance since, from the

moment cellular disruption occurs, release of cellular contents, oxygen exposure and induced

physiological, biochemical and microbiological reactions lead to accelerated product decay. As to

minimize the cellular damage induced by wounding, peeling and cut operations should be done,

manually or mechanically, using sharp knives diminishing cellular crush (Laurila and Ahvenainen,

2002; Portela and Cantwell, 2001). Several studies have demonstrated that minimizing the

WHY MINIMAL PROCESSING AFFECTS FRESH-CUT CARROT QUALITY AND WHICH ARE THE STANDARD PRESERVATION TREATMENTS?

28

imposed cellular damage during fresh-cut vegetable production benefits the induced physiology of

the tissues and decreases product deterioration and abrasion peeling, mostly used in carrot and

potato processing, should be avoided (Rico et al., 2007a; Rico et al., 2007b; Ragaert et al., 2006;

Laurila and Ahvenainen, 2002; Barry-Ryan and O’Beirne, 1998; Ahvenainen, 1996; Bolin and

Huxsoll, 1991).

Figure 11 General unit operations in the production of fresh-cut shredded carrot. (Diagram created

based on Alegria et al., 2009; Moreti et al., 2007; Hager and Howard, 2006; Klaiber et al., 2005; Francis et al., 1999; Barry-Ryan and O’Beirne, 1998; Bolin and Huxsoll, 1991; Garg et al.,1990)

Unit operation Aims & Recommendations


29

Carrot peeling during minimal processing improves the appearance of the final product (Kenny and

O’Beirne, 2010), since browning reactions are prevented. It is known that phenol oxidizing

enzymes, such as polyphenol oxidase (PPO, EC 1.10.3.1.), are present at higher concentrations in

peels than in more inner carrot tissues, which was correlated with the respective browning potential

(Chubey and Nylund, 1969). Additionally, carrot peeling provides significant microflora reduction, of

about 2 Log10 according to Garg et al. (1990), as it removes a highly contaminated tissue and helps

to ensure that microbial limits are kept under control as to prevent quality loss of the fresh-cut

product. Nonetheless, FC carrot quality is affected by peeling method (mechanical vs. manual):

Kenny and O’Beirne (2010) reported that as wounding severity increased (manual to mechanical

method: abrasion), the shorter was the product shelf-life since it enhanced respiration rate,

damaged color, and induced higher ascorbic acid losses. On the other hand, these authors also

reported that severe peeling increased total phenol and antioxidant levels.

Concerning cut operation, Barry-Ryan and O’Beirne (1998) studied the effect of slicing carrot

manually (razor blade) or mechanically, and within this latter one, using sharp or blunt blades. The

results clearly demonstrated that the physical damage caused by slicing induced physiological

stress, enhanced microbial growth and changes in products appearance and aroma due to higher

cell permeability, higher levels of exudate and higher respiration rates which were dependent on

the blades sharpness (razor blade > sharp machine blade > blunt machine blade). The authors

concluded that FC carrot quality and shelf-life could be extended by minimizing physical damage

through the use of a sharp blade.

In general, total counts of microbiological populations on minimally processed vegetables after

processing are known to range from 3.0 to 6.0 Log10 units (Ragaert et al., 2007). Surface

decontamination treatments during minimal processing acquire a primordial role in the stability,

safety and overall quality of minimally processed vegetables (Hagenmaier and Baker, 1998). Carrot

falls into the low-acid (pH 5.8-6.0) and high humidity food category and, as a fresh-cut, the large

number of cut surfaces provides ideal conditions for microbial outgrowth. Decontamination is

usually done by immersion of the cut product into chlorine solutions (200 ppm up to 5 minutes), in a

proportion of 3 L.kg-1

(Ahvenainen, 2000) followed by a rinsing step to minimize the presence of the

decontamination agent from the products surface (5 ppm).

The packaging operation is another imposed barrier for preserving quality of FC products and

involves modified atmosphere packaging systems (MAP). To shredded carrot, flexible oriented

polypropylene (OPP) films are used and more than providing a physical barrier between the

product and surroundings, this operation is important to control products physiological activity

during the respective shelf-life.

2.2 QUALITY DECAY

The main quality changes in FC products (Figure 12) are mostly the result of wounding operations

(and respective intensity) which lead to fast quality losses namely due to a) Internal tissues


30

exposure to oxygen, leading to oxidative reactions; b) Physical tissue damage leading to cellular

disruption which promotes the release and intermixing of enzymes and substrates; c) Induction of

physiological responses in the cut tissues, namely increase in respiration and ethylene production

rates; and d) Rapid microbial outgrowth as nutrients become readily available providing favorable

conditions for microorganisms to proliferate increasing even the risk of eventual pathogenic

contamination (Oliveira et al., 2012; Oms-Oliu et al., 2010; Hodges and Toivonen, 2008; Soliva-

Fortuny and Martín-Belloso, 2003; Watada and Qi, 1999).

Figure 12 Main changes and consequences induced by minimal processing.

Quality suitable for fresh-cut shredded carrot consumption is limited to only 4 to 5 days as the

result of a series of dynamic factors that compromise products fresh-like characteristics

(Amanatidou et al., 2000; Barry-Ryan et al., 2000; Barry-Ryan and O’Beirne, 2000). Minimal

processing operations lead to increases in non-microbial and microbial spoilage during storage with

negative impact on sensory quality (Jacxsens et al., 2003) as summarized in Table 6.


31

Table 6 Main quality changes in fresh-cut carrot.

Quality trait

Cause Effect

Color

Pigment leaching during washing procedures (Alegria et al., 2009; Vandekinderen et al., 2008; Sant’Ana et al., 1998);

Changes in the phenolic metabolism (Cisneros-Zevallos et al., 1995; Howard et al., 1994; Howard and Griffin, 1993; Bolin and Huxsoll, 1991);

Lignin deposition on cut surfaces (Howard et al., 1994; Howard and Griffin, 1993; Bolin and Huxsoll, 1991; Tatsumi et al., 1991);

Superficial dehydration (Barry-Ryan et al., 2000; Cisneros-Zevallos et al., 1995; Howard et al., 1994; Bolin and Huxsoll, 1991;).

Superficial whitening

Texture

Increased endogenous and/or microbial enzymatic activities leading to tissue softening (Toivonen and Brummel, 2008; Nguyen-The and Carlin, 1994);

Decrease in texture (softening)

Lignification and/or superficial dehydration leading to tissue hardening (Toivonen and Brummel, 2008; Barry-Ryan et al., 2000).

Increase in texture (hardening)

Aroma and flavor

Sugar leaching during washing procedures (Alegria et al., 2009; Vandekinderen et al., 2008; Klaiber et al., 2004; Selijåsen et al., 2001; Carlin et al., 1989);

Accumulation of isocoumarin and 6-methoxymellein (Cantwell et al., 1989; Sarkar and Phan, 1979);

Excessive microbial outgrowth, especially lactic acid bacteria, pseudomonads and yeasts (Kakiomenou et al., 1996; Nguyen-The and Carlin, 1994; Carlin et al., 1990b; Carlin et al., 1989).

Losses in perceived sweetness;

Development of bitter flavor

Losses in characteristic aroma / Development of off-odors

Nutritional value

Nutrient leaching during washing procedures (Alegria et al., 2009; Vandekinderen et al., 2008; Klaiber et al., 2004; Selijåsen et al., 2001; Sant’Ana et al., 1998; Carlin et al., 1989);

Oxidation reactions promoted by wounding over the phenolic and carotenoid composition (Soliva-Fortuny and Martín-Belloso, 2003, Talcott et al., 2001; Talcott and Howard, 1999; Li and Barth, 1998).

Loss in nutritional value

2.2.1 Physiological and biochemical changes

Respiration rate increases are directly related to injury severity (Saltveit, 2004a) and several

biochemical phenomena are triggered as a response to wound-induced respiration. Reported

responses include synthesis of ATP-dependent phosphofructokinase involved in carbohydrate

breakdown leading to pyruvate, enhanced aerobic mitochondrial respiration and oxidative reactions

leading to browning (Saltveit, 1997; Rolle and Chism, 1987).

Associated to the respiratory activity is the ethylene metabolism. Additionally to its physiological

function, ethylene is also involved in plant responses to both biotic and abiotic stresses (Iqbal et al.,

2013; Saltveit, 1999). Ethylene has been described as a mediator of the wound signal, inducing

expression of defense genes (Watanabe and Sakai, 1998). The effects of wound-induced ethylene

are dependent on the commodity and respective sensitivity to ethylene, but stimulation of

respiratory rates, enzymatic activities, increase in cell permeability and loss of cellular


32

compartmentation have been referenced as main effects (Saltveit, 2002; Wang et al, 2002; Morgan

and Drew, 1997; Watada et al., 1990; Yu and Fang, 1980).

In what concerns FC carrot, the wounding intensity and storage temperature significantly influence

the products respiration rate as it can be seen in Table 7. As reported by Carlin et al. (1989) and

MacLachlan and Stark (1985), the respiration rate of FC carrot, particularly in the shredded format,

raises 2 to 8 times as compared to the whole commodity.

Table 7 Carrots respiration rate as affected by wounding intensity and temperature. (Adapted from

Gorny, 1997)

Respiration rate (mg CO2.kg-1.h-1)

Wounding intensity

Temperature (C) Whole peeled Sliced Sticks Shreds

0 - 5 11 15

5 9-12 13 19 24

10 17-21 25 42 46

23 54 72-81 - 108-126

Considering the production, distribution and commerciality temperature range (0 to 10 C), this

significant increase in respiration rate in FC carrot as a result of minimal processing is critical to

products quality and indicates that the packaging system should be well designed as the used films

should have high O2 and CO2 permeability in order to prevent the establishment of anaerobic

conditions favorable to microbial proliferation. Regarding ethylene, it has been shown that

concentrations above 0.1 ppm induce also the production of isocoumarin, related to the

development of bitter taste in FC carrot during the respective low temperature storage (Talcott and

Howard, 1999b; Lafuente et al., 1996).

Color is one of the main attributes that characterizes the freshness of most vegetables, and can

even influence sweetness perception and pleasantness (Clydesdale, 1993). Carrot is an example

as to demonstrate two distinct biochemical processes that compromise FC color, whitening and

browning (Bolin and Huxoll, 1991 a,b; Cisneros-Zevallos et al., 1997; 1995; Chubey and Nylund,

1969).

As carrots’ experience stresses, biotic or abiotic, synthesis of phenolic compounds and wound

barriers, namely lignin, are induced (Talcott and Howard, 1999; Babic et al., 1993; Howard et al.,

1994). Phenolic compounds play a key role in plant’s defense mechanisms protecting the tissues

against microbial/oxidative damage and are also involved in the healing process, namely lignin

biosynthesis. Lignin is the generic term for a large group of aromatic polymers resulting from the

oxidative combinatorial coupling of 4-hydroxyphenylpropanoids (Boerjan et al., 2003) which are

deposited predominantly in cell walls, making them rigid and resistant. However, lignin deposition

in the cut areas leads to carrots superficial whitening along with activation of oxidative enzymes,

such as lypoxygenase and peroxidase that incur in phenolic and carotene degradation (Howard


33

and Dewi, 1996; Howard and Griffin, 1993; Whitaker, 1991). Moreover, Alegria et al. (2010) and

Howard et al., (1994) found a positive correlation between peroxidase activity and superficial

whitening.

Cisneros-Zevallos et al. (1995) proposed a mechanism describing white discoloration development

on peeled carrots, involving both physical and physiological responses to wounding. The physical

response, reversible, is expressed as a color change due to surface dehydration while the

physiological response, observed during storage, involves the activation of the phenolic

metabolism and lignin biosynthesis resulting in an irreversible color change.

Another set of important reactions that lead to FC color changes are the browning reactions. Within

these reactions, enzymatic browning has been the focus of much research. FC products enzymatic

browning is mainly attributed to the activity of polyphenol oxidase (PPO). This enzyme participates

in the oxidation of phenolic compounds from which the formation of o-quinones which are further

polymerized to complex brown pigments (Garcia and Barrett, 2002, Nicolas et al., 1993). The

enzymatically induced color variation relates to the metabolized phenolic compounds and the

browning intensity can be related to the tissues richness in phenolics and PPO availability (Abreu

et al., 2011; Garcia and Barrett, 2002).

In FC carrot, browning reactions during product storage are uncommon since peels are removed

during processing. The importance of peels in this color change is a reflection of its richness in

PPO and high phenolic concentrations (Chubey and Nylund, 1969; Zhang and Hamauzu, 2004)

and since this tissue is removed during minimal processing, reports on FC carrot browning are

scarce.

Independently of the color change, whitening or browning, a common feature is present in both

mechanisms: the wound-induced phenolic synthesis trough the activation and increase of PAL

activity. Phenolic degradation is also promoted by enzymatic oxidation leading to changes in the

respective bioavailability or biological activity (Tomás-Barberán and Espín, 2001). Nonetheless,

since fresh-cut products consist of living tissues, still physiologically active, are able to synthetize

phenolic compounds (wound-induced synthesis) during the products shelf-life and thus lessen this

degradation trend and increase FC antioxidant capacity during product storage (Simões et al.,

2011; Reyes et al., 2007; Ruiz-Cruz et al., 2007; Toivonen and DeEll, 2002). The accumulation of

phenolic compounds is however dependent on several factors such as wounding intensity

(Surjadinata and Cisneros-Zevallos, 2012; Heredia and Cisneros-Zevallos, 2009), initial phenolic

concentration (Reyes et al., 2007), atmospheric gas composition during storage (Jacobo-

Velázquez and Cisneros-Zevallos, 2009) and also storage temperature (Padda and Picha, 2008).

Further considerations about the wound-induced responses will be undertaken in 3.

The tissues’ physical anatomy, particularly cell size, shape and packing, cell wall thickness and

strength, the extent of cell-to-cell adhesion, together with turgor status determine the firmness of a

vegetable tissue. The physical damage imposed during minimal processing cause’s subcellular

compartmentalization and the mixing of substrates and enzymes initiate reactions that normally do

not occur. Enzymatic textural changes can therefore occur since cell-wall pectic compounds are


34

readily available, for instance, demethylesterification by pectin methylesterase (PME, EC 3.1.1.11)

and pectin polymers hydrolyzes by polygalacturonase (PG, EC 3.2.1.15). PME catalyzes the

demethylesterification of galacturonic acid units of pectin, generating free carboxyl groups and

releasing protons (Giovane et al., 2004). Further depolymerisation of the demethylesterified pectin

can occur as a result of polygalacturonase activity. Interestingly, PME was first identified in carrots

(Fremy, 1840 cit in Giovane et al., 2004) more than a century ago when pectin modification to

pectic acid was observed in carrot juice. PG has as preferential substrate demethylated pectin, and

is therefore able to depolymerise the low methoxy pectin obtained by the action of PME, thus

showing the dynamic action of both enzymes (Luo, 2006) that lead to tissue softening. If excessive,

softening contributes to limit FC products shelf-life. Besides softening, and particularly in carrot,

increase in firmness can also be observed as a result of the lignification process triggered by

wounding operations (Toivonen and Brummel, 2008).

Non-enzymatic textural changes can also occur and usually are related to water loss (dehydration)

resulting in a loss of turgor and crispness. These losses are enhanced in FC since peel and sub-

epidermal layers are absent in the products exposing inner tissues (Toivonen and Brummel, 2008)

and facilitate exudation.

Other enzymatic reactions triggered by minimal processing and lead to the development of off-

odors are fatty-acid oxidation. Oxidation of unsaturated fatty acids to carbon dioxide by

lipoxygenase induces the release of volatile compounds that compromises the fresh-like aroma

(Rolle and Chism, 1987).

2.2.2 Microbiological changes

Fresh-cut vegetables harbour lower numbers of microorganisms than unwashed whole vegetables,

as a result of washing in chlorinated water. However, wounding operations and temperature abuse

during storage results in increased mesophilic aerobic counts (Nguyen-The and Carlin, 1994).

Microbial development can be considered as one of the major causes leading to FC quality

deterioration (Koek et al., 1983) and Jay (1994) reported that about 20% of FC products are

rejected on this basis. Microbial development during FC storage can lead to the accumulation of

secondary metabolites that are responsible for changes in aroma (ethanol and lactic acid

production) and texture as a result of released pectinolytic enzymes (Nguyen-The and Carlin,

1994). Spoilage of fresh-cut vegetables by bacteria is characterized by brown or black

discoloration, production of off-odors and loss of texture. Fermentative spoilage by lactic acid

bacteria or yeasts is also observed in several FC fruits and vegetables (James and Ngarmsak,

2010). A great diversity in number is also found in FC products and usually counts of 103 to 10

9

cfu.g-1

, including the eventual presence of pathogens, are found (Hagenmaier and Baker, 1998;

Nguyen-The and Carlin, 1994).

The microorganisms associated with FC products vary in accordance with produce type (raw

material) and storage conditions. This association demonstrates the importance of the raw material

quality and the importance of temperature as to determine the nature of the microflora present in


35

the product. Besides the native microflora, FC products are also susceptible to contamination

during processing. Vegetable pH, nutrient content and availability, storage temperature and RH are

also factors that significantly influence the prevalence of a microbial group over other (Rosa, 2002).

Although some vegetable hold in their composition compounds with antimicrobial activity (e.g.,

phenolics, glucosinolates and carbonyl compounds), the respective effect in controlling microbial

development is minimal (Cherry, 1999; Lund, 1992).

The native population found in FC products is mainly composed (80-90%) of Gram negative

bacteria belonging to the Pseudomonas, Enterobacter and Erwinia genera (Francis et al., 1999;

Nguyen-The and Carlin, 1994; Marchetti et al., 1992). Within these, Pseudomonas are more

competitive and about 57% of the total bacterial flora found in FC belongs to this genera (King et

al., 1991). The findings of Nguyen-The and Carlin (1994) and Koek et al. (1983) confirm this

prevalence, 5 to 10 times regarding other genera, and in fresh-cut carrot this genera is also most

expressive contributing to respective spoilage (Nguyen-The and Carlin, 1994). The spoilage

caused by these bacteria is characterized by the production of tissue-degrading enzymes like

pectolytic cellulases, xylanases, glycoside hydrolases and lipoxygenase, which contribute to FC

softening and also yellowing of green vegetables during storage (James and Ngarmsak, 2010;

Nguyen-The and Carlin, 1994).

Beside the high counts in Pseudomonas genera, FC carrots in particular and as well in other

vegetables (e.g. lettuce and cabbage), high counts in lactic acid bacteria (LAB) are also found.

These gram-positive bacteria are traditionally known as fermentative organisms and are associated

with fermented food products and with food spoilage. Souring of the product, gas production, lactic

and acetic acid production and slime formation are some of the effects observed as a result of

lactic acid bacteria fermentative metabolism and ability to proliferate under anaerobic conditions

(Ragaert et al., 2007). Carlin et al. (1990) found LAB counts in the order of 108 cfu.g

-1 in FC carrot

which was lower than the counts found for Pseudomonas. However, Kakiomenou et al. (1996)

found lactic acid bacteria to be the predominant organisms (followed by pseudomonads and

yeasts) in samples of FC shredded carrot and observed a pH drop as a result of lactic, acetic,

tartaric, citric and succinic production associated to LAB growth. These authors also isolated

Leuconostoc mesenteroïdes as the main homofermentative species causing FC shredded carrot

spoilage and correlated it with slime formation on the shreds.

In FC vegetables, the molds are less frequent than bacteria and yeast due to vegetable intrinsic

properties such as a slightly acid to neutral pH (Magnuson et al., 1990) and usually, the found

molds belong to the Sclerotinia, Mucor, Aspergillus, Cladosporium, Phoma and Rhizopus genera.

In contrast, numerous yeast species have been identified in FC products and respective counts can

sometimes be as high as the ones found regarding bacteria (Nguyen-The and Carlin, 1994). The

identified yeast species include Candida spp., Cryptococcus spp., Rhodotorula spp., Trichosporon

spp., Pichia spp. and Toruslaspora spp. and are associated with whole fresh produce and also FC

products spoilage (Nguyen-The and Carlin, 1994).


36

Although not their natural habitat, pathogenic microorganisms find suitable conditions to growth in

fruits and vegetables due to the richness and availability of nutrients. Due to the possible

contamination of raw materials, FC products can therefore be considered as a vehicle for the

transmission of bacterial, parasitic and viral pathogens capable of causing human illness, as

reported by Beuchat (1996) and Nguyen-The and Carlin (1994). The pathogens most frequently

linked to FC products-related outbreaks include bacteria (e.g. Salmonella, Escherichia coli), viruses

(Norwalk-like, hepatitis A), and parasites (Cryptosporidium, Cyclospora) (Francis et al., 1999;

Beuchat, 1998; Tauxe et al., 1997; Nguyen-The and Carlin, 1994). Among these, bacteria are of

the greatest concern regarding reported cases and gravity of illness (James and Ngarmsak, 2010).

Presence of Listeria monocytogenes, Aeromonas hydrophila, Salmonella, Escherichia coli

O157:H7, Yersinia enterocolitica and Campilobacter jejuni in FC products are considered as the

leading causes of FC products-related outbreaks in the USA (Olsen et al., 2000; Francis et al.,

1999; Beuchat, 1998; Nguyen-The and Carlin, 1994). In 2002, the EU Scientific Committee on

Food (2002) reported that the prevalence of foodborne pathogens on fruit and vegetables and their

involvement in outbreaks were not well documented from a European perspective, and as a result,

Abadias et al. (2008) published a study focusing on the FC market in Spain and found that within

300 analyzed FC samples, 4 tested positive for the presence of Salmonella and two harbored L.

monocytogenes. None of the samples tested positive for E. coli O157:H7, pathogenic Y.

enterocolitica or thermotolerant Campylobacter (Abadias et al., 2008).

Then again, despite high mesophilic and LAB counts found during FC carrots shelf-life (Alegria et

al., 2010; Klaiber et al., 2005 a; Nguyen-The and Carlin, 1994), very few outbreaks were linked

directly to carrots and it was found that FC carrot appear to have minimal microbial risk associated

with their consumption (Erickson, 2010; Abadias et al., 2008).

2.2.3 Nutritional and bioactive changes

Ahvenainen (1996) stated that focus should be given to the nutritional changes caused by minimal

processing and respective outcome during the products shelf-life. Several studies have, during the

following years, tried to fill this gap and emphasis was given to vitamin, sugar, amino acid, fat and

fiber contents as well as in the phytochemical composition (Antunes et al., 2013; Martínez-

Hernández et al., 2013; Jacobo-Velazquez et al., 2012; Surjadinata and Cisneros-Zevallos, 2012;

Abreu et al., 2011; Heredia and Cisneros-Zevallos, 2009 a,b; Vandekinderen et al. 2008; Reyes et

al., 2007; Bett et al., 2001; Lamikanra and Watson, 2001; Seljåsen et al., 2001; Lamikanra et al.,

2000; Agar et al., 1999; Buta et al., 1999; Gil et al., 1998; Rocha et al., 1998; Palmer-Wright and

Kader, 1997). Within those studies, a general decrease in nutritional quality is found as a result of

minimal processing and the main identified reasons were:

- Leaching of pigments, sugars, acids, vitamins and essential fatty-acids;

- Oxidation reactions, either enzymatic, induced by the presence of free radicals or by chlorine treatment;

- Enzymatic hydrolysis of structural macromolecules;

- Substrate consumption as a result of increased physiological and microbiological activities.


37

In FC carrot, reports on increased sugar leaching as a result of washing procedures are found

(Ruiz-Cruz et al., 2007; Klaiber et al., 2004; Carlin et al., 1990) and losses are further increased

during storage which was well correlated with metabolic changes (Seljåsen et al., 2001) and with

fermentative processes induced by MAP (Kato-Noguchi and Watada, 1997 a) and/or microbial

growth (Carlin et al., 1990). The described losses in FC carrot sugar content leads to changes in

the products sweet taste perception which could compromise respective acceptability (Seljåsen et

al., 2001).

Regarding processing effects over dietary fiber composition, no leaching of dietary fiber into the

processing water has been reported with blanching, boiling and canning of carrots (Nyman et al.,

1987). However, Chantarro et al. (2008) found that, to the production of an antioxidant dietary fiber

powder from carrot peels, blanching would result in the increase of total dietary fiber yield and in

the insoluble to soluble ratio of an antioxidant dietary fiber powder production.

Processing and storage have significant effects over FC carrot bioactive markers (phenolics and

carotenoids). Although studies focusing simultaneously on minimal processing and storage effects

on FC carrot bioactive markers are, to our knowledge, not found, literature covering independently

the above mentioned effects is.

Carotenoid pigments are relatively stable in their natural environment, but processing operations

may contribute to increase pigments degradation (Rigal et al., 2000). Concerning carotenoid

content, Vandekinderen et al. (2008) reported that main losses during carrot minimal processing

were attributed to compound leaching to the wash waters (decontamination and rinsing operations)

and further acknowledged that the impact of sodium hypochlorite on carotene content was

dependent on the used concentration (5 vs. 200 ppm) from which higher concentrations were

responsible for greater losses. During product storage, carotenoid content of shredded carrot is

reduced (up to 65%) and respective decrease is influenced by decontamination treatments, as

shown by Ruiz-Cruz et al. (2007). The decrease behavior in carotenoid content is commonly

related to oxidative changes namely those triggered by wounding (exposure of cut tissues to light

and oxygen) and can eventually be associated to losses in the products nutritive value as well.

However, Lana et al. (2011) reported an irregular trend in carotenoid concentration during storage

and, despite carotenoid losses induced by minimal processing operations, no significant changes

were determined during storage.

Technological processing (e.g., thermal treatments, enzyme treatments, fermentation, radiation

treatments) is known to induce changes in the phenolic composition of fruits and vegetables.

Usually, processing induces phenolic degradation, chemically or enzymatically, or induces changes

in the composition, impairing the products quality. These changes can even promote increased

release of phenolics from the respective matrices which, in turn, can influence the bioavailability

and respective biological activity (Tomás-Barberán and Espín, 2001).

After processing the phenolic content of FC grated carrot is decreased and respective losses

attributed to leaching effects during water treatments (Vandekinderen et al., 2008). Moreover, Ruiz-

Cruz et al. (2007) found that the initial phenolic losses in shredded carrot were dependent on used


38

decontamination agent (sodium hypochlorite, peroxyacetic acid, acidified sodium chloride vs.

unwashed shredded carrot).

To carrot, wounding operations included in minimal processing induce a series of cascade-

responses, that lead to the general increase in phenolic content as a stress response (Jacobo-

Velazquez et al., 2012; Surjadinata and Cisneros-Zevallos, 2012; Heredia and Cisneros-Zevallos,

2009 a,b; Klaiber et al., 2005 b). However, during storage changes in phenolic content depict an

interesting behavior: a significant increase is observed in the early stages of storage (3-6 days)

which is followed by a consistent decrease to the end of considered storage period (Ruiz-Cruz et

al., 2007; Babic et al., 1993; Klaiber et al., 2005 b). This behavior has been also described in other

FC fruits and vegetables (e.g. broccoli, apple, purslane, lettuce) and is related to a first tissue

response to wounding (phenolic synthesis) and subsequent phenolic oxidation (Amodio et al.,

2014).

2.3 PRESERVATION TREATMENTS BY DEFAULT

Minimal processing embodies the hurdle technology concept as introduced by Leistner and Rödel

(1976) to the fullest. Within this concept, microbial safety and stability as well as nutritional quality

during products shelf-life can be achieved by successful design and selection of adequate hurdles

(preservative factors), such as low temperature storage (Leistner, 2000). Many different treatments,

conventional and/or emerging, applied as single or in combination, have been tested to avoid

accelerated decay in FC products, addressing the physiological, biochemical and microbiological

changes induced by minimal processing and worsened during storage. The main steps throughout

the processing chain of minimal processing of shredded carrot involving preservative treatments

are the decontamination and modified atmosphere packaging operations (refer to Figure 11) and,

in this section, focus on those operations will be given.

2.3.1 Decontamination

Intact commodities are somewhat resistant to the proliferation of decay microorganisms seeing as

natural barriers [both physical (epidermis) and biochemical (phytoalexins)] prevent microbial

contamination and growth. However, in fresh-cut products the physical barriers are ruptured during

processing (peeling) which allows enough microbial breach to overcome the innate biochemical

defenses (Barros and Saltveit, 2013). As a result, appropriate measures are needed to control of

microbial outgrowth during FC products shelf-life, namely the introduction of a decontamination

step during FC processing to reduce initial microbial load (Siddiqui et al. 2011; Oms-Oliu et al.,

2010; Artés et al., 2009; Rico et al., 2008; Beuchat, 1998). Currently, to achieve such objective,

processors rely on the use of chemical decontamination agents such as chlorine.

The available published research indicates that the use of chlorinated water as well as most of the

more recent sanitizing agents such as chlorine dioxide, ozone, and peroxyacetic acid, are however

insufficient to achieve suitable microbial reduction (above 90% of initial microbial load) (Allende et


39

al., 2006a; Sapers, 2003; Beuchat et al., 1998; Guerzoni et al., 1996). Furthermore, these

treatments can lead to undesirable quality changes, negatively affecting the nutritional and sensory

quality of the product (Laurila and Ahvenainen, 2002). The decontamination efficiency associated

with the chemical agents is variable and dependent on the indigenous microorganisms and on the

physical-chemical and morphological characteristics of the vegetables.

In the fresh-cut industry, one of the most used chemical agents for decontamination purposes is

sodium hypochlorite (Simons and Sanguansri, 1997). FC carrot is no exception and

decontamination is commonly carried out by immersion of cut tissues into chlorinated water. The

usual concentration (as active chlorine, HIPO) ranges from 50 to 200 ppm and the typical treatment

duration is from 1 to 5 minutes (Sapers, 2001; Beuchat, 1998).

As Seymour (1999) reported, when product decontamination is done using HIPO solutions in the

narrow dosage of 100 to 125 ppm, the respective efficiency is in the range of 1 to 2 Log10 cycles.

However, over the last years, the use of such method for decontamination purposes is questioned

since it has been shown that chlorine has a limited effect for control of the microbial load, including

pathogenic bacteria, raising concerns about its use to assure the safety of FC products (Abadias et

al., 2008; Kim et al., 1999; Xu, 1999; Cena, 1998). The low associated efficiency has been

attributed to the HIPO consumption by the high organic and mineral load present on cut products

surface, being therefore unavailable for decontamination purposes (Parish et al., 2003; Sapers,

2001). Moreover, issues such as the proportion ratio of cut product to HIPO solution volume,

effective concentration of active chlorine, contact time and solution pH significantly influence the

achieved decontamination efficiency of HIPO solutions and should be design specifically by

product.

The associated low efficiencies could also be related to the fact that these hydrophilic solutions are

unable to reach the vegetable surface (hydrophobic in nature) where microorganisms could be

protected against the lethal effects of HIPO by a biofilm (Kim et al., 1999; Nguyen-The and Carlin,

1994). Moreover, the vegetables heterogeneous morphology provides difficult access sites

challenging the decontamination solution contact with the product surface (Seymour et al., 2002).

In regard to pathogenic flora, HIPO solutions hold low efficiency in eliminating this flora and also

minimize the natural competition effect (Abadias et al., 2008; Parish et al., 2003; De Roever, 1998)

raising concerns regarding the fresh-cut vegetables safety.

Additionally to the microbiological limitations, HIPO solutions can also induce sensorial changes in

the product, namely in aroma perception, giving place to an off-odor usually identified as

“disinfectant” (Alegria et al., 2010; 2009), loss of nutrients (Sant’Ana et al., 1998) and color

changes (by oxidation or leaching) which could lead to consumer product rejection.

To the best of our knowledge there is no mandatory limit regarding HIPO dosage in these products,

but chlorine residual amount should not exceed 5 ppm as admissible in potable water. Besides this

regulation, HIPO use should be limited since, when in contact with organic matter, formation of

carcinogenic chlorinated by-products such as chloramines and trihalomethanes (Xu, 1999; Wei et

al., 1999; Dychdala, 1991) is a risk and calls attention to several safety concerns regarding


40

chemical hazard to both human and environment. These current apprehensions have led to an

increased restriction on chlorine use whereas in some European countries, including Germany,

The Netherlands, Switzerland and Belgium, its use for washing fresh-cut products is banned

(Beltrán et al., 2005).

In the search for effective decontamination treatments, development of alternative treatments with

minimal impact to products fresh-like quality characteristics has risen over the last years.

Treatments such as UV-C, light pulses, electrolyzed water, power ultrasound, thermal treatments

(mild heat or heat shock), ionizing radiation and high pressure are some examples of alternative

procedures that have been evaluated FC products (Goméz et al., 2011; González-Aguilar et al.,

2010; Oms-Oliu et al., 2010; Alothman et al., 2009; Artés et al., 2009; Gil et al., 2009; Ölmez and

Kretzschmar, 2009; Rico et al., 2007; Allende et al. 2006a).

2.3.2 Modified atmosphere packaging

Modified atmosphere packaging (MAP) can be defined as the manipulation of the environmental

conditions, namely (and most used) by increasing CO2 levels and decreasing O2 levels inside the

package (Shewfelt, 1986). MAP can either be achieved passively or actively. Passively achieved

MAP involves packaging the product in a semi-permeable film, usually a bag, sealing it and, due to

the respiration metabolism of the product, oxygen levels decrease and carbon dioxide increases

until it reaches a steady-state. Ideally, this equilibrium should provide the desirable effects without

the establishment of anoxic (anaerobic) conditions. Passive MAP depends not only on the product

respiration, but also on temperature, products fill weight in the package, total respiring area and, of

course, on the packaging film O2 and CO2 transmission rates (Allende et al., 2004). Active MAP,

involving also the use of semi-permeable films but, before sealing, the existing gas in the package

is replaced by an ideal gaseous mixture of oxygen, carbon dioxide and nitrogen. Package sealing

should be done as soon as possible so that no changes to this atmosphere are introduced. Also

considered as active MAP are the uses of ethylene, oxygen or carbon dioxide

adsorbers/absorbers.

To the minimal processing technology, the simultaneous use of modified atmosphere packaging,

usually using O2/CO2 concentrations of 5-10%/2-5%, and low temperature storage are hurdles that

can be applied to extend the quality and shelf life of minimally processed products by minimizing

stress reactions (Kader et al., 1989; Labuza and Breene, 1989; Barry-Ryan et al., 2000; Farber et

al., 2003). Nonetheless, in carrot, reports on MAP storage are contradictory: Bruemmer (1988)

stated that MAP storage of carrot sticks is not beneficial since harvested carrots are physiologically

too mature for senescence control. Kato-Noguchi and Watada (1996, 1997a) reported that

accelerated glycolysis and increased ethanol and acetaldehyde levels are achieved when a < 2%

O2 concentration is used in fresh-cut carrot storage. On the other hand, Carlin et al. (1990b), in

minimally processed carrot, found that with a 2% to 10% O2 and 10% to 40% CO2, sugar content is

best maintained but excessive growth of lactic acid bacteria would favor the product spoilage when

CO2 concentrations above 30% were reached. Izumi et al. (1996) and Kakiomenou et al. (1996)


41

reported that the use of anoxic conditions (0.5% O2) was beneficial to minimally processed carrot

sensorial quality, but there was an increased risk to the development of anaerobic pathogens.

Higher sensorial quality and longer shelf-life was also achieved in purple carrots with a MAP

treatment of 90% N2, 5% O2, 5% CO2 but no significant effect was observed in orange carrots as

reported by Alasalvar et al. (2005). Alternative MAP, like combinations of high O2 (50%) high CO2

(30%) were also tested in minimally processed carrot (slices) and resulted in a longer product

shelf-life, up to 2-3 days when compared to passive MAP and was at least as efficient as an active

MAP of 1% O2 / 10% CO2 (estimated shelf-life of 8 to 12 days).

The use of semi-permeable flexible polymeric films involves several drawbacks to due their

structure and permeability. Fonseca et al. (2000) highlight seven weaknesses regarding the use of

flexible polymeric films: “(i) films are not strong enough for large packages, (ii) films permeability

characteristics change unpredictably when films are stretched or punctured, (iii) some films are

relatively good barriers to water vapor, causing condensation inside packages when temperature

fluctuations occur and consequently increasing susceptibility to microbial growth, (iv) films

permeability may be affected by water condensation, (v) the uniformity of permeation

characteristics of films is not yet satisfactory, (vi) films permeability is too low for high respiring

products, and (vii) products that require high CO2/O2 concentrations may be exposed to

anaerobiosis because of the high ratio of CO2 to O2 permeability coefficients” (Fonseca et al.,

2000).

The emergent interest in finding adequate packaging solutions namely for high respiration

products, such as fresh-cut carrot, led to the development of alternative MAP systems. Emond and

Chau (1990, cit in Fonseca et al., 2000) introduced the concept of perforation-mediated MAP (PM-

MAP). As for conventional passive MAP, the involved gases in PM-MAP are O2 e CO2, and it has

been considered as ideal to several fresh vegetables like broccoli, spinach and cauliflower (Ratti et

al., 2002) and some studies considering the use of PM-MAP in fresh-cut products has also proven

beneficial to shredded grated or sliced carrot (Edelenbos et al., 2009), baby spinach leaves

(Allende et al., 2004) and shredded Galega kale (Fonseca et al., 1999). Seljåsen et al. (2004)

suggested that storage temperature and packaging material type had the greatest influence on the

sensory quality of carrots since package should ensure adequate ventilation under low temperature

storage conditions has to maintain the product quality during the entire commercial circuit (from

producer, distribution chain till the final consumer). Avoidance of anaerobic conditions in ventilated

packages (provided by perforations) was, according to Seljåsen et al. (2004), of extreme

importance to ensure a high quality product since it prevented increased production of ethanol with

repercussions to carrots sensorial attributes (ethanol flavor and “sickeningly” sweet taste). Hurme

et al. (1995) have also shown that the use of micro-perforated packaging films increased grated

carrot shelf-life by at least 4 days in regard to non-perforated packages. Edelenbos et al. (2009),

showed that the use of PM-MAP provided a higher sensory quality of minimally processed carrot,

with high terpene flavor quality and, after 8 days of storage (5 C), oxygen levels of >9% and <13%

in CO2 were achieved, while samples packaged with non-perforated films, the atmospheric


42

composition decreased to about 0% O2 and up to about 45% CO2, leading to significant changes in

the products sensory profile.

43

3 HOW ABIOTIC STRESSES CAN IMPROVE FRESH-CUT PRODUCTS QUALITY?

3.1 ABIOTIC STRESSES FEATURES

One of the most outstanding functions known to plant cells is the respective ability to respond to its

own defense against imposed stresses, either biotic or abiotic. This induced response is achieved

due to the vegetable cell plasticity in respective genomics, proteomics and metabolomics which

ultimately lead to the modulation of certain defense metabolic pathways (Aghdam et al., 2013;

Timperio et al., 2008). The plant's adaptive mechanisms to both biotic and abiotic stresses respond

in a similar manner, namely by triggering a primary defense mechanism and by inducing the over-

expression of specific proteins as consequence of each stress (Timperio et al., 2008), as is

illustrated in Figure 13.

Figure 13 Specific and common stress induced responses in plants. Adapted from Pandey et al., 2011

and Timperio et al. (2008). ([1]- Jonak et al., 2000; [2]- Christopher et al., 2004; [3]- Harms et al., 1998; [4]- Suzuki and Mittler, 2006; [5]- Amme et al., 2006; [6]- Murata et al., 2007).

HOW ABIOTIC STRESSES CAN IMPROVE FRESH-CUT PRODUCTS QUALITY?

44

It has already been established that different protein families are known to be associated with

plant's response to stresses by being newly synthesized, accumulated or decreased, playing a

major role in the plant’s antioxidant defense system as well. Focusing on abiotic stresses, these

are able to induce protein dysfunctions namely by changing proteins levels, solubility or structure

(Timperio et al., 2008), where cell adaptation to maintain cellular homeostasis under stressful

conditions is essential to the respective survival. To achieve this aim, one protein family involved in

the stress response stands out, heat shock proteins (HSP) (Aghdam et al., 2013; Timperio et al.,

2008).

Heat shock proteins constitute a stress-responsive family of proteins first detected as a plant

response to high temperature by the respective accumulation (Ritossa, 1962), characteristic that

gave them their name. Nover (1991 cit in Aghdam et al. 2013) and Vierling (1991) concluded that

these proteins played an important role in plants’ heat tolerance (thermotolerance) since, under

normal conditions, these proteins are practically undetectable but rapidly accumulate as a

response to heat shock. It was also found that the HSPs synthesis and accumulation rates were

proportional to the temperature and exposure time to the stress (Saltveit, 2002, Waters et al., 1996;

Howarth, 1991). Moreover, accumulation of HSPs not only induces protection against the stress

that causes their biosynthesis but also against any subsequent stressful situation (Chen et al.,

1988; Lin et al., 1984). However, HSPs gene expression and protein accumulation is not triggered

by heat shock alone, factors such as fruit ripening (Neta-Sharir et al., 2005; Ding et al., 2001;

Medina-Escobar et al., 1998; Lawrence et al., 1997), low temperature (Lopez-Matas et al., 2004;

Lubaretz and Zur Nieden, 2002; Ukaji et al., 1999) and oxidative stress (Jofre et al., 2003; Lee et

al., 2000; Banzet et al., 1998; Eckey-Kaltenbach et al., 1997) are some of the environmental and

developmental factors that induce HSPs accumulation. In fact, oxidative stress, a secondary stress,

boosts the levels of reactive oxygen species (ROS) which stimulates synthesis and accumulation of

HSPs (Sevillano et al., 2009), showing that, during plant’s evolutionary progression, vegetable cells

were able to reach a high level of control over ROS toxicity and use these molecules as signals to

the induction of HSPs as part of their defense mechanism (Timperio et al., 2008). According to the

above, one can state that plants have developed a highly sensitive and specific system to monitor

the respective surrounding environment (Lamikanra et al., 2005).

3.2 WOUNDING

In fresh-cut fruit and vegetable products wounding is intrinsic to minimal processing arising mainly

from peeling and cut operations and the wound-induced response is dependent on tissue

characteristics, maturity of the fruit or vegetable of interest, the coarseness or sharpness of the

cutting equipment used and the temperature at which the cutting is done (Toivonen and DeEll,

2002).

Wounding is one of many abiotic stresses that trigger a cascade of signals that migrate to non-

wounded tissues and induce a series of physiological responses (Saltveit, 2000; Ke and Saltveit,

1989). As plants are exposed to agents that cause tissue damage, a cascade of mechanisms


45

towards tissue regeneration are activated (Rakwal and Agrawal, 2003; Ryan, 2000) and most

responses occur in a time frame of minutes to hours. These responses include production/release,

perception and the transcription of specific signals to activate wound-related defense genes (Zhao

et al., 2005; Cantos et al., 2001a; Léon et al., 2001, Orozco-Cardenas et al. 2001).

An initial response to wounding is the increase in accumulation of ascorbate synthase, ascorbic

acid and ethylene (Toivonen and Hodges, 2011; Reyes et al., 2007). Wound-induced synthesis and

accumulation of phenolic compounds through the phenylpropanoid pathway as is up-regulated by

phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) (Ke and Saltveit, 1989) and stands out as a

crucial defense mechanism against wounding stresses (Heredia and Cisneros-Zevallos, 2009a;

Dangl et al., 2000; Dixon and Paiva, 1995; Matern and Grimmig, 1994). PAL enzyme metabolizes

the first committed step of plants phenylpropanoid metabolism, and is considered as a precursor of

vast number of compounds with different biological functions such as lignins, flavonoids,

coumarins, stilbenes and benzoic acid derivatives (Dixon and Paiva, 1995; Hahlbrock and Scheel,

1989) and is also associated with wound-induced ethylene production.

The stimulation of the phenylpropanoid pathway can be experimentally achieved by using

resistance elicitors or by exposure to specific stress conditions, namely the respective wounding

intensity (Cisneros-Zevallos, 2003; Saltveit, 2000). However, the wound-signaling process in plants

is complex and involves a multiplicity of molecules with regulatory activities, where “linear”

signaling pathways can form up a network allowing signals to overlap and interconnect which is

known as cross-talk between wound-signaling pathways (Rakwal and Agrawal, 2003). Methyl

jasmonate (MeJa) is, as ethylene, a phytohormone responsible for the perception and transcription

of wound signals and the related pathway is the octadecanoid pathway (Rakwal and Agrawal,

2003; Mueller et al., 1993) and, according to the findings of Saniewski et al. (1987) and Campos-

Vargas and Saltveit (2002), a positive correlation is found between plants exposure to exogenous

MeJa (stress) and synthesis of phenolic compounds, presumably as an integral part of the plant’s

defense system (Heredia and Cisneros-Zevallos, 2009a). In Figure 14 is a schematic

representation of the wound-induced responses leading to phenolic synthesis during carrots

healing process, as proposed by Jacobo-Velázquez and Cisneros-Zevallos (2012).


46

Figure 14 Wound-induced phenolic synthesis during the wound healing process. (Adapted from

Jacobo-Velázquez and Cisneros Zevallos, 2012).

Phenolic compounds can act as antioxidants by the respective interference in autocatalytic (chain)

oxidative reactions (primary oxidations) or by quenching free radicals (secondary oxidations)

(Gordon, 1990). These compounds are also known by the respective ability to respond to

phytopathogenic infections and promotion of plants regenerative processes by forming defensive

barriers namely by the increase in the production of compounds such as lignin and suberin

(Howard et al., 1994; Hoffman and Heale, 1987; Garrod et al., 1982). Moreover, the antioxidant

properties of phenolic compounds stimulate the need to implement strategies to increase

respective levels in vegetable tissues (Cisneros-Zevallos, 2003) and thus contributing to the

already known health benefits associated with fruits and vegetables consumption.

As a response to wounding stress, it was found that other metabolites also increase in

concentration. As examples, isocoumarin in carrots (Lafuente et al., 1996), anthocyanins in midribs

of red pigmented lettuce (Ferreres et al., 1997), methanethiol, allyl isothiocyanates and dimethyl

disulfide in cabbage (Chin and Lindsay, 1993) and aldehydes and alcohols in cut peppers (Wu and

Liou, 1986.

It is possible that both wounding and hormonal stresses share a common signaling molecule that

amplifies the tissues response to the stress (Heredia and Cisneros-Zevallos, 2009 b). ROS are a

suitable candidate since are considered as signaling molecules of the wounding stress (Toivonen

and Hodges, 2011) and, also, wounding is responsible for the increase of respiratory rates. Several

studies have shown that rises in respiratory rates induces an increase in ROS levels (Murphy and

DeCoursey, 2006; Brookes, 2005; Apel and Hirt, 2004; Mittler, 2002) leading to a cascade-loop of

events, including a rise in phytohormones concentration which consequently also triggers an

increase in the respiratory rate (Saltveit, 1999) implying again a rise in ROS levels which therefore

amplifies the response. This response amplification can also be achieved through a positive ROS

feedback loop by the continuous increase of NADPH oxidase activity resulting in even higher ROS

levels (Afanas’ev, 2006; Brandes, 2005; Mittler et al., 2004). Whatever the response mechanism, a


47

common trait is that cross-talking between stress signaling molecules is observed (Zhao et al.,

2005; Rakwal and Agrawal, 2003) resulting in a synergic effect of the different applied stresses to

induce the plants defense system (Heredia and Cisneros-Zevallos, 2009 b).

3.3 HEAT SHOCK

The cell’s competence to sense and respond to high temperatures is a common phenomenon of

adaptation to environmental stress in all organisms. Heat shock can be defined as the effect of

subjecting, in the case, plants to a higher temperature (10 C) than that of ideal development. Two

main reactions occur in the cell as a response to heat shock: induction of HSPs and synthesis

suppression of most other proteins (Milioni et al., 2001). Heat stress induces disturbances in

cellular homeostasis and can lead to damaging effects on plant metabolism, primarily by acting on

protein complexes (loss of quaternary structure) and secondarily by uncoupling biosynthetic

pathways since it interferes with respective electron transfer inducing a higher energy state with

subsequent ROS formation (Timperio et al., 2008; Suzuki and Mittler, 2006).

At a molecular level, elevated temperatures trigger the expression, synthesis and accumulation of a

well-defined set of HSPs while suppressing the synthesis of most other genes (Ashburner and

Bonner, 1979 cit in Milioni et al., 2001). It is thought that polypeptide misfolding and protein

denaturation mediate the heat shock signal transduction and that regulation of HSPs expression is

achieved by the activation of the heat-shock transcription factor (HSF) which, in turn, recognizes

the conserved DNA-binding sites at the promoter region of most hsp genes, the heat-shock

element (HSE) (Milioni et al., 2001).

The de novo synthesis and accumulation of HSPs enables plants with thermotolerance (Saltveit,

2005; Vierling, 1991) preventing, for instance, chilling injuries (Saltveit, 2002; 1991). Acting as

chaperones, HSPs protect heat sensitive cellular organelles and membranes (Vierling, 1991).

Several studies corroborate the association between high temperature induction of HSPs with a

protective effect. It was suggested that a heat treatment promoted the enhancement of HSPs gene

expression during peach ripening and development (Zhang et al., 2011). Also, it was found that a

hot air treatment (38 C/10 h) induced HSP70 expression, giving rise to the activities of antioxidant

enzymes such as superoxide dismutase and catalase, reducing electrolyte leakage and

malondialdehyde (MDA) content (lipid peroxidation product) which lead to grape berry chilling injury

tolerance (Zhang et al., 2005). The association between heat shocks and stimulation of chilling

injury resistance were also demonstrated by Sevillano et al. (2010) in cherimoya fruit, He et al.

(2012) in banana, and Polenta et al. (2007, cit in Aghdam et al., 2013) in tomato and grapefruit, to

name a few.

Heat treatments are recognized as having beneficial effects during postharvest storage. Used as a

traditional quarantine treatment for disinfestation and disinfection of fruits and vegetables (Lurie,

1998), heat treatments (e.g. fresh produce immersion in hot-water baths) are used to control

insects, postharvest pathogens, to prevent chilling injury, and overall product decay (Jing et al.,


48

2010; Kim et al., 2009; Fallik, 2004; Loaiza-Velarde et al, 2003; Delaquis et al., 1999; Loaiza-

Velarde et al., 1997). In food processing, the use of heat treatments has as primordial goals

reducing the contaminating microbial load and inhibiting enzymatic activities which lead to

significant changes of the physical-chemical characteristics of a given product. However, heat

treatments promote also changes, to a certain extent, of the products fresh-like sensorial

characteristics (color, texture, flavor and nutritional value). Those changes are dependent on the

applied thermal intensity and treatment objective (Wiley, 1994) and, therefore this treatment is not

usually thought for minimal processing purposes.

Nonetheless, the use of heat treatments as postharvest treatments brings forward beneficial effects

at a physiological level which could be used in minimal processing, as long as its intensity does not

compromise the fresh-like quality principle. As a result, to overcome or at least minimize these

undesirable quality changes is the high temperature short time (HTST) concept. It is based on the

fact that the inactivation of microorganisms mainly depends on the treatments temperature,

whereas many undesirable quality changes depend mostly on the duration of the treatment

(Ohlsson, 1980).

In that sense, heat treatments influence over enzymatic catalysis, namely over PAL (Saltveit, 2000)

and peroxidases (POD, EC 1.11.1.x) (Alegria et al., 2010) activities, prevents accumulation of

isocoumarin, discoloration reactions (by preventing oxidation reactions) and development of

undesirable aromas (Howard et al., 1994). Another favorable effect of heat treatments with

significance to minimal processing is related to its effects in promoting the activity of pectin methyl

esterase (PME) which enables maintenance of cell wall integrity preventing possible softening

phenomenon that may rise during fresh-cut products storage (Vu et al., 2004). Reduced respiration

rates as a result of heat treatments have also been identified and are considered as a most

beneficial effect with impact in fresh-cut products quality during its shelf-life: Rico et al. (2008)

found that steaming (100 C/10 s) fresh-cut lettuce lead to significant microflora reduction and also

resulted in lower respiration rates during products storage. Reduced respiration was also found by

Odumeru et al. (2003) in fresh-cut shredded iceberg lettuce as a result of a 47 C/30–180 s heat

treatment. Serrano et al. (2004) also found that a 45 C/10 min heat treatment resulted in a

significant reduction of plums respiration rates.

Pittia et al. (1999) have studied the effects of a blanching treatment (95 C/3 min) over the

microbiological and enzymatic stability of minimally processed pear (cubes) and have concluded

that it was efficient to the products quality maintenance during storage. In fresh-cut Rocha pear

(quarters), Abreu et al. (2011) found that a mild-heat pre-treatment (35 C/20 min) was efficient in

preventing browning reactions during products storage due to reduced phenolic contents and

changes in polyphenol oxidase (PPO) kinetic behavior. Also, it allowed for maintenance of products

firmness and led to higher sensorial acceptance in regard to untreated fruits increasing products

shelf-life by 6 days.

Lamikanra and Watson (2007) evaluated the biochemical effects of hot water treatment

(60 C/60 min) on fresh-cut cantaloupe melon during storage. The authors found that the hot water


49

pre-treatment before processing resulted in shelf-life extension of the cut fruit since it reduced

respiration rate, reduced enzymatic activities and increased fruit firmness and cohesiveness.

Obando et al. (2010) found that a pre-cut heat treatment of 50 C/15 min was most effective to

preserve the quality of the ‘Ryan Sun’ peaches in wedge shape due to the treatments effect in

decreasing cut fruits respiration rate, maintaining flesh firmness of wedges and lowering the

browning reaction extent during products storage. Lemoine et al. (2009) found that the application

of a heat treatment (hot air, 48 C/3 h) was appropriate to prevent fresh-cut broccoli senescence

diminishing tissue damage and increasing overall quality of the product during storage (0 C).

Several works have reported that, in lettuce, the use of short heat shock treatments significantly

reduces the wound-induced increase in PAL activity, the accumulation of phenolic compounds and

thus preventing tissue browning (Loaiza-Velarde and Saltveit, 2001; Saltveit 2001; Loaiza-Velarde

et al. 1997). Saltveit (2000), while addressing the effects of a heat shock (45 C/90 s) on the

wound-induced changes in phenolic metabolism of iceberg lettuce leaves found a hierarchical

order in the induced response. It was found that lettuce leaves simultaneously exposed to heat

shock and wounding would preferentially induce the synthesis of HSP rather than PAL, suggesting

that the response to some stresses are preferentially expressed in regard to other stresses. More

recently, Campos-Vargas et al. (2005) showed that a heat shock of 45 C/2 min applied in lettuce

delayed the wound-induced increase in PAL activity, but did not delay the increase in wound-

induced PAL mRNA. These findings lead to the conclusion that PAL activity is reduced due to

either a reduction in translation of wound-induced PAL mRNA or by increasing the turnover of the

induced PAL protein as a result of heat shock.

In fresh-cut carrot, immersion of the roots in hot water (100 C/45 s – Alegria et al., 2010;

50 C/2 min - Klaiber et al., 2005a) prior to shredding has proven beneficial as to effectively reduce

the initial microbial load and keeping microbial levels under control during low temperature storage

without compromising the products sensorial quality. Howard et al. (1994) also found that steaming

carrots prior to processing allowed for color maintenance during storage since it prevented lignin

deposition on carrots surface. Also related to heat treatments effects on color maintenance is the

partial inhibition of POD (Alegria et al., 2010, Howard and Griffin, 1993). Regarding the respiration

rate, Alegria et al. (2010), while using a 100 C/45 s heat treatment also found lower

decreases/increases in O2/CO2 levels during storage when compared to chlorinated shredded

carrot.

The use of heat treatments in combination with other treatments can also be beneficial to quality

maintenance of fresh-cut products. These benefits could arise from heat treatments ability to

potentiate the action of chemical agents and increasing the decontamination efficiency and quality

maintenance of fresh-cut products. For instance, the works by Klaiber et al. (2005a), in carrot, and

Delaquis et al. (1999), in lettuce, have shown that combining heat treatments with hypochlorite are

able to delay the development of spoilage microflora while preserving the products initial color.


50

3.4 UV-C RADIATION

In food production the use of UV-C radiation/light has as main objective achieving microbial stability

(reduction of the initial microbial load and/or microbial development control). UV-C is a surface

decontamination methodology (Yaun et al., 2004) that has been approved by the code of Food and

Drug Administration (FDA) in the USA (Rhim et al., 1999) on food products to control surface

microorganisms, and holds as main advantages the fact that it does not leave residues or require

extensive safety equipment (Yousef and Marth, 1988; Wong et al., 1998; Yaun et al., 2004). The

lethal effect of UV-C is related to its ability to cause severe damage to the microbial DNA (Lucht et

al., 1998; Kuo et al., 1997). It was observed that UV–C doses from 0.5 to 20 kJ.m−2

induced

pyrimidine dimers, changing the DNA helix and blocking microbial cell replication (Nakajima et al.,

2004; Lado and Yousef, 2002). Cells are then unable to repair the radiation damage which leads to

cell death and sub-lethally injured cells can suffer of mutations. This effectiveness seems to be

independent of treatments temperature (in the range of 5 C to 37 C) but it is dependent on

products superficial structure (Lado and Yousef, 2002; Bintsis et al., 2000; Gardner and Shama,

2000). Reports on the effectiveness of UV-C treatments in reducing microbial loads on the surface

of fresh-cut F&V and particularly over pathogens are available (Schenk et al., 2007; Fonseca and

Rushing, 2006; Allende et al., 2006 a,b).

Despite the known limited ability of UV light to penetrate food surfaces several studies have

demonstrated UV potential to reduce bacterial contamination (Allende et al., 2006 b; López-Rubira

et al., 2005; Allende and Artés, 2003 a,b; Erkan et al., 2001; Liu et al., 1993) in F&V, however

dependent on applied dose. Moreover, it is also known that Gram-negative bacteria are more

sensitive to UV-C than Gram-positive (Gayán et al., 2012) which can lead to the assumption that

the UV lethal effect could be higher regarding pseudomonads than lactic acid bacteria (Gram

positive), predominant contaminating flora in carrot.

More than its decontamination effect, UV-C is recognized as an abiotic stress, inducing plants

defense mechanism, and can also influence cell permeability increasing electrolyte, amino acid and

carbohydrate leakage which, in turn, could stimulate bacterial growth (Artés et al., 2009). There is

therefore the need to find a balance between the desirable effect of reducing microbial

contaminants without damaging the product (Ben-Yehoshua and Mercier, 2005).

One induced response of UV-C is the increase of phytoalexins levels. Phytoalexins are

antimicrobial/antioxidant substances that are synthesized as a result pathogen infection and rapidly

accumulate at the infection area (Ahuja et al., 2012; Hammerschmidt and Dann, 1999). Beside this

primary function, phytoalexins act as elicitors of other defense mechanisms leading to changes in

cell wall, increasing the synthesis and activity of several enzymes and by increasing antioxidant

levels. These changes can ultimately be regarded as holding potential to increase the health

benefits attributed to fruits and vegetables (Jiang et al., 2010; González-Aguilar et al., 2007;

Cisneros-Zevallos, 2003).


51

From the responses induced by UV-C, attention has been focused on induced synthesis and/or

accumulation of phytochemicals/antioxidants in the plant, leading to increased antioxidant capacity.

The extent of this response depends on the dose applied, exposure time, the sensitivity of the

antioxidant/phytochemicals towards irradiation, and the raw material used, namely the radiation

effect on other food constituents directly or indirectly related to the synthesis of these compounds

(Alothman et al., 2009). To no surprise, UV-C interferes with PAL activity levels and two possible

outcomes can be observed, the respective increase or decrease. These outcomes are however

dependent on the applied dose as reported by Nigro et al. (2000). The authors observed an

increase in PAL activity at low UV-C doses (0.50 kJ.m-2

), while high doses (2.50 kJ.m-2

) lowered

respective activity levels. Shown in Table 8 is a brief collection of studies concerning the use of UV-

C treatments and respective effects.

Table 8 Compilation of the major reported effects of UV-C irradiation in fruits and vegetables.

Product UV-C dose Outcome Reference

Blueberries 2 or 4 kJ.m-2

Cultivar dependent:

cv. Bluecrop: no change in total phenolic content; increases in total anthocyanins content and FRAP values;

cv. Collins: no changes

Perkins-Veazie et al., 2008

Broccoli 8 kJ.m-2 Increases in total phenolic and ascorbic acid contents, and in antioxidant capacity

Lemoine et al., 2007

Broccoli florets

4-14 kJ.m-2 Lower total phenolic and total flavonoid content;

Higher antioxidant capacity

Costa et al., 2006

Fresh-cut ‘Red Oak Leaf’ and ‘Lollo rosso’ lettuces

0.4 - 8.14 kJ.m-2

Reduction in psychrotrophic and coliform bacteria;

Reduced yeast growth;

Stimulated growth of lactic acid bacteria (due to reduced competitive flora)

Allende and Artés, 2003a,b

Fresh-cut ‘Red Oak Leaf’ lettuce

Two sided irradiation;

1.18 - 7.11 kJ.m-2

Reduction in natural microflora;

Tissue softening and browning were induced after 7 days

Allende et al., 2006b

Fresh-cut carrot (different cut formats/wound intensities)

UV-B;

141.4 mJ.cm-2

Increase in soluble phenolics;

Increase in 5-O-caffeoylquinic acid;

Increase in antioxidant capacity;

Increase in PAL activity;

Maintenance in carotenoid content;

Du et al., 2012

Fresh-cut carrot (shredded)

60 W × 50 cm

distance x t (min)

Increases in phenolic content, particularly chlorogenic acid, antioxidant capacity and PAL activity were positively correlated with UV-C exposure times

Surjadinata Cisneros-Zevallos, 2005

Fresh-cut mangoes

2.46 and 4.93 kJ.m-2

Increased total phenolic and total flavonoid content González-Aguilar et al., 2007b

(continues)


52

Table 8 (cont.) Compilation of the major reported effects of UV-C irradiation in fruits and vegetables.

Product UV-C dose Outcome Reference

Onion 0.44 x 104 to19.1 x 104

erg.mm-1 Reduction in postharvest rots

Lu et al., 1988

Papaya 0.2 - 2.4 kJ.m-2

Inhibition of conidial germination of Colletotrichum gloeosporioides and/or mycelial growth;

Fruit scald was observed

Cia et al., 2007

Peaches 7.5 kJ.m-2

Reduction of host resistance to brown rot;

Increased PAL activity;

Ripening delay;

Ethylene production suppression

Stevens et al., 1998

Peppers 7 kJ.m-2

Lower total phenolic content;

Higher antioxidant capacity;

No change in carotenoid content

Vicente et al. (2005)

Pomegranate arils

0.56 - 13.62 kJ.m-2

No changes in respiratory rates as a result of all tested UV–C doses;

Some doses were able to reduce mesophilic, psychrotrophic, LAB and enterobacteriaceae counts; No observable effect over the yeast and molds group

López-Rubira et al. 2005

Strawberries 0.25 and 1.0 kJ.m-2

Increased anthocyanin content Baka et al., 1999

Strawberries 0.43 - 4.30 kJ.m-2

Increases in antioxidant capacity and in anthocyanins and phenolic contents correlated with increased dose

Erkan et al., 2008

Strawberry 0.05 - 1.50 J.cm-2

Significant delay in fungal growth;

Higher doses (1.00 and 1.50 J.cm-2) induced calyx browning and leaf drying

Marquenie et al., 2002

Sweet cherry 0.05 - 1.50 J.cm-2

No effect on fungal growth Marquenie et al., 2002

Sweet potato 3.6 kJ.m-2 Increased resistance to Fusarium root rot;

Increased PAL activity;

Stevens et al., 1999

Table grape 0.125 - 4 kJ.m-2 Reduced postharvest grey mold (Botrytis cinerea) development;

Increase in epiphytic yeasts (including yeast-like fungi) and bacteria

Nigro et al., 1998

Table grapes cv. Napoleon

254 nm; 30 min (1780-2300 µw.cm2); 254 nm

Increased resveratrol content

Cantos et al., 2000; Cantos et al., 2001b

Tomato 1.3 - 40 kJ.m-2

Treatments induced resistance to black mold (Alternaria alternata), gray mold (Botrytis cinerea), and Rhizopus soft rot (Rhizopus stolonifer);

Ripening delay (color and firmness maintenance)

Liu et al., 1993

Zucchini squash slices

254 nm; 10 and 20 min

Reduced microbial activity;

Increase in respiration rate;

No change in ethylene production

Erkan et al., 2001


53

As a postharvest treatment, UV-C treatments have proven beneficial as to reduce respiratory rates

(Baka et al., 1999), to control rot development (Erkan et al., 2001) and to delay ripening and

maturation of fruits and vegetables such as apples, citrus fruits, tomato, peaches, grapes and

guavas (Perkins-Veazie et al., 2008). In the studies by Erkan et al. (2008) and González-Aguilar et

al. (2007) it was shown that UV-C treatments can effectively increase the antioxidant capacity of

strawberries and fresh-cut mango, respectively. Concerning firmness, several studies in

mushrooms, peppers and tomato (Jiang et al., 2010; Vicente et al., 2005; Maharaj et al., 1999)

have shown that this abiotic stress has the ability of delaying tissue softening by interfering in the

activity of pectinolytic enzymes (Barka et al., 2000). Nonetheless, the UV-C dose plays a

fundamental role over the desired beneficial stress response. Concerning this matter, Dhallewin et

al. (2000) have demonstrated that doses of 0.5 kJ.m−2

were beneficial to the quality of “Star ruby”

grapefruit while doses of 1.5 kJ.m−2

led to unwanted tissue browning and/or necrosis. Also,

Gonzalez-Aguilar et al. (2001) found that doses of 4.93 kJ.m−2

induced favorable changes to fresh-

cut mangos quality while doubling the dose would lead to fruit damage. In strawberries, 0.25 kJ.m−2

treatments led to longer fruit storability while 1 kJm−2

treatments led to severe fruit damage (Baka

et al., 1999).

Beside assurance of dosage optimization, it was also shown by Dhallewin et al. (2000) that the

beneficial UV-C stress effects are also dependent on harvest timing, meaning produce maturity. In

that study, the author found that grapefruit harvested in a pre-commercial maturation was more

susceptible to UV-C damage than fully mature fruits. This finding has immediate consequences

regarding fruit and vegetable processing and requires a previous study concerning the optimal UV-

C dose to be used in a narrow postharvest period.

In fresh-cut carrot, the use of UV-B and UV-C radiation has proven to enhance the wound-induced

phenolic synthesis and respective antioxidant capacity (Du et al., 2012; Surjadinata and Cisneros-

Zevallos, 2005). Regarding the use of UV-C, Surjadinata (2006) stated that its use promoted

increases up to 15% over the wound-induced accumulation of phenolic compounds after storing

carrots 4 days at 15 °C.

From the available literature there is evidence that the use of UV-C radiation is a promising

preservation method that offers several advantages to food processors as it does not leave any

residue, does not have legal restrictions, is easy to use and lethal to most types of microorganisms

(Bintsis et al., 2000). Moreover, this abiotic stress has an impact on the secondary metabolism of

fresh produce which mainly leads to increase synthesis of phytochemicals with nutraceutical

activity (Cisneros-Zevallos, 2003), making it an interesting and challenging method to be used by

the fresh-cut industry. Additionally, finding the best conditions, doses, and combination treatments

for different hurdle technologies is considered another challenge that the application of UV-C

radiation faces.

PART III – MATERIAL & GENERAL

METHODS

57

1 PLANT MATERIAL

Carrots (Daucus carota L.) from cv. Nantes of about 140–180 mm length and of 40 mm diameter

(upper end) were obtained at Mercado Abastecedor da Região de Lisboa, MARL (Lisboa, Portugal)

and carrots from cv. Navajo were purchased at a local supermarket (HEB, College Station, TX,

USA). Upon arrival to the laboratory, carrots were sorted, washed to remove excessive dirt, dried

and maintained at 5 C (±1 C) until use (< 16 h unless otherwise stated).

2 SAMPLE PREPARATION

2.1 WOUNDING INTENSITIES

To evaluate the wounding effects on induced phenolic synthesis, 2 wound intensities were tested,

low wounding (LW) and high wounding (HW). Two LW and two HW intensities were considered

given the nature of tissue type and evaluation of wounding operations included in the fresh-cut

carrot production. The LW and HW intensities considered for the evaluation of tissue type were

obtained by peeling or cut using a Grindomix GM200 (3000 rpm x 1 second). The LW and HW

intensities considered for the evaluation of inherent minimal processing wounding operations were

obtained by peeling or shredding using a pilot scale vegetable slicer (Dito Sama MV-50 equipped

with a CX.21, knife J7-8, Dito Sama, Aubusson, France). The wound intensity value (A/W) was

calculated by dividing the resulting wound area (m2) over the weight (kg) of the carrot cuts, and

peel thickness was measured using Kuhnke video seam monitor VSM III (v4.2) (Manfred Kuhnke,

Berlin, Germany) and remaining measurements with a slide caliper. Cut dimensions (mean±SD)

and wounding intensity values are shown in Table 9 with the identification of studies.

Table 9 Carrot cut dimensions and respective wounding intensity values.

Study Sample type Wounding intensity

Sample dimension (mm)

Wound intensity value (A/W; m2.kg-1)

Phenolic synthesis as affected by tissue type and wounding intensity

Peel tissue

Low (LW) Length: 152.00±10.88; Width: 10.25±1.68; Thickness: 0.73±0.11

1.8

High (HW) Length: 12.12±2.64 Width: 9.55±1.14; Thickness: 0.73±0.11

2.1

Inner tissue

Low (LW) Length: 204.67±3.94; Diameter: 40.88±1.99

0.1

High (HW) Length: 8.07±1.62; Width: 5.06±1.60; Height: 3.30±1.14

2.3

Phenolic synthesis as affected by peel removal and shredding

Whole carrot Peeled Length: 199.27±20.42; Diameter: 38.87±7.49

0.1

Shredded carrot Unpeeled / Peeled Length: 59.14±8.55; Width: 5.36±0.79; Height: 5.36±0.53

2.7

MATERIAL & GENERAL METHODS

58

2.2 INDUCED PHENOLIC SYNTHESIS STUDIES

Whole carrots (3 carrots with or without peel, 3 to 5 independent replicates) were placed 4-L clear

glass jars (closed and vented every 8 h to avoid CO2 accumulation as previously described by

Reyes et al., 2007) and “air” stored for the designated storage period at 5 C (Cryocell RS600SE,

Aralab – Equipamentos de Laboratório e Electromecânica Geral Lda., Portugal).

Cut carrot peel/inner tissues and shredded carrots (with or without peel), were prepared using a

Grindomix GM200 (3000 rpm x 1 second) and a pilot scale vegetable slicer (Dito Sama MV-50

equipped with a CX.21, knife J7-8, Dito Sama, Aubusson, France), respectively. Sample aliquots of

125 g (3 to 5 independent replicates) were placed 4-L clear glass jars and treated as previously

described.

2.3 SHELF-LIFE STUDIES

Minimal processing operations were carried out according to the industrial practice. Cv. Nantes

carrots were peeled using a sharp stainless steel vegetable peeler, treated according to respective

experimental design, shredded (Dito MV-50 shredder) and packaged in portions of 100 or 125 g

using bags made from films of 35 μm bioriented polypropylene2 (200 x 110 mm) (Amcor Flexibles

Neocel – Embalagens Lda., Lisboa, Portugal). Two films were used: film A (commonly used in FC

carrot production and taken as standard) and film B (as alternative packaging solution). The

permeability of film A was with oxygen (OTR) and carbon dioxide (CTR) transmission rates of

1100 cm3.m

-2.24 h

-1.atm

-1 and 3000 cm

3.m

-2.24 h

-1.atm

-1 at 23 ºC, respectively. Film B (PPlus -

35PA120) had the same characteristics of film A plus laser established micro-perforations at

120 µm intervals, ensuring significant changes in the film permeability. The bags were heat-sealed

and stored at 5 ºC (S600 Pharma, Fitoclima or a Cryocell RS600SE, Aralab – Equipamentos de

Laboratório e Electromecânica Geral Lda., Portugal) for the designated “MAP” storage period.

The industrial fresh-cut carrot standard consists of processed carrot as described (using film A as

packaging solution), including the following procedures: post-cut decontamination with a 200 mg/L

(ppm) free chlorine solution for 1 min (5 C), rinsing (potable water, 5 C) and a water removal step

(manual centrifuge). After these procedures, samples were treated as previously described. The

complete standard technological flowchart of fresh-cut shredded carrot was already shown in

Figure 11 of section 2.1 of the Literature Review.

2 Films were kindly provided by a fresh-cut carrot processor, Campotec, Torres Vedras, Portugal.


59

3 HEAT SHOCK AND UV-C STRESS TREATMENTS PROCEDURES

3.1 SINGLE APPLICATION

3.1.1 Heat shock

Heat shock (HS) samples were prepared by immersion of whole peeled carrots (unless otherwise

stated) in hot-water at previously optimized pre-cut conditions of 100 C for 45 s (Alegria et al.,

2010; 2009; Alegria, 2007) as follows: Potable tap water was heated to the specified temperature

(100 C ± 1 C) using a thermostatically-controlled pilot scale sterilizer and temperature was

monitored continuously with T-type thermocouples. Treatment time (45 s) started immediately after

carrot submersion. Heat treated samples were then cooled in ice-cold potable water (0 C) for

5 min, paper dried and processed as described in the “Sample preparation” section according to

study objectives.

3.1.2 UV-C

The UV-C treatment included in the “Preliminary study of hear shock and UV-C stress effects on

fresh-cut carrot quality” was carried out using a rudimental UV-C apparatus at room temperature.

The UV-C apparatus consisted of two reflector racks placed longitudinally and each one was

equipped with unfiltered germicidal emitting lamps (λ = 254 nm) (TUV 15W/G15 T8, Phillips, The

Netherlands). Lamps were located 15 cm above the illumination area. A wooden box (43 cm (w) x

50 cm (l) x 24 cm (h)) covered with a reflector inner layer (food grade aluminum foil) and supported

by a metal framework enclosed the UV-C lamps, reflectors and treatment area, providing UV

protection for the operator. Prior to use, lamps were turned on and allowed to stabilize for a period

of time not less than 15 min. In order to determine the UV-C irradiation intensity of the lamps, a

HD2102.2 photoradiometer (Delta Ohm, Padova, Italy) equipped with a LP471 UVC probe (Delta

Ohm, Padova, Italy), was used.

Whole peeled carrots were placed in a single layer on the illumination area at the fixed distance

and rotated manually (180) at half the treatment duration in order to ensure even exposure to UV

light of both carrot hemispheres. Treatment time was of 2 min which was based on previous

studies of our work group (Simões, 2010) and the applied UV-C dose was calculated from a mean

of 10 readings (0.78 ± 0.36 kJ.m-2

). After treatment, carrots were held at 5 ºC for 24h until

shredding operations. After shredding, samples were processed as described in section 2.3 using

film A as packaging film.

All other UV-C treatments were conducted in a temperature controlled environment using a S600

Pharma chamber (Fitoclima, Aralab – Equipamentos de Laboratório e Electromecânica Geral Lda.,

Portugal) equipped two UV-C reflector racks. Each reflector rack was equipped with three


60

TUV 18W unfiltered germicidal emitting lamps (59 x 2.8 cm, = 254 nm, Phillips, The Netherlands),

placed at 14 cm intervals and with a 12.5 cm distance to the chamber walls. Each lamp rack was

placed at a distance of 20 cm of the illumination area (one above and one below) as shown in

Figure 15.

Figure 15 Schematic representation of UV-C lamp distribution in the S600 Pharma chamber.

The use of the S600 Pharma chamber to conduct UV-C treatments was appealing since it made

possible to maximize products exposure to the UV source (two reflector racks) and respective

application under controlled refrigerated conditions (5 C) upholding the low temperature

maintenance premise during all stages of FC production. Nonetheless, it is known that temperature

is a critical factor that influences UV germicidal effect and should be kept between 20 C and 24 C

(Memarzadeh et al., 2010). As a result, the temperature influence (5 C, 15 C and 25 C) on UV-C

dose was evaluated since radiation exposure time could be affected by the temperature change.

The results are shown in Figure 16 and reveal that the UV-C dose was directly proportional to the

exposure time and temperature. According to the found proportionality, longer exposure times were

to be considered to achieve similar UV-C doses to those obtained at room temperature.


61

Figure 16 Effects of temperature and radiation exposure time on UV-C dose.

In conclusion, UV-C treatments were conducted in the S600 Pharma chamber at 5 C and with the

chamber door closed to provide UV protection for the operator. Prior to use, lamps were turned on

and allowed to stabilize for a period of time not less than 15 min. UV-C doses (kJ.m-2

) were

monitored with an HD2102.2 photoradiometer (Delta Ohm, Padova, Italy) equipped with an LP471

UVC probe (Delta Ohm, Padova, Italy) placed on the illumination area.

Pre-cut samples were prepared as follows: carrots were peeled and placed in single layer on the

illumination area and treated according to the established doses (exposure times). Subsequently,

the pre-cut samples were held at 5 C for 24 h until shredding operations. After this hold-up, carrots

were processed as previously described in the “Sample preparation” section according to study

objectives.

Post-cut samples were prepared as follows: carrots were peeled and immediately shredded. 125 g

portions were distributed in a single layer on a tray covered with a reflector layer (food grade

aluminum foil) to maximize UV light exposure, at a distance of 20 cm from the UV-C lamp rack, and

exposed to the designated UV-C dose. After treatment, shredded carrots were processed as

described in the “Sample preparation” section according to study objectives.

3.2 COMBINED APPLICATION

Whole peeled carrots were submitted to a 100 C/45 s heat shock as previously described (section

3.1.1) and, after shredding, submitted to a 2.5 kJ.m-2

UV-C dose (section 3.1.2). After treatments,

samples were processed as described in the “Sample preparation” section according to study

objectives.

5 ºC 15 ºC 25 ºC

0.5 1 2 4 8

Radiation exposure time (min)

0

1

2

3

4

5

UV

-C d

os

e (

kJ.m

-2)


62

4 ANALYTICAL PROCEDURES

4.1 HEADSPACE GAS ANALYSIS

Headspace gas samples were taken with a hypodermic needle through an adhesive septum

previously fixed on the sample bags and were analyzed for oxygen and carbon dioxide

concentrations (%) using a Checkmate 9900 O2/CO2 gas analyzer (PBI-Dansensor, Ringsted,

Denmark). On each sampling day, 3 to 5 bags were analyzed per sample type.

4.2 TOTAL PHENOLIC CONTENT

Protocol I

(Used in all studies except for the “Preliminary study of hear shock and UV-C stress effects on

fresh-cut carrot quality”)

Carrot tissue (5 g) was homogenized with methanol (20 mL) using a Yellow line DI 25 basic

polytron (IKA-Werke GmbH&Co.KG, Staufen, Germany) and left overnight at 5 C. Homogenates

were centrifuged at 29000 g for 15 min at 5 C (Sorvall RC5C, rotor SS34, Sorvall Instruments,

Du Pont, Wilmington, Delaware, USA) and the clear supernatant (methanolic extract) was used for

total phenolic content determination using the method described by Swain and Hillis (1959).

Methanolic extracts (150 μL) were diluted with nanopure water (2400 μL) in test tubes, followed by

the addition of 0.25 N Folin-Ciocalteu reagent (150 μL). The mixture was incubated for 3 min, and

then, 300 μL of 1 N Na2CO3 was added. The final mixture was incubated for 2 h at room

temperature in the dark. Spectrophotometric readings at 725 nm were collected using an ATI

Unicam UV/VIS 4 spectrophotometer (Unicam Sistemas Analíticos, Lisboa, Portugal). The

extraction was performed in each of the samples replicates and three independent measures were

taken per sample replicate. Total phenolic content (TPC) were quantified as mg chlorogenic acid

from a standard curve developed for this standard (0–0.35 mg.mL-1

) and results expressed as mg

chlorogenic acid equivalents/100 g of fresh tissue (mg CAE.100 g-1

).

Protocol II

(Used only in the “Preliminary study of hear shock and UV-C stress effects on fresh-cut carrot

quality”)

Total phenolic content was determined using the Folin-Ciocalteu reagent (Singleton and Rossi,

1965). Grind carrot (10 g) was homogenized with 70% methanol (1:1, w:v) using a Yellow line DI

25 basic polytron. After centrifugation (4000 g x 20 min; 4K15 SIGMA® Laboratory Centrifuges,

rotor n.11150, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at 4 C, 0.1 mL

aliquot of the supernatant (3 replicates) is mixed with 5 mL of diluted Folin-Ciocalteu reagent (1/10,

v/v) and 4 mL of 7.5% Na2CO3. The mixture is then placed in a water-bath, at 45 C, for 15 min,

after which the absorbance at 765 nm was measured (ATI Unicam UV/VIS 4 spectrophotometer),


63

using gallic acid as standard. A standard curve was prepared using gallic acid as standard

(concentration ranging from 0 to 600 mg.L-1

) and results expressed as gallic acid equivalents per

100 g of product (mg GAE.100 g-1

). Triplicates were performed for each sample type.

4.3 PHENOLIC PROFILE AND CHLOROGENIC ACID QUANTIFICATION

The same extracts used for the TPC assay (Protocol I) were used for determining the phenolic

profiles (minimum of three independent extracts for each sample type) using an HPLC method

described by Petitjean-Freytet et al. (1991), with some changes as follows. Data acquisition and

integration procedures were carried out using Waters Millennium 3.2 software (Milford, MA, USA).

Separation was done in an Alliance System from Waters (Waters 2690 Separations Module,

Milford, MA, USA) equipped with a photodiode array (PDA) detector (Waters 996, Milford, MA,

USA). The column used to separate the phenolic compounds was a 3.9 mm x 300 mm, 4 µm, C18

reverse-phase column (Waters Nova Pack, Milford, MA, USA) operated at room temperature. The

injection volume was 20 µL. Two mobile phases were used: water adjusted to pH 2.3 with formic

acid as solvent A and acetonitrile:water (80:20) also adjusted to pH 2.3 as solvent B. Both solvents

were filtered and degassed before use. The gradient system was 0/88, 5/88, 10/85, 35/70, 40/50,

50/50, 88/55, 88/60 (min/% solvent A) with a flow rate of 1 mL.min-1

. Prior to injection, samples

were filtered through a 0.2 µm nylon syringe filter. Commercial standards of chlorogenic acid,

ferulic acid and p-hydroxybenzoic acid were used for peak identification when possible, by

comparing respective retention times and UV–VIS spectra. Quantification of chlorogenic acid was

based on a developed external standard curve (5-100 µg.mL-1

).

4.4 TOTAL CAROTENOID CONTENT

Carotene content is determined according to Talcott and Howard (1999). In semi-dark conditions,

2 g fresh sample was mixed with 20 mL of an acetone/ethanol (1:1) solution containing 200 mg.L-1

butylated hydroxytoluene (BHT) into a plastic jar. The mixture was then homogenized in a polytron

(20000 rpm x 1 min, Yellow line DI 25 Basic polytron) until uniform consistency. The remaining

puree from homogenizer was washed into the plastic jar with the least amount possible of solvent.

The homogenate was then filtered through a Whatman #4 filter and further washes with

acetone/ethanol solvent are followed until no further color change is observed (avoid exceeding a

volume of 100 mL). The filtrate was then transferred to a 100 mL-graduated cylinder and solvent

was further added to a final volume of 100 mL. The mixture is then transferred to an amber

Erlenmeyer flask and 50 mL of n-hexane were added to the sample. The mixture was shaken and

allowed to stand (in dark) for 20-30 minutes to allow separation to occur. 25 mL of nanopure water

was further added and allowed to stand until complete phase separation. An aliquot of carotenoid

solution (hexane phase) was transferred into a glass cuvette and spectrophotometric readings at

470 nm were collected using an ATI Unicam UV/VIS 4 spectrophotometer. A standard curve was

prepared using -carotene as standard (concentration ranging from 0 to 15 mg.L-1

) and results


64

expressed as -carotene equivalents per 100 g of product (mg -carotene eq.100 g-1

). Triplicates

were performed for each sample type.

4.5 -CAROTENE CONTENT

-carotene content was determined using the carotenoid solution prepared as described previously

(total carotenoid content) by HPLC. Data acquisition and integration procedures were carried out

using Waters Millennium 3.2 software (Milford, MA, USA). Separation was carried out in a Nova-

Pak Silica column (3.9 mm x 150 mm; 4 µm) operated at room temperature. Samples were injected

into the system by means of a Rheodyne manual sample injector (Model 7125) equipped with a

sample loop of 50 µL (injection volume) and the mobile phase consisted of a mixture of n-

hexane:isopropanol (99:1, v/v) which was previously was filtered and degassed. The flow rate was

set at 1 mL.min-1

(isocratic) and total run time was of 5 min. A commercial standard of -carotene

was used for peak identification, by comparing respective retention times and UV–VIS spectra

(Waters 2996 Photodiode array detector) and respective quantification was based on a developed

external standard curve (0-10 µg.ml-1

).

4.6 ANTIOXIDANT CAPACITY

Protocol I

Antioxidant capacity (AOx) was determined according to the procedure described by Arnao et al.

(2001), using the ABTS radical cation decolorization assay, as follows: an ABTS mother solution

was prepared from the equitative mixture of a 15 mM ABTS solution and a 4.9 mM K2S2O8 solution

and allowed to react (room temperature) for 12 h. Afterwards, this solution was diluted with ethanol

to give an absorbance near 1.1 (±0.02) at 734 nm (ATI Unicam UV/VIS 4 spectrophotometer).

Hydrophilic antioxidant capacity

The same extracts used for the TPC assay (Protocol I) were used for determining the hydrophilic

antioxidant capacity (AOxH). A 150 µL sample aliquot (hydrophilic sample extract) was mixed with

2850 µL of the ABTS diluted solution and allowed to react for 2 h in the dark and readings at

734 nm were collected. Blanks were prepared using methanol as reference. Standard curves were

prepared daily using Trolox (in methanol; concentrations ranging 0 to 15 M) as reference and

results expressed as mg Trolox equivalents per 100 g of product (mg TE.100 g-1

).

Lipophilic antioxidant capacity

Carrot tissue (5 g) was homogenized with dichloromethane (25 mL) using a Yellow line DI 25 basic

polytron. Homogenates were allowed to stand until complete phase separation and the organic

phase (carotenoid extract) collected and used to measure the respective antioxidant capacity

(AOxL). A 150 µL sample aliquot (lipophilic sample extract) was mixed with 2850 µL of the ABTS

diluted solution and allowed to react for 2 h in the dark and readings at 734 nm were collected.


65

Blanks were prepared using dichloromethane as reference. Standard curves were prepared daily

using Trolox (in dichloromethane; concentrations ranging 0 to 15 M) as reference and results

expressed as mg Trolox equivalents per 100 g of product (mg TE.100 g-1

).

Protocol II

(Used only in the “Phenolic synthesis as affected by peel removal and shredding” trial and in the

“Heat shock and UV-C combined stress effects on the wound-induced dynamics” study)

Antioxidant capacity (AOx) was determined according to the procedure described by Re et al.

(1999), using the ABTS radical cation decolorization assay as follows: an ABTS mother solution

was prepared from the equitative mixture of a 7 mM ABTS solution and a 2.45 mM K2S2O8 solution

and allowed to react (room temperature) for 12 h. Afterwards, a dilution from this solution was

made to give an absorbance near 0.7 (±0.02) at 734 nm (ATI Unicam UV/VIS 4

spectrophotometer). The same extracts prepared for the total phenolics assay were also used for

the AOx assay. A 100 µL sample aliquot (hydrophilic sample extract) was mixed with 3000 µL of

the ABTS diluted solution and allowed to react for 7 min at 30 C and readings were continuously

monitored at 734 nm to determine ABTS inhibition. The inhibition rate is then converted to

µg Trolox equivalents per 100 g of product (µg TE.100 g-1

) by extrapolation from a standard curve

(prepared daily) using Trolox as reference, with final concentrations ranging 0 to 15 M.

4.7 PHENYLALANINE-AMMONIA LYASE (PAL) ACTIVITY

The PAL activity assay was adapted from Ke and Saltveit (1986). A 4-g sample was added with

0.4 g polyvinylpolypyrrolidone (PVPP) and homogenized with 16 mL of 50 mM borate buffer (pH

8.5) containing -mercaptoethanol (400 μL.L-1

) under low-light conditions and at low speed to

prevent protein denaturation (Yellow line DI 25 basic polytron). Homogenates were filtered through

4 layers of cheesecloth, centrifuged at 29000 g for 15 min at 4 C (Sorvall RC5C, rotor SS34,

Sorvall Instruments) and the supernatants collected (PAL assay solution). PAL activity

determination was performed by pipetting 3357 μL of the borate buffer and 500 μL of 100 mM L-

phenylalanine substrate solution into a 10 mL test tube. The reaction was started by the addition of

1143 μL of PAL assay solution. Blank reactions were prepared as described using nanopure water

instead of PAL assay solution. Spectrophotometric readings at 290 nm were registered before and

after 1 hour of incubation at 40 C in a spectrophotometer (ATI Unicam UV/VIS 4) previously

blanked with borate buffer. Each sample replicates was extracted taking three independent

measures per sample replicate. One unit of PAL activity (U) was defined as the amount (μmol) of t-

cinnamic acid synthesized per hour using a t-cinnamic acid standard curve (0.0 – 0.15 μmol.mL-1

).

4.8 POLYPHENOL OXIDASE (PPO) ACTIVITY

Polyphenol oxidase (PPO) activity level was assayed in homogenates prepared as follows: 10 g

sample was added with 0.1 g PVPP and homogenized (Yellow line DI 25 basic polytron) in a 1:2


66

(w:v) ratio with 0.1 M phosphate buffer (pH 6.5) at low speed in an ice-bath to avoid excess heating

as to prevent protein denaturation. Homogenates were filtered through 4 layers of cheesecloth,

centrifuged at 29000 g for 15 min at 4 C (Sorvall RC5C, rotor SS34, Sorvall Instruments). The

resulting crude extract was used without further purification. PPO activity was assayed

spectrophotometrically by a modified method based on Siriphanich and Kader (1985) and Galeazzi

et al. (1981). The reaction mixture contained an aliquot of crude extract and 2.9 mL substrate

solution (40 mM catechol in 50 mM phosphate buffer, pH 6.5) to a final reaction volume of 3.0 mL.

The rate of catechol oxidation was followed at 420 nm for 1 min. An enzyme activity unit was

defined as an increase of 0.1 in absorbance per minute per mL.

4.9 PEROXIDASE (POD) ACTIVITY

Peroxidase (POD) activity was determined according to Bifani et al. (2002) with some

modifications. Enzyme extraction was performed at 4 °C and the reagents and equipment were

cooled to that temperature before use. POD was extracted (1:4, w:v) by homogenizing (20 000

rpm×1 min, DI 25 Basic Yellow polytron, IKA Werke GMBH&Co. KG, Germany) carrot tissues in a

0.1 M NaCl (extraction solution). Homogenates were centrifuged (29000 g x 15 min at 4 C; Sorvall

RC5C, rotor SS34, Sorvall Instruments) and the supernatant collected and used as crude extract.

POD activity was determined spectrophotometrically (ATI UNICAM UV/VIS 4) at 470 nm from the

rate of H2O2 decomposition with guaiacol serving as hydrogen donor at pH 6.5 (0.1 M phosphate

buffer). The unit definition of POD activity is given as the amount of crude enzymatic extract that

causes an increase of 0.1 in absorbance per minute per mL.

4.10 DIETARY FIBER CONTENT

Total dietary fiber content was determined by the enzymatic-gravimetric method described in the

AOAC method 985.29 (AOAC, 1990) and the insoluble dietary fiber according to the AOAC method

991.42 (AOAC, 1992). Soluble fiber content was calculated as the difference between total and

insoluble fiber contents.

4.11 PH

The pH was measured (room temperature) in the homogenized of carrot samples in distilled water

(1:1, w:v) using a pH meter (Crison Micro pH 2001, Crison Instruments, Spain). One measurement

per sample replicate was made.

4.12 SOLUBLE SOLIDS CONTENT

The soluble solids content (SSC), expressed as ºBrix, was measured in the homogenized of carrot

samples in distilled water (1:1, w:v) using a digital refractometer (DR-A1, ATAGO Co Ltd., Japan)


67

equipped with a thermostatic water bath set at 20ºC. One measurement per sample replicate was

made.

4.13 COLOR

Color measurements were performed in a colorimeter (Minolta Chroma Meter CR 300, Osaka,

Japan) by measuring the CIE L*a*b* parameters (C illuminant). The instrument was calibrated

using a standard white tile (L*=97.10, a*=0.19, b*=1.95). A total of 15 measurements were made

per sample and the L*a*b* data was then transformed to the respective Hue angle, whiteness index

(WI, Eq. 1) and the total color difference (E) was estimated as shown in Eq. 2, where: Li, ai and bi

correspond to the respective L*, a* and b* values of raw material, and Lf, af and bf correspond to

the respective sample L*, a* and b* values of considered sample at day X.

𝑊𝐼 = 100 − √(100 − 𝐿∗)2 + 𝑎2 + 𝑏2 (Bolin and Huxsoll, 1991) Eq. 1

∆𝐸 = √(𝐿𝑓∗ − 𝐿𝑖

∗)2

+ (𝑎𝑓∗ − 𝑎𝑖

∗)2

+ (𝑏𝑓∗ − 𝑏𝑖

∗)2 Eq. 2

4.14 SENSORIAL ANALYSIS

Analytical-descriptive tests were used to discriminate the sensory quality attributes of shredded

carrot. A panel of 8/10 semi-trained panelists (members of our Department) was required to

differentiate changes in color, fresh-like appearance and fresh-like aroma using a 5 point numeric

rating scale as follows:

Color: 1 - Nonwhite; 2 - Slightly white; 3 - Moderate white; 4 - Severe white; 5 - Extreme

white, where anchor 1 correponds to the perception of the fresh-cut untreated carrot color

immediately after cutting;

Fresh-like appearance: 1 - Excellent/fresh appearance; 2 - Moderate; 3 - Limit of

saleability; 4 - Poor; and 5 - No fresh appearance, where anchor 1 correponds to the

perception of the fresh-cut untreated carrot appearance immediately after cutting;

Fresh-like aroma: 1 - Very intense, 2 - Intense; 3 - Moderate; 4 - Low; and 5 - Absent,

where anchor 1 corresponds to the perception of the fresh-cut untreated carrot aroma

immediately after cutting. Panelists are also asked to identify the presence of any off-odors

in the comments section.

Samples of shredded carrot were served in plastic dinner plates marked with three-digit code

numbers and presented in a randomized order. Panel members were already familiarized with the

product and scoring system since they were experienced on shredded carrot sensory evaluation

(Alegria et al., 2010; 2009; Alegria, 2007). Panelists were asked to evaluate samples during

storage, scoring the level of difference in perceived intensity between the sample and the fresh

reference (freshly untreated shredded carrot) with respect to each attribute.


68

Shredded carrot samples were also evaluated for overall acceptance to determine product

sensorial rejection index using a five point hedonic rating scale:

Rejection Index: 1 = Excellent freshly cut, 2 = Good, 3 = Limit of marketability, 4 = Poor

and 5 = Unusable.

Panelists were also asked to enclose any relevant comment under the comments area. The mean

scores for attribute intensity and rejection index were calculated. The applied cut-off for the overall

acceptance was fixed at anchor 3 (limit of marketability) of the rejection index and scores above 3

indicated unacceptable samples.

4.15 MICROBIOLOGICAL RESPONSES

Total mesophilic aerobic count (TAPC) was performed according to EN ISO 4833: 2003.

Yeasts & molds (Y&M) were determined using Rose Bengal Chloramphenicol Agar by surface

inoculation and incubated at 25 ºC during 5 days. Lactic acid bacteria count (LAB) was assessed

according to ISO 15214: 1998. Microbial counts were expressed as Log10 (cfu.g–1

). For assessment

of initial microbial load reduction, mean counts for each treatment were subtracted from the mean

counts of the raw material to give an average Log10 reduction in the attached microbiological

groups.

5 STATISTICAL ANALYSIS

5.1 ANALYSIS OF VARIANCE

Data from the studies were subjected to analysis of variance (one-way or factorial ANOVA) using

the StatisticaTM

v.8 Software (StatSoft Inc., 2007). Statistically significant differences (p<0.05)

between samples were determined according to Tukey Honestly Significant Difference (HSD) test,

unless otherwise stated. Pearson correlation coefficients were also generated between the studied

responses.

In shelf-life studies, the decontamination efficiency of tested treatments was calculated as the

difference of respective microbial group (TAPC, LAB and Y&M) of raw material, and the counts

determined on samples. The found differences were then submitted to variance analysis and

statistically significant differences (p<0.05) between samples were determined according to Tukey

HSD test.

5.2 RESPONSE SURFACE METHODOLOGY AND MODEL FITTING

Response surface methodology (RSM) based on a two-variable central composite rotatable design

(CCRD) was used as a function of two factors. The complete design consisted of three sets of

experimental points: (1) a traditional factorial design with 2k points, k being the number of


69

independent variables (factors) with coded levels + 1 and - 1; (2) a star of 2k points, coded as +

and - on the axis of the system at a distance of =[2k]1/4

from the origin, that accounts for non-

linearity; (3) central points, which are replicated to provide an estimate of the lack of fit of the linear

statistical model obtained as well as of the pure error of the experiments (Montgomery, 1991). To

this totally randomized design, 4 central points were added and the resulting matrix is shown in

Table 10.

Table 10 Experimental design matrix used the response surface methodology trials.

Coded factors

Run A B

5 - 0

3 1 -1

12 (C) 0 0

6 0

13 (C) 0 0

11 (C) 0 0

2 -1 1

9 (C) 0 0

1 -1 -1

4 1 1

8 0

7 0 -

14 (C) 0 0

10 (C) 0 0

The experimental data obtained was fitted to a second-order polynomial equation (Eq. 3) to predict

each dependent variable (Y) through a stepwise multiple regression analysis using StatisticaTM

v.8

Software (StatSoft Inc., 2007). In accordance, the statistical analysis of the second-order model

equation expressed by Eq. 3 was performed in the form of the variance (variance analysis,

ANOVA). This analysis included the determination of lack of fit and significance of the effects of

each independent variable (UV-C dose – UV and storage time – St).

ji

2

1j

2

jjjjiij

2

1j

jj0 bbbbY XXXX Eq. 3

Y – Predicted response;

b0 – Coefficient for intercept;

bi – Coefficient of linear effect;

bjj – Coefficient of quadratic effect;

bij – Coefficient of interaction effect;

Xi and Xj – Coded independent variables.

The criteria for eliminating a variable from the full regression equation was based on the multiple

regression coefficients, following the criterion proposed by Montgomery (1991) in which the

difference between r2 and r

2adj values should be < 0.1. The standard error estimate, significance F-


70

test, and derived p values were also evaluated for variable elimination. The F-ratio

regression/residuals was used to evaluate the statistical significance of the variance attributed to

the model effectiveness and the F-ratio lack-of-fit/pure error to evaluate the lack of fit associated

with experimental errors. Three-dimensional response surface plots were generated by using

plotting software (StatisticaTM

v.8 Software, StatSoft Inc., 2007).

5.3 HIERARCHICAL CLUSTER AND PRINCIPAL COMPONENT ANALYSIS (PCA)

Hierarchical cluster was run using StatisticaTM

v.8 Software (StatSoft Inc., 2007). The clustering

process involved three steps: data standardization; assessment of a dissimilarity measure among

samples; and the use of a grouping technique. In the present study, data standardization was done

in order to provide null means and variance equal to one for each sample. The Euclidean distance

was used as a dissimilarity distance (Sneath and Sokal, 1973). Ward’s method was used as the

grouping technique (Ward, 1963), which involves an agglomerative clustering algorithm starting out

at the leaves (n clusters of size 1) and continues to the trunk until all the observations are included

into one cluster which is referenced as most appropriate for quantitative variables.

Principal component analysis, a multivariate exploratory technique, was also run using StatisticaTM

v.8 Software (StatSoft Inc., 2007). All variables were mean centered and standardized (scaled) to

unit variance prior to analysis, i.e. correlation matrix. The principal components were obtained by

computing the eigenvalues and eigenvectors of the studies data correlation matrix (Jackson, 1991).

For each component of the PCA, a score for each sample was calculated as a linear combination

for each quality variable and the contribution of each variable to the PCA score was deduced from

the parameters loading for the factor. A bi-dimensional representation of this multidimensional set

was made for the principal components that accumulated a significant percentage of original

information, above 70%, which is considered sufficient to define a good model for qualitative

purposes (Larrigaudière et al., 2004).

5.4 MODELING MICROBIAL CHANGES

The cell load data of the mesophilic microbial group (TAPC), collected during the storage of the

products were modelled according to Gompertz equation (Eq. 4) modified by Zwietering et al.

(1990):

𝑦 = 𝑘 + 𝐴 ∗ 𝑒𝑥𝑝 {−𝑒𝑥𝑝 [(𝜇𝑚𝑎𝑥 ∗ 𝑒

𝐴) ∗ (𝜆 − 𝑆𝑡) + 1]} Eq. 4

where y is the Log10 cfu.g-1

, k the initial level of the dependent variable to be modelled (TAPC), A

the maximum bacteria load attained at the stationary phase, μmax the maximal growth rate

(Log10 cfu.g-1

per day), λ the lag time (days) and St is the storage time. The experimental data

were modelled through the Non-Linear Regression Procedure (using least squares estimation and


71

Levenverg–Marquart method, for minimizing the sum of squares of the deviation between

experimental values and the ones predicted by the mathematical model) of the statistic package

StatisticaTM

v.8 Software (StatSoft Inc., 2007). The parameter’s precision was evaluated by

confidence intervals at 95%, and the quality of regression was assessed by the determination

coefficient (R2), and randomness and normality of residuals (Hill and Grieger-Block, 1980), thus

allowing best fit model parameters.

The microbiological shelf-life was calculated as the time necessary to attain a total bacterial count

of 7.5 Log10 cfu.g-1

, within the recommended threshold interval proposed by HPA (106-10

8 cfu.g

-1;

Health Protection Agency, 2009) and also in accordance with the French regulation (5 x107 cfu.g

-1;

Ministere de l’Economie des Finances et du Budget, 1988), by using Gompertz parameters

according to the modified equation proposed by Zwietering et al. (1991) (Eq. 5):

𝑆ℎ𝑒𝑙𝑓 − 𝑙𝑖𝑓𝑒 = 𝜆 −𝐴 ∗ {𝑙𝑛 [−

𝑙𝑛(7.5) − 𝑘𝐴

] − 1}

𝜇𝑚𝑎𝑥 ∗ 𝑒 Eq. 5

5.5 MODELING PHENOLIC CONTENT CHANGES

The total phenolic content (TPC) data collected during storage of the products were modelled

according to the equation proposed by Amodio et al. (2014) (Eq. 6).

𝑇𝑃𝐶 = 𝑇𝑃𝐶0 ∗ 𝑒𝑥𝑝(−𝑘2 ∗ 𝑆𝑡) +𝑘1 ∗ 𝑇𝑃𝐶𝑝𝑟𝑒0

(𝑘1 − 𝑘2)∗ [𝑒𝑥𝑝(−𝑘2 ∗ 𝑆𝑡) − 𝑒𝑥𝑝(−𝑘1 ∗ 𝑆𝑡)] Eq. 6

where TPCpre and TPC are the precursor and phenolic content, respectively, k1 is the rate constant

for the de novo synthesis of phenolic compounds, k2 is the rate constant of their oxidation, and St is

the storage time (days). For the description of the changes in phenolic content as a function of time

for the fresh-cut product, TPC0 was experimentally measured using the methodology reported

above (section 4.2 – Protocol I), and k1, k2 and TPCpre0 were estimated.

The experimental data were modelled through the Non-Linear Regression Procedure (using least

squares estimation and Levenverg–Marquart method, for minimizing the sum of squares of the

deviation between experimental values and the ones predicted by the mathematical model) of the

statistic package StatisticaTM

v.8 Software (StatSoft Inc., 2007). The parameter’s precision was

evaluated by confidence intervals at 95%, and the quality of regression was assessed by the

determination coefficient (R2), and randomness and normality of residuals (Hill and Grieger-Block,

1980), thus allowing best fit model parameters.

PART IV - DEVELOPED RESEARCH

STUDIES

75

1 FRESH-CUT CARROT: RAW MATERIAL BIOACTIVE QUALITY AND DECAY

PROFILE

In this topic highlight is on the main aspects that determine fresh-cut carrot quality under the

influence of the raw material intrinsic characteristics and of minimal processing per se:

Carrots chemical composition, particularly its bioactive composition, varies according to the

respective cultivar, harvest period and tissue type. To evaluate cultivar, crop season and

tissue type effects on carrots’ bioactive quality two studies were carried out (studies 1.1

and 1.2)

Fresh-cut carrots bioactivity could be improved during storage by triggering phenolic

synthesis as a stress response to wounding. This mechanism is known to be dependent on

wounding intensity, tissue type and storage temperature. To characterize the ability of

carrot tissues (peel and inner tissues) to synthetize phenolic compounds according to

wounding intensity and to quantify the effects of peeling and shredding operations two

studies were carried out (studies 1.3 and 1.4).

Research on FC carrot quality changes usually focus on the effects of one particular

minimal processing operation (e.g. peeling, decontamination, packaging systems) or just

on the changes occurring during storage. A comprehensive approach (study 1.5) was

pursued to ponder the impacts of both minimal processing (multiple operations) and

storage on the overall quality of FC carrot.

1.1 MORPHOLOGICAL DISTRIBUTION OF BIOACTIVE COMPOSITION ALONG CV. NANTES

CARROT TISSUES FROM TWO CROP SEASONS

To assess the morphological distribution (peel, cortical and vascular parenchyma) of bioactive

markers (phenolic and carotenoid contents), related antioxidant capacity and enzymes involved in

its synthesis and degradation (PAL, PPO, POD), six sample types (triplicates) were set up: carrot

peel, cortical and vascular (central stele) tissues were collected as visually recognized (Figure 17)

from two cv. Nantes crop seasons (5 kg), spring (S) and fall (F). Respective weights were taken

against carrots total weight to estimate individual tissue proportion (%). Sample identification is

shown in Table 11.

FRESH-CUT CARROT: RAW MATERIAL BIOACTIVE QUALITY AND DECAY PROFILE

76

Figure 17 Collected tissues: a) peel; b) cortical parenchyma; c) vascular parenchyma (central stele as

identified in Figure 9 [pp. 21]).

Immediately after tissue excision, samples were analyzed for total phenolic (TPC – Protocol I) and

carotenoid (TCC) contents, antioxidant capacity (AOx – Protocol I, both hydrophilic and lipophilic

components) and enzymes involved in its synthesis and degradation (PAL, POD and PPO

activities). The individual contribute of each tissue from the two crop seasons on evaluated

responses were established from sample mean comparison (ANOVA, Tukey HSD test, p=0.05).

Table 11 Sample identification and description.

Tissue type Sample identification

Peel* Peel_i

with i=(S, F) representing Spring crop (S, harvested in May-June) and Fall crop (F, harvested in October-November), respectively.

Cortical parenchyma Cortical_i

Vascular parenchyma Vascular_i

*Peel tissues were 0.73±0.11 mm, measured using a Kuhnke video seam monitor VSM III (v4.2).

The raw material (whole roots) from both crop seasons was also characterized and used to

estimate the tissues contribute (%) to each evaluated response. The results are shown in Table

App. 1 (appendix 1)

1.1.1 Results and discussion

1.1.1.1 Bioactive markers & related enzymes

The contents of bioactive markers (TPC and TCC) and enzymes involved in its synthesis and

degradation (PAL, PPO and POD activities) of the individual cv. Nantes carrot tissues in two crop

seasons are shown in Table 12 which includes also tissues proportion. Peels stand as the least

representative tissue to whole carrot (9 to 12%), followed by the vascular (10 to 16%) and

cortical parenchyma (71 to 78%), independently of crop season.


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As for the phenolic bioactive marker (TPC; Table 12), significantly higher contents were found in

peels: 3 to 4 times regarding the cortical parenchyma (p<0.05) and 7 to 8 times regarding the

vascular parenchyma (p<0.05) from spring and fall crops, respectively. These relations clearly

indicate a grading in cv. Nantes carrot phenolic concentration from peel > cortical >vascular

parenchymas, irrespective of crop season (Table 12). Reports on an uneven phenolic compound

distribution along carrot tissues are found (Zhang and Hamauzu, 2004) and peels are regarded as

an extremely rich tissue in antioxidant phenolics, which agrees with our findings. In fact, carrot peel

phenolic content is sufficiently high to be considered as a source of extractable phenolics to the

production of antioxidant food ingredients (Chantarro et al., 2008), opening an opportunity window

to an extra income for fresh-cut carrot processors since, instead of a waste, peels were to be

regarded as a by-product.

Carrot total carotenoid content (TCC, Table 12) varied (p<0.05) between 8.2 and 15.1 mg -

carotene eq.100 g-1

among carrot samples. Carrot tissue samples from spring crop showed higher

(p<0.05) levels than from fall season and, within the same season, significant differences were only

registered between cortical parenchyma and peel tissues. Similar (p>0.05) carotenoid content was

found between vascular tissues and with both peel and cortical tissues in the spring crop while, in

the fall crop, respective content was only similar to that of the cortical tissue. Chantarro et al.

(2008) reported that the -carotene content in fresh carrot peels was similar to the reported content

by Negi and Roy (2001) in carrot flesh (20 mg.100 g-1

dry weight) which is, considering that -

carotene is the prevalent carotenoid in carrot, distinct from our findings.

Table 12 Bioactive markers content and activity levels of enzymes involved in its synthesis and degradation of cv. Nantes carrot as influenced by tissue type (peel, cortical and vascular parenchyma) and crop season (spring [S] and fall [F]).

Sample identification

Tissue proportion

(%)

TPC

(mg CAE.100 g-1)

TCC

(mg -carotene eq.100 g-1)

PAL

(U.100 g-1)

PPO

(U.g-1)

POD

(U.g-1)

Peel_S 12.1b±0.5 232.7e±20.4 12.9bc±0.8 57.6b±0.7 141.4d±15.6 479.1c±63.5

Cortical_S 71.4d±1.7 72.1c±5.3 15.1d±1.6 25.4c±0.6 31.4a±2.3 72.1b±6.4

Vascular_S 16.4c±2.1 35.1a±2.7 13.4cd±0.8 20.9a±1.4 34.6a±4.4 10.7a±1.3

Peel_F 8.5a±0.6 151.3d±4.0 8.2e±0.5 57.6b±0.7 103.7c±5.0 111.0b±8.5

Cortical_F 77.4e±3.0 37.3a±0.4 11.6ab±1.1 22.4a±0.6 14.7b±1.9 12.7a±0.6

Vascular_F 10.1ab±1.8 17.9b±0.5 10.7a±0.8 20.9a±1.4 22.3ab±0.9 5.7a±0.3

Units expressed on respective tissue fresh weight (FW) basis. Values represent the mean of three replicates±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

Irrespective crop season (p>0.05), peel tissues had 2 to 3 times higher (p<0.05) PAL activity

levels than remaining tissues. Cortical and vascular tissues PAL activity levels varied between 21

and 25 U.100 g-1

, significant only in carrot samples from spring crop (Table 12). As for PPO activity,

peel tissues had significantly higher activity levels (about 5 times) than cortical or vascular tissues,

which had similar (p>0.05) levels, regardless of crop season (Table 12). Concerning POD activity


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and apart from crop season, again peel tissues had significantly higher activity levels than cortical

or vascular tissues. On the other hand, PPO activity levels between cortical and vascular tissues

was significantly influenced by crop season, and only samples from the spring season registered

differences (p<0.05, Table 12).

Bearing in mind the specific tissue function these results are not surprising: peels (or epidermis)

are the most exterior layer that protects the root from environmental stresses and the high phenolic

content found in this peripheral tissue could be considered as an indicator of a more specialized

defense mechanism with the ability to synthetize secondary metabolites as a stress response.

Moreover, the higher levels in PAL activity found in peel are not surprising since respective activity

allows phenolic synthesis and accumulation as a defense response (Dixon and Paiva, 1995). On

the other hand, phenolics are also oxidized in defense-related mechanisms becoming substrates to

PPO and POD, polymerized into dark pigments in the case of PPO and converted into lignin in the

case of POD, reactions easily observed in carrot peel.

Considering the estimated carrot tissues proportions (peels – 9 to 12%; cortical parenchyma – 71

to 74%; vascular parenchyma – 10 to 16%) and the quantification of the bioactive responses in the

raw material (whole root; Table App. 1, appendix 1) of each crop season, the individual contribute

of each tissue to carrots bioactive markers (phenolic [TPC] and carotenoid [TCC] contents) as well

as to the activity levels of the enzymes involved in its synthesis and degradation was found to be

independent of crop season (Table App. 2, appendix 1). As a result, the mean contributes of the

individual tissues from both seasons were used to estimate respective distribution along the root

tissues (Figure 18).

Figure 18 Schematic representation of a carrots’ cross section and respective tissue contributions to cv. Nantes carrot bioactive responses.

From the individual contribute of each tissue on evaluated responses (Figure 18) it is possible to

state that peel, yet with a small tissue proportion (11%) to carrot as a whole, significantly

contributes to carrots total phenolic content accounting for about 30% of the total carrot

concentration. Nonetheless, the major contributor to carrots total phenolic content was the cortical


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parenchyma contributing with 62% (Figure 18) due to its massive representativity in carrot (about

75%). The vascular parenchyma has the lowest TPC contribution to carrots total, with about 5%. In

the work by Zhang and Hamauzu (2004), significant differences were found concerning the

phenolic content of different carrot tissues from two cultivars (cv. Chibagosun and cv. Hitomigosun)

and concluded that peels were responsible for about 50% of the total phenolic content found in

both carrot cultivars. Regarding carrot TCC, the cortical parenchyma was also the major contributor

(75%; Figure 18), which is not surprising given this tissue specificity known as carrots storage unit

since the respective cells gather highest concentrations of starches, sugars and carotenoids

(Waldron et al., 2003). Similar contributes (p>0.05) were found regarding peel and vascular tissues

to carrots TCC.

Concerning enzymes, to carrots’ total PAL activity level the same sorting as for TPC contributes

was found: cortical parenchyma (64%) > peel (21%) > vascular parenchyma (10%). For both

oxidative enzymes, peels exhibit close PPO and POD activity levels to that determined in the

cortical parenchyma: 40% vs. 52% (p<0.05) and 50% vs. 48% (p>0.05), respectively (Figure 18).

Once more, the vascular tissue has a negligible contribute for carrots total PPO (12%) and POD

(2%) activities, which can also be related to its specific function, serving as channels for water and

nutrients to the upper plant (xylem) and photosynthesis metabolites from the upper plant to the root

(phloem) (Purves, Orians and Heller, 1995).

1.1.1.2 Antioxidant capacity

Contributing to carrots antioxidant capacity are both hydrophilic and lipophilic compounds, namely

phenolics (hydrophilic component) and carotenoids (lipophilic component). As previously shown,

the concentration of these compounds varies significantly along cv. Nantes carrot tissues and

between crop season, therefore differences in antioxidant capacity are expected. As a result, to

assess carrot tissues total antioxidant capacity (AOxT) as influenced by crop season, both

hydrophilic (AOxH) and in lipophilic (AOxL) antioxidant extracts were assessed and the results are

shown in Figure 19.

The total antioxidant capacity varied significantly according to tissue type (Figure 19) and carrot

peels showed significantly higher (p<0.05) antioxidant capacity levels than cortical (2 to 3 times)

and vascular tissues (4 to 5 times). Moreover, higher (p<0.05) AOxT levels were found in carrot

peel from the spring crop season regarding fall crop.


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Figure 19 Antioxidant capacity (total [AOxT], hydrophilic [AOxH] and lipophilic [AOxL] components) on different carrot tissues (peel, cortical and vascular) from two crop seasons (spring and fall). Error

bars represent the confidence interval at 95%.

To AOxT, the hydrophilic component (AOxH) was the predominant contributor as expressed by the

high positive and significant correlation found between these responses (AOxT vs. AOxH; r2=0.9,

p<0.05) and higher (p<0.05) AOxH capacities were found in carrot peel regarding cortical and

vascular tissues, irrespective of crop season (Figure 19). The lipophilic contribution (AOxL) to the

total tissue antioxidant capacity was lower than the hydrophilic component (AOxT vs. AOxL, r2=0.7,

p<0.05) but, much like AOxH, it is also possible to define a grading order in tissue AOxL, from

peel > cortical > vascular tissues, regardless crop season (Figure 19). Furthermore, from these

results it can be stated that carrot antioxidant capacity is mainly supported by its phenolic content

(AOxT vs. TPC; r2=0.9, p<0.05) than by its carotenoid content (AOxT vs. TCC; r

2<0.1, p>0.05). In

fact, considering the evaluation of the whole root antioxidant capacity from both crop seasons,

AOxT of 59.9±0.6 and of 95.5±4.4 mg TE.100 g-1

(p<0.05; Table App. 1, appendix 1) were

determined in spring and fall crops, respectively, from which it was found that 60% was due to the

phenolic hydrophilic component, irrespective of crop season.

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1.2 CHARACTERIZATION OF THE TOTAL PHENOLIC CONTENT IN TWO CARROT CULTIVARS

(NANTES VS. NAVAJO) AND IN TWO CV. NANTES CROP SEASONS (SPRING VS. FALL)

To characterize the differences and inherent variability in total phenolic content from two carrot

cultivars, all mean TPC (protocol I), covering 4 years (2010-2013) from raw material

characterization of cv. Nantes (N=69) and cv. Navajo (N=21) were gathered and compared

(unequal N HSD test, p=0.05). Furthermore, in the attempt to find if the established differences in

carrot TPC from two cv. Nantes crop seasons was included within the cultivars’ variability, the TPC

results from the raw material characterization of spring and fall seasons were plotted against the

full TPC appraisals of cv. Nantes.

1.2.1 Results & Discussion

1.2.1.1 Total phenolic content

From the raw material mean TPC characterization of both cv. Nantes and cv. Navajo, it was found

that cv. Navajo has a 2-fold higher phenolic content (143.8±11.4 mg CAE.100 g-1

) than cv. Nantes

(73.5±16.8 mg CAE.100 g-1

) (Table 13). The found difference could be related to distinct genetic

background as Seljåsen et al. (2013) pointed out, reporting that differences up to 10 times in

phenolic content could be found among carrot cultivars.

Table 13 Total phenolic content of cv. Nantes and cv. Navajo carrot.

Cultivar N TPC (mg CAE.100 g-1)

Mean(±SD) Median Minimum Maximum

Nantes 69 73.5a±16.8 75.2 42.4 111.6

Navajo 21 143.8b±11.4 140.1 123.2 172.6

In the mean(±SD) column, different letters represent significant differences at p=0.05 (unequal N HSD test).

Carrots total phenolic content can be described as a cultivar characteristic according to Augspole

et al. (2012) who found that cv. Nantes hybrids (Nantes/Berlikum, Nantes/Maestro, Nantes/Forto,

Nantes/Bolero and Nantes/Champion) had differences in phenolic content of the same order as in

our study (2 fold). In another study, Nicolle et al. (2004) while comparing 20 carrot cultivars, from

white to purple in color, reported that phenolic content varied significantly between cultivars,

following a grading-like pattern related to color, from white (3.3-3.4 mg GAE.g-1

dry weight), yellow

(4.3-4.4 mg GAE.g-1

dry weight), orange (3.3-6.0 mg GAE.g-1

dry weight) and purple (9.6-

16.9 mg GAE.g-1

dry weight). Furthermore, Leja et al. (1997) found that among cv. Joba, cv.

Kazan, cv. Berlanda and cv. Vita Longa carrots, the phenolic content varied significantly with cv.

Berlanda and cv. Joba representing the cultivars with highest and lowest contents. Besides total

phenolic content, Leja et al. (2013), Matějková and Petříková (2010) and Nicolle et al. (2004) also

demonstrated that total carotenoid and vitamin (C, E) contents as well as antioxidant capacity are

distinct between carrot cultivars.


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Within cv. Nantes it was previously established (Part III, section 1.1) that the TPC contents of

carrots from spring crop (82.1±2.1 mg CAE.100 g-1

) was 2 times higher (p<0.05) than that of fall

crop season (46.8±2.0 mg CAE.100 g-1

) (Table App. 1, appendix 1). Environmental conditions are

considered to be most influential to carrots bioactive composition (Manach et al., 2004) and the

differences found between crop seasons regarding TPC could therefore be related to differences in

light exposure, temperature and precipitation. Is has been shown that low cultivation temperature

(9 C vs. 21 C) influences significantly carrots sweetness (increasing fructose and glucose

contents and decreasing sucrose), lowering dry matter content and decreasing -carotene content

(Rosenfeld et al., 1998). In addition, solar exposition, namely increase in UV radiation, has been

shown to be correlated with increased phenolic content (Seljåsen et al., 2013) and also carotene

content (Evers et al., 1997).

However, the significant difference between cv. Nantes TPC from spring and fall crop seasons is

well fitted within cv. Nantes variability as respective mean values are included between the

minimum (42.4 mg CAE.100 g-1

) and maximum (11.6 mg CAE.100 g-1

) TPC mean values of cv.

Nantes (Table 13). The TPC variability in cv. Navajo, cv. Nantes and cv. Nantes from spring and

fall seasons is shown in Figure 20.

Figure 20 Variability plot of total phenolic content (TPC) of cv. Navajo (N=21), cv. Nantes (N=69) and cv. Nantes from fall (N=1) and spring (N=1) crop seasons.

It can be observed from Figure 20 that the TPC variability of cv. Nantes and cv. Navajo is distinct,

supporting the 2-fold (p<0.05) difference found between cultivars. The differences found between

the TPC levels of spring and fall cv. Nantes crop seasons fit within the expectable cultivar variability

leading to the conclusion that crop season influence over TPC is not a significant factor as also

pointed by Leja et al. (1997). In effect, year-to-year variations have been reported to hold little

Raw Data Group Means

Navajo Nantes Nantes (Fall) Nantes (Spring)

Cultivar

0

20

40

60

80

100

120

140

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influence over carrots phenolic content, since insignificant climate changes are encountered within

an annual crop season (Kramer et al., 2012; Søltoft et al., 2010).

1.3 PHENOLIC SYNTHESIS AS AFFECTED BY TISSUE TYPE AND WOUNDING INTENSITY

For the evaluation of peel and inner carrot tissues ability to accumulate phenolic compounds as a

stress response, four sample types, peels (P) and inner (In) tissues with low intensity (LW) and

high intensity (HW) wounding stress, were prepared from a lot of 10 kg of carrot as described in

Part II, section 2.1. Low intensity samples were established form peel tissues removed in strips

from the length of the roots by means of a sharp stainless steel vegetable peeler and remaining

tissues considered to constitute inner tissue samples. High intensity wounding samples (peels and

inner tissues) were set by means of a Grindomix GM200 (3000 rpm x 1 second). Sample

identification is shown in Table 14.

Table 14 Sample description, wound intensity values (A/W) and identification.

Description A/W (m2.kg-1) Sample identification

Peel tissues submitted to low wounding stress 1.8 LW_P

high wounding stress 2.1 HW_P

Inner* tissues submitted to low wounding stress 0.1 LW_In

high wounding stress 2.3 HW_In

*Inner tissues were constituted by both cortical and vascular parenchymas.

Three independent replicates per sample were prepared and “air” stored at 5 C (Part III, section

2.2). Total phenolic content (TPC – protocol I), PAL and polyphenol oxidase (PPO) activity levels

and CIELab color were measured during storage on five sampling dates, i.e., 0, 3, 7, 10 and 14

days. The influence of tissue type and wounding intensity on evaluated responses were

established from sample mean comparison (ANOVA, Tukey HSD test, p=0.05).



Changes in TPC of carrot peels (P) and inner (In) tissues subjected to two wounding intensities

(low and high wounding intensity, LW and HW, respectively) during low temperature storage are

shown in Figure 21.

The initial phenolic content (day 0) of peels was, as expected, 5 times higher (p<0.05, 137-

199 mg CAE.100 g-1

vs. 26-40 mg CAE.100 g-1

) than in inner tissues (Figure 21), regardless of

wounding intensity. During storage, phenolic accumulation in peel tissues was independent of

wounding intensity and greater (p<0.05) than that registered in inner tissues. In the later tissues,

the induced phenolic synthesis was significantly influenced by stress intensity and, during storage,


84

significant accumulation was only determined in inner tissue samples submitted to high wounding

intensity.

Figure 21 Changes in total phenolic content (TPC) in carrot peel and inner tissue samples as

influenced by wounding intensities during low temperature storage (5 C, 14 days). Error bars represent

the 95% confidence interval. [ LW_P: peel tissues submitted to low wounding stress; HW_P: peel tissues

submitted to high wounding stress; LW_In: inner tissues submitted to low wounding stress; HW_In: inner tissues submitted to high wounding stress].

In both peel samples, LW_P and HW_P, significant phenolic accumulation was registered until day

10 (maximum TPC levels of 630 and 696 mg CAE.100 g-1

, respectively), with no further changes to

day 14 (p>0.05). Regarding inner tissues and in comparison to peel tissues, no significant

differences in TPC levels were determined in samples subjected to a low intensity wounding

(LW_In samples; 26.9 – 49.5 mg CAE.100 g-1

, p>0.05) while in a high wounding stress (HW_In

samples), inner tissue samples registered significant and continuous phenolic accumulation from

day 3 reaching TPC levels of 400 mg CAE.100 g-1

at day 14 (Figure 21).

Taking into account the common significant phenolic increase time interval (from day 3 to 10) in

both tissue types, phenolic accumulation in peel tissues was 2 times faster than in inner tissues

since increase rates of 77 and 37 mg CAE.100 g-1

per day were found in peel and inner tissues,

respectively. This difference brings forward the possibility that peels could have a higher ability to

synthetize phenolics than inner tissues and it could be assumed that peels are a more specialized

tissue regarding plants defense mechanisms. Despite the higher phenolic synthesis ability of peels,

the tissue proportion in whole carrot is low (10%) and therefore the individual contribute to the

phenolic synthesis dynamic in fresh-cut carrot could be diluted in regard to the massive proportion

of inner tissues (90%).

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1.3.1.2 PAL activity

Phenolic accumulation in stress situations is well documented in regard to the induction of the

phenylpropanoid metabolism, particularly in what concerns the activation of PAL enzyme. It is

known that a wound signal is generated as a response to physical wound and is translated into a

physiological response (Campos-Vargas et al., 2005; Saltveit, 2004; Kang and Saltveit, 2003). The

precise nature of the signal is still unknown, but it seems to propagate into adjacent, non-injured

tissues (Kang and Saltveit, 2003), inducing the transcription of specific mRNAs, stimulating de

novo synthesis of PAL and respective increase in activity (Choi et al., 2005; Ke and Saltveit, 1989).

More recently, Jacobo-Velázquez et al. (2011) proposed a model in which ATP and reactive

oxygen species (ROS) act as signaling molecules to induce the activation of PAL triggering the

phenylpropanoid metabolism in carrot.

PAL activity changes during low temperature storage of carrot peels (P) and inner (In) tissues

subjected to two wounding intensities (low [LW] and high [HW] wounding intensity) are shown in

Figure 22.

The initial PAL activity levels in peels were 5 to 14 times higher (p<0.05) than those estimated in

the inner tissues (Figure 22), confirming that carrot tissues carrot tissues can be distinguished not

only regarding phenolic content but also by the respective initial PAL activity level.

Figure 22 Changes in PAL activity in carrot peel and inner tissue samples as influenced by

wounding intensities during low temperature storage (5 C, 14 days). Error bars represent the 95%

confidence interval. [ LW_P: peel tissues submitted to low wounding stress; HW_P: peel tissues submitted to

high wounding stress; LW_In: inner tissues submitted to low wounding stress; HW_In: inner tissues submitted to high wounding stress].


0 3 7 10 14

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86

The significant increase in PAL activity of all samples to day 3 is a clear indicator of a stress

response (wounding) which, in this case was tissue dependent (p<0.05): peel samples (LW_P and

HW_P) observed increases of 1.1 to 1.4 times the initial level, while increases of 2.9 and 3.4 times

the initial level were found in inner tissue samples (LW_In and HW_In, respectively). However, in

inner tissue samples subjected to a low intensity wounding (LW_In samples), the respective PAL

activity decreased afterwards with no significant changes during the remaining storage period,

corroborating the determined non-significant change in TPC levels during storage (Figure 21).

Increases in PAL activity of peel samples subjected to a low intensity wounding (LW_P samples)

were only detected to day 3 where maximum activity levels were found (500 U.100 g-1

) and

maintained (p>0.05) until day 7, decreasing (p<0.05) thereafter (Figure 22). Nonetheless,

significant phenolic accumulation was registered until day 10 in these samples (Figure 21) which

suggests that although PAL synthesis is interrupted phenolic synthesis still occurs. As for peels

subjected to a high intensity wounding stress (HW_P samples), the maximum activity level

(360 U.100 g-1

) was reached after 10 days of storage coinciding with the maximum TPC level

found in these samples. Inner tissue samples subjected to high wounding intensity stress (HW_I

samples) showed continuous and significant increases in PAL activity levels, reaching also its

maximum at day 10, with similar activity levels to those found in peels submitted to a low wounding

stress (LW_P samples; p>0.05, 480 U.100 g-1

).

Considering the changes in PAL activity of peel tissue samples, the higher peel ability to phenolic

accumulation is once more brought forward and it could be related to the initial PAL activity levels

(Figure 22) which, in turn, influence the respective phenolic accumulation rate. That is to say that

peels could be a more specialized tissue to respond to wounding since they have a more prepared

mechanism to phenolic synthesis. It is also a possibility that inner tissues would need more time to

accumulate phenolics at a similar rate to that of peels since there is first the need to prepare the

necessary mechanism for phenolic synthesis to occur, namely by de novo synthesis of PAL

enzyme.

Independently of tissue type, PAL activity reaches its maximum activity level for TPC

concentrations varying between 250 and 400 mg CAE.100 g-1

, and starts to decrease when a TPC

concentration above 500 mg CAE.100 g-1

is reached. As a result, there is the possibility that this

TPC level could induce PAL retro-inhibition in carrot tissues. Boerjan et al. (2003) suggested that

decrease in PAL activity may be due to auto-regulation mechanisms and/or to the inhibition of PAL

expression at a transcriptional and post-translational level by cinnamic acid, i.e., by feedback

modulation. In fact, the relation found between PAL activity and TPC in both tissues during storage

suggests that when this relation is around 1, phenolic synthesis starts to decline until no significant

phenolic accumulation is detected since high phenolic levels are reached (Table App. 4, appendix

1). The found relations support the raised hypothesis that PAL activity might be regulated by

phenolic levels.


87

1.3.1.3 PPO activity

Changes in polyphenol oxidase (PPO) activity of carrot tissue samples are shown in Figure 23.

Irrespective of stress intensity, peel tissues had higher (p<0.05) PPO activity levels than inner

tissues (400-480 U.100 g-1

vs. 350-360 U.100 g-1

), with one exception (LW_P at day 0) These

results agree with the findings of Chubey and Nylund (1969) who found that carrot superficial

tissues have higher concentrations of phenol oxidizing enzymes than inner tissues. Changes in

PPO activity of inner tissue samples subjected to low and high wounding stress (LW_In and HW_In

samples) were non-significant during storage, except at day 14, where a significant decrease in

activity was determined in both samples (Figure 23).

Figure 23 Changes in PPO activity in carrot peel and inner tissue samples as influenced by

wounding intensities during low temperature storage (5 C, 14 days). Error bars represent the 95%

confidence interval. [ LW_P: peel tissues submitted to low wounding stress; HW_P: peel tissues submitted to

high wounding stress; LW_In: inner tissues submitted to low wounding stress; HW_In: inner tissues submitted to high wounding stress].

During storage and despite the distinct (p<0.05) initial PPO level, peel tissues submitted to low and

high wounding intensity stress (LW_P and HW_P samples) registered a significant PPO activity

increase (p<0.05) to day 3, decreasing only at day 14. The significant difference found in initial

TPC levels (Figure 21) between these samples might be related with the respective PPO levels

(Figure 23) since synthesized phenolic compounds could be used as PPO substrates.

1.3.1.4 Color

The lower (p<0.05) L* values and respective higher hues (p<0.05) found in peel samples regarding

inner tissue samples (Table 15) are suggestive that peels are more susceptible to browning than

inner tissues which is in agreement with the findings of Chubey and Nylund (1969).


0 3 7 10 14

Storage time (days)

100

200

300

400

500

600

700

PP

O (

U.1

00

g-1

of

tissu

e)


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Table 15 Changes in color (L* value, hue angle and total color difference) in carrot peel (_P) and inner (_In) tissue samples as influenced by wounding intensities (Low: LW; High: HW) during low

temperature storage (5 C, 14 days).

Sample id. Storage time

(days, 5 C) L* Hue E

LW_P

0 55.1f±4.0 60.4hi±1.8 10.0bc±4.1

3 60.7g±2.8 60.6hij±1.9 8.0b±6.2

7 50.5de±2.6 59.6fgh±1.7 12.6cd±4.3

10 48.2cd±1.5 61.6ij±1.8 15.6de±3.7

14 47.2c±2.5 59.2efgh±2.2 19.2ef±4.7

HW_P

0 46.6bc±1.5 62.3j±0.7 14.8d±2.8

3 44.4ab±1.4 60.1hi±1.3 21.5f±2.5

7 43.8a±1.5 60.0ghi±2.5 20.8f±2.0

10 43.1a±1.3 59.1efgh±1.4 22.1f±1.2

14 42.2a±1.4 59.8ghi±1.0 22.7f±2.1

LW_In

0 55.9f±1.6 58.0cdef±0.8 2.2a±1.1

3 60.4g±2.2 54.1a±1.3 8.9bc±2.0

7 61.6g±1.8 56.8bcd±0.4 6.3b±1.8

10 61.9g±1.8 56.7bcd±0.6 6.8b±1.2

14 62.6g±1.6 55.5ab±0.7 8.5b±1.0

HW_In

0 51.3e±1.0 58.3defg±1.3 6.6b±2.3

3 51.2e±0.9 57.5cde±0.6 7.2b±1.6

7 50.8e±1.2 57.8cde±0.9 7.2b±2.3

10 50.7e±1.4 56.7bcd±1.1 8.9bc±2.2

14 50.1de±1.2 56.3bc±1.3 8.8b±2.4

Values represent the mean of 15 measurements per sample ± SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

During storage, both peel tissue samples, registered significant decreases in luminosity (L* value)

which could be associated with peels higher susceptibility to enzymatic browning than inner tissues

(Table 15). In inner tissue samples subjected to low intensity wounding stress (LW_In samples), an

increase (p<0.05) in L* values during storage was observed to day 3 (Table 15) which is usually

associated surface whitening, a well-documented defect of shredded carrot (Barry-Ryan and

O’Beirne, 1998), which could be attributed to superficial dehydration. Nonetheless, during storage

no further changes in these samples were determined. In homologous samples subjected to a high

intensity wounding this defect was not detected since respective L* values were maintained during

storage (p>0.05).


89

The significantly higher color differences (E; Table 15) determined in peel tissue samples at day 0

(E of 10.0 and of 14.8, p<0.05, for LW_P and HW_P samples, respectively) indicate a higher

deviation from raw material color than that of inner tissue samples (E of 2.2 and of 6.6, p<0.05, for

LW_In and HW_In samples, respectively). During storage, the E evolutions of peel samples

(LW_P and HW_P) also reflect the intense browning that occurred in these tissues: E from 10.0 to

22.7 (p<0.05). Inner tissue samples subjected to low intensity wounding stress (LW_In samples), a

significant increase in E values to day 3 (from 2.2 to 8.9) was registered but no additional E

changes (p>0.05) were found during storage. Regarding inner tissues samples subjected to high

wounding stress, the determined non-significant E changes suggest that the color of these

samples remained relatively constant during the entire storage period. The highlighted color

differences between peel and inner tissue samples submitted to high intensity wounding can be

observed in Table 16.

Table 16 Photo record of peel (HW_P) and inner tissue (HW_In) samples submitted to of high intensity

wounding during storage (5 C, 14 days).

1.4 PHENOLIC SYNTHESIS AS AFFECTED BY PEEL REMOVAL AND SHREDDING

It is aimed to quantify the effects of the wounding stresses involved in the fresh-cut carrot

production, peeling and shredding, and the respective balance between the removal of a phenolic-

rich tissue (peel) and the respective wound-induced synthesis under low temperature storage

conditions (5 C), while a mimetic approach to the fresh-cut shredded carrot reality.

To comply with the study objectives, carrots (10 kg) were equally divided into four groups as to

comply with a full factorial design considering peeling and shredding as independent variables:

whole unpeeled carrots (sample id.: W); whole peeled carrots (sample id.: Wpeeled); unpeeled

shredded carrot (sample id.: S); and peeled shredded carrot (sample id.: Speeled). Five independent

replicates (of three carrots for whole and 125 g for shredded samples) were prepared for each

sample type set as described in Part III, section 2.2 and “air” stored at 5 C. Determination of total

Storage time (days, 5 C)

Sample id. 0 3 7 10 14

HW_P

HW_I


90

phenolic content (TPC – Protocol I), PAL activity level, quantification of chlorogenic acid and

antioxidant capacity (AOx – Protocol II) were carried out on four sampling dates, i.e., 0, 3, 7 and 10

days (at 5 C).


The effects of peeling and shredding on the phenolic content (TPC), phenylalanine ammonia lyase

(PAL) activity, antioxidant capacity (AOx) and chlorogenic acid (CA) responses during storage are

shown in Table App. 5 (appendix 1). The discussion of peeling and shredding as main effects is

endorsed by the non-significant effect of the interaction between independent variables (PxS) and,

to the changes of evaluated responses during storage, shredding was the most influential factor, as

shown by the highest F values (Table App. 5, appendix 1).

1.4.1.1 Total phenolic content and chlorogenic acid quantification

Peeling always leads to the significant reduction (p<0.05) of TPC (day 0, Figure 24) where

decreases of, respectively, 39% and 34% (p>0.05) in whole peeled (Wpeeled samples) and shredded

peeled samples (Speeled samples) were estimated. This trend shows the already discussed

significant contribute of peels (30%, Part IV, section 1.1) to carrots initial phenolic content.

However, despite that contribute and as previously hypothesized (Part IV, section 1.3), shredding

increases significantly the superficial area of the product which further increases the low peel

representativity in carrot (10% of carrots total weight) diluting the tissue individual contribute to the

phenolic content of shredded carrot samples.

During low temperature storage (Figure 24), peeled whole carrot samples (Wpeeled samples)

registered a TPC increase, from 57.0 to 77.8 mg CAE.100 g-1

(p<0.05), while no changes (p>0.05)

in TPC were assessed in unpeeled whole carrot samples (W samples). The registered significant

TPC increase in peeled whole carrot samples (Wpeeled samples) indicates that peeling has an

important effect in the stress-induced phenolic accumulation. However, the initial impact of peel

removal is far exceeded when an extreme wounding stress such as shredding is applied,

confirming also that the inner tissues have the ability to synthetize phenolics under an intense

wounding stress.


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Figure 24 Effect of peel removal and shredding on TPC changes of carrot samples during low

temperature storage (5 C, 10 days). Error bars represent the 95% confidence interval. [ W: whole unpeeled

carrots; Wpeeled: whole peeled carrots; S: unpeeled shredded carrot; Speeled: peeled shredded carrot].

In peeled whole carrot samples and unpeeled shredded carrot samples (Wpeeled and S samples,

respectively) where peeling and shredding act as single stress effects, increases of 0.4 and 1.2

times in the initial content were found during storage (Figure 24). The proportional response to

wound intensity in carrot was already demonstrated by Heredia and Cisneros-Zevallos (2009 b)

who reported that phenolic synthesis followed an increase order from sliced, pie-cut and shredded

carrot tissues while intact tissues (whole and unpeeled) had no significant change in TPC during

storage (15 C, 12 days).

More recently, Surjadinata and Cisneros-Zevallos (2012) reported that intense wounding such as

shredding induced a ~2.5-fold increase in soluble phenolics after 4 days of storage at 15 C in

three carrot cultivars (cv. Navajo, Legend and Choctaw). It is noteworthy that significant phenolic

accumulation during the expected product shelf-life was still achieved during low temperature

storage (5 C). Surjadinata and Cisneros-Zevallos (2012) worked at a temperature of 15 C in order

to promote wound responses, while this study was conducted at 5 C to meet the conditions

recommended for quality maintenance of fresh-cut products during respective storage (distribution

and commercialization).

During storage, the behavior of TPC increase was similar in unpeeled (S samples) and peeled

shredded (Speeled samples) carrot samples despite the significant differences registered until day 7

(Figure 24). While significant increases in TPC were registered during the entire storage period in

peeled shredded carrot samples (Speeled samples), this trend was only registered up to the 7th day in

unpeeled shredded carrot samples (S samples). The induced phenolic synthesis attributed to

shredding was sufficient to significantly overcome the phenolic levels (p<0.05) determined in the

W Wpeeled S Speeled

0 3 7 10

Storage time (days)

0

25

50

75

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raw material (sample W at day 0; 93.2 mg CAE.100 g-1

), by 60% and by 40% regarding

unpeeled (S samples) and peeled shredded (Speeled samples) carrot samples, respectively, after 7

days of storage (usual shelf-life of this product). By day 10, no significant differences were found

between these samples (p>0.05). This result was to be expected seeing as the individual peels

contribute to the phenolic accumulation of shredded carrot was significantly diluted as the inner to

peel tissues ratio significantly increases in shredded carrot.

Amodio et al. (2014) proposed a model where phenolic changes during storage are affected by two

mechanisms: phenolic synthesis and phenolic oxidation. Even though the complete description of

the phenylpropanoid metabolism would be very complex, the authors found a reasonable simplified

mechanism to describe the phenolic changes in fresh-cut products. The proposed model takes into

account the de novo biosynthesis of phenolics promoted by PAL (stress induced phenolic

synthesis) and the degradation of phenolics to oxidized compounds during storage, and the

associated equation is described in Part III, section 5.5 identified as Eq. 6.

To evaluate the peel influence on phenolic changes during low temperature storage, the TPC data

of unpeeled (S samples) and peeled (Speeled samples) shredded carrot samples were fitted to Eq. 6

and the model projections are shown in Figure 25 (model parameters included in Table App. 6 and

model regression evaluation in Table App. 7, appendix 1).

Figure 25 Changes in TPC and model fitting to the proposed equation by Amodio et al. (2014) applied to unpeeled (S) and peeled (Speeled) shredded carrot samples during low temperature storage

(5 C, 10 days). [ S: TPC data of unpeeled shredded carrot; TPC model fit of unpeeled shredded carrot;

Speeled: TPC data of peeled shredded carrot; TPC model fit of peeled shredded carrot].

Similar TPC increase behaviors are found from the model projections of unpeeled (S samples) and

peeled (Speeled samples) shredded carrot samples (Figure 25) where it is found that the shredding

Data: S Speeled

Model fit: S Speeled

0 3 7 10

Storage time (days)

0

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

g-1

)

Raw material


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effects were sufficient to match and surpass the raw material initial phenolic level

(93.2 mg CAE.100 g-1

) within the considered storage period. The unpeeled shredded carrot (S

samples) model suggest a less pronounced phenolic accumulation rate than the one described by

the peeled shredded carrot (Speeled samples) model which could be related to differences in

respective phenolic degradation / oxidation rates due to the presence / absence of peel.

Nonetheless, the found behaviors for either sample type suggest that significant phenolic

degradation would not be found during the tested “air” storage conditions (5 C; 10 days). For

model purposes and considering the experimental set up, a longer storage period should be

considered as to observe the expected behavior from a fresh-cut product, with an initial phenolic

increase stage followed by respective decrease occurring at a later storage period (Amodio et al.,

2014).

Usually carrots respond to wounding stresses by synthesizing hydroxycinnamic acids, namely

chlorogenic acid (CA) and derivatives (Jacobo-Velázquez and Cisneros-Zevallos, 2012; Heredia

and Cisneros-Zevallos, 2009), ferulic acid and p-hydroxybenzoic acid (Surjadinata and Cisneros-

Zevallos, 2012) which influence the respective antioxidant capacity. The changes in CA content of

carrot samples during storage as a result of peeling and shredding are shown in Figure 26.

A significant decrease was registered in the CA content of whole unpeeled carrot samples (W

samples) from day 0 (16.6±5.5 mg.100 g-1

) to 3 (10.1±3.1 mg.100 g-1

) without apparent reason.

Considering the peeling effect, a significant decrease is registered at day 0 in all peeled samples

(Wpeeled and Speeled) supporting also that peels significantly contribute to carrots CA content. The

significant increase registered during storage in whole peeled carrots (Wpeeled samples), from

4.2±0.3 (day 0) to 13.1±0.5 (day 10) reveals that the non-significant increase in TPC levels of these

samples could be due to CA accumulation as a response to the peeling wounding stress. Even so,

the CA accumulation level during storage was greater when a more intense wounding stress as

shredding was imposed.

During storage and in regard to the respective initial CA contents, increases in 2.0 and 3.9 times

were found in unpeeled (S samples) and peeled (Speeled samples) shredded carrot samples (Figure

26). Without any differences (p>0.05), both of these samples registered significant phenolic

accumulation to overcome the initial impact of peeling (removal of a phenolic rich tissue) and

shredding (increase in inner tissues superficial area) and reach the raw material CA content

(sample W at day 0) after 3 (S samples) and 7 days (Speeled samples) of storage.


94

Figure 26 Effect of peel removal and shredding on chlorogenic acid (CA) changes of carrot samples

during low temperature storage (5 C, 10 days). Error bars represent the 95% confidence interval. [ W:

whole unpeeled carrots; Wpeeled: whole peeled carrots; S: unpeeled shredded carrot; Speeled: peeled shredded carrot].

The significant increases in CA of both shredded samples during storage are consistent with the

findings of Surjadinata and Cisneros-Zevallos (2012) and confirm that chlorogenic acid was the

prevalent synthesized antioxidant phenolic compound (90%, Figure 27). The accumulation of

chlorogenic acid suggests that its synthesis is responsible for the overall increase in TPC levels

and antioxidant capacity, as also confirmed by the high correlations coefficients found between

chlorogenic acid content and total phenolic content (R2 = 0.90).

W Wpeeled S Speeled

0 3 7 10

Storage time (days)

0

5

10

15

20

25

30

Ch

loro

ge

nic

ac

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.10

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Figure 27 Typical HPLC-DAD chromatogram (shown at 340 nm) of methanolic extracts from (a)

whole carrot (raw material; W sample at day 0) and (b) peeled shredded carrot stored at 5 C for 10 days (Speeled sample at day 10). Peak id.: #1 – chlorogenic acid; #2 – p-hydroxybenzoic acid; #3 – ferulic acid.

Trace amounts of ferulic acid (Rt = 27.302 min; max = 236.3; 323.7 nm) and p-hydroxybenzoic acid

(Rt = 9.320; max = 217.5; 255.1 nm) were only detected in shredded samples after 10 days of

storage (Figure 27). During storage, enhancement in the concentration of ferulic acid in stressed

carrot was already described by Jacobo-Velázquez et al. (2011) in sliced and pie-cut carrot and

Surjadinata and Cisneros-Zevallos (2012) reported also to find increments in p-hydroxybenzoic

acid in shredded carrot samples apart from the increase in ferulic acid. The trace amounts of ferulic

acid found could be explained by its possible use in the production of lignin and suberin during the

plants healing process as suggested by Jacobo-Velázquez and Cisneros-Zevallos (2012).


Changes in PAL activity as a function of peeling and shredding during low temperature storage

(5 C; 10 days) are shown in Figure 28. To day 3, all samples registered significant PAL activity

increase however higher in samples submitted to shredding. The higher PAL activity increase in

shredded samples confirms that the induced response (phenolic synthesis) by peeling (Wpeeled

samples) is surpassed by the high intensity wounding stress of shredding (S and Speeled samples).

The unanticipated increase in PAL activity to day 3 of unpeeled whole carrot (W samples) could be

justified by the stress caused during sample preparation since no other stress was applied. From

day 3 to 10, no further significant changes in PAL activity were registered in whole samples (peeled

or unpeeled).


96

Figure 28 Effect of peel removal and shredding on PAL activity changes of carrot samples during

low temperature storage (5 C, 10 days). Error bars represent the 95% confidence interval. [ W: whole

unpeeled carrots; Wpeeled: whole peeled carrots; S: unpeeled shredded carrot; Speeled: peeled shredded carrot].

PAL activity levels of both shredded samples reached their maximum at day 7 of storage, with

increases (p<0.05) of 8.5 and 9.9 times the initial records in unpeeled (S samples) and peeled

(Speeled samples) samples, respectively (Figure 28). The significant difference found between

sample PAL maximum activity levels can be interpreted as a proportional response to wounding

intensity (shredding vs. peeling x shredding). Although a significant decrease in PAL activity was

observed to day 10 in both sample types, the activity level registered in peeled (Speeled samples)

shredded carrot samples was still sufficient to sustain significant phenolic accumulation from day 7

to 10 (Figure 24). The decrease behavior in PAL activity during storage was already described in

fresh-cut products like lettuce (Choi et al., 2005; López-Galvéz et al., 1996), jicama (Aquino-

Bolaños et al., 2000) and shredded carrot (Jacobo-Velázquez et al., 2011), and it is suggested to

be a result of feedback modulation (Boerjan et al., 2003) or due to the diversion of the synthetic

capacity of the cell to the production of other proteins (Saltveit, 2000).

W Wpeeled S Speeled

0 3 7 10

Storage time (days)

0

50

100

150

200

250

PA

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The changes in antioxidant capacity (AOxH) during low temperature storage (5 C, 10 days) are

shown in Figure 29.

Figure 29 Effect of peel removal and shredding on antioxidant capacity (AOxH) changes of carrot

samples during low temperature storage (5 C, 10 days). Error bars represent the 95% confidence interval. [

W: whole unpeeled carrots; Wpeeled: whole peeled carrots; S: unpeeled shredded carrot; Speeled: peeled shredded carrot].

Determined antioxidant capacity showed to be positively and significantly correlated to phenolic

accumulation (TPC vs. AOxH; R2 = 0.84, p<0.05) and, to this response, shredding was the most

influential effect (p<0.05; Table App. 5, appendix 1) as indicated by the 10 times higher F value

regarding peeling (FShredding = 63.098 vs. FPeeling = 6.223). Due to the positive and significant

correlation found between TPC and AOxH, the considerations taken in regard to TPC can be

applied: intense wounding such as shredding (S and Speeled samples) promotes the increase of

antioxidant compounds (particularly phenolics) to levels suitable to assure maintenance of the raw

material AOxH level (sample W at day 0) after 7 days of storage. Moreover, from the found

correlation between AOxH and CA content (R2 = 0.80, p<0.05) it can be suggested that the

induced CA synthesis and accumulation is the main responsible for the overall increase in

shredded carrot antioxidant capacity.

W Wpeeled S Speeled

0 3 7 10

Storage time (days)

200

400

600

800

1000

AO

xH

(

g T

E.1

00

g-1

)


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1.5 STANDARD MINIMAL PROCESSING AND STORAGE EFFECTS ON FRESH-CUT CARROT

QUALITY

The aim of this study was to characterize the time-decaying sequential pattern that lead to fresh-

like quality losses of FC carrot and to quantify the effects of selected minimal processing unit

operations on the respective bioactive content and its influence during the standard shredded

carrot 7-day shelf-life.

The experimental design was based on a completely randomized design, with 3 independent

replicates per sample type, considering the evaluation of bioactive (total phenolic [TPC – Protocol I]

and carotenoid [TCC] content and respective antioxidant capacity [AOx – Protocol I]), physical-

chemical (pH, soluble solids content [SSC]) and

microbial (TAPC) responses in samples collected

during minimal processing: after peeling,

decontamination and packaging and during 7 days of

“MAP” storage at 5 C, the estimated shelf-life given

from industrial processors.

A lot of 20 kg of carrots was obtained (Part III, section

1) and minimally processed as described on Part III,

section 2.3. Samples were collected during fresh-cut

shredded carrot production, as identified in Figure 30,

and the packaged product (100 g; using film A as

packaging film) was also analyzed during the

subsequent “MAP” storage of 7 days (0, 3, 5 and 7

days at 5 C). During the products shelf-life the same

responses were evaluated adding headspace gas

analysis [O2/CO2 (%) evolution] and sensorial analysis

[color; fresh-like appearance and aroma; and sample

acceptability], which were also determined at day 1 of

storage. Fiber content estimation (total, insoluble and

soluble fractions) was also determined in the raw

material and in the minimal processed product at days

0 and 7.


1.5.1.1 Headspace gas analysis

One of the most important traits of fresh-cut products is maintenance of an active physiology,

namely its respiratory metabolism which has an inverse relation with the respective shelf-life. The

wounding stress imposed during minimal processing induces a substantial increase in carrots

Figure 30 Minimal processing unit operation flow chart and identification of sample collection site.


99

respiration rate (Alegria, 2007). The wound-induced respiration stimulates significant metabolic

changes leading to prolonged increase in respiration which is also stimulated by the

increase/accumulation of ethylene (Saltveit, 2004 a).

Changes in atmosphere composition (O2 and CO2), achieved passively by the product respiration

during respective shelf-life are shown in Figure 31. As it can be observed, a complete inversion of

the packages gaseous composition was attained within one day of storage at 5 C, where O2 levels

decreased significantly to 1.1% and CO2 raised up to 14.8% (p<0.05). Further O2 decreases

(p<0.05) were registered to day 3, to levels of 0.2% which were relatively maintained (p>0.05)

during the remaining storage period, while consistent CO2 increases, significant from day 3

onwards, were registered during the entire storage period, reaching levels as high as 27.6%.

Figure 31 Atmosphere composition (O2 and CO2 concentrations, %) evolution within the packages of

fresh-cut carrot during products shelf-life (5 C, 7 days). Error bars represent the confidence interval at 95%.

Several studies demonstrated in different fresh-cut carrot formats (baby, slices, sticks and shred)

that temperature and gas composition have a great influence on the sensory quality of the product

with significant impact on products decay, weight loss, white discoloration and microbial growth

(Simões et al., 2011; Alasalvar et al., 2005; Izumi et al. 1996; Babic et al., 1993). Once the shift

from aerobic to anaerobic conditions is achieved, fermentation is, of course, responsible for

significant ethanol, acetaldehyde and lactic acid accumulation accountable for off-odors/flavors,

tissue damage and pH drop (Kakiomenou et al., 1996; Lee et al., 1995).


The effects of selected minimal processing unit operations and low temperature storage on TPC

changes can be observed in Figure 32. Regarding the raw material TPC level

(85.3±2.2 mg CAE.100 g-1

), peel removal contributed significantly for the determined 44% TPC

O2 CO2

0 1 3 5 7

Storage (days)

0

5

10

15

20

25

30

35

Ga

s c

on

ce

ntr

ati

on

(%

)


100

decrease (48.0±2.5 mg CAE.100 g-1

) found after peeling operation. Yet, the phenolic content was

further reduced (p<0.05) by an additional 10% during processing, namely after the post-cut chlorine

decontamination procedure (40.1±3.0 mg CAE.100 g-1

).

Figure 32 Minimal processing unit operation effects on total phenolic content and respective

influence during product storage (5 C, 7 days). Error bars represent the confidence interval at 95%

considering the MP operation analysis and Storage analysis independently.

The significant TPC decrease found after decontamination could find explanation within the

procedure itself: shredded carrot is immersed into chlorinated solutions and leaching phenomena

are expected during the operation. These results are in agreement with the findings of

Vandekinderen et al. (2008) who reported significant decreases (25%) in phenolic levels of peeled

grated carrot samples after chlorine decontamination (200 ppm/5 min) attributing that decrease to

leaching phenomena. The higher reported phenolic decrease (25% vs. 10%) could be related to

the substantial dissimilarity regarding treatment time: in the present study, shredded carrot was

immersed in chlorinated water (200 ppm) for 1 min, while Vandekinderen et al. (2008) used a

longer treatment time (200 ppm / 5 min) which could promote solute leaching into the solution.

Overall, during processing a 59% significant decrease in TPC (35.1±1.7 mg CAE.100 g-1

determined in samples after packaging) was found regarding the raw material and such phenolic

level was maintained (p>0.05) during 5 days at 5 C (sample TPC values ranging from 35 to

31 mg CAE.100 g-1

, Figure 32). From day 5 to 7, a significant phenolic increase was determined to

levels of 54.0 mg CAE.100 g-1

(p<0.05) however insufficient to recover from the losses promoted

during minimal processing (Figure 32).

By the end of the considered storage, a still significant 37% difference between product and raw

material was found. Since it was demonstrated (Part IV, section 1.4) that induced phenolic

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synthesis/accumulation occurs as a response to wounding operations during low temperature

storage (5 C) and that response is sufficient to match the raw material level, it could be assumed

that newly synthetized phenolics are being consumed as antioxidants countering the oxidizing

nature of HIPO and respective accumulation. Adding to this result could be also the available

oxygen levels inside the packages since in the previous studies samples were placed in jars vented

periodically (“air” storage) in opposition to foreseeable oxygen consumption in the packaged

product as a result of respiration and microbial development.

Gas composition inside the packages has also been reported to have negative consequences in

what concerns phenolic synthesis since, when excessively low O2 levels are reached, a decrease

in phenolic accumulation is registered in fresh-cut products, including carrot (Alasalvar et al., 2005;

Klaiber et al., 2005 b; Tudela et al., 2002; Babic et al., 1993). The low O2 levels registered just after

one day of storage (Figure 31), did not irreversible blocked phenolic synthesis as a significant TPC

increase to day 7 was determined (Figure 32), but could have significantly influenced the achieved

phenolic levels.

1.5.1.3 Total carotenoid content

Initial carotenoid content was of 18.2 mg -carotene eq.100 g-1

(Figure 33). No significant changes

in TCC were registered after peeling since no differences were found between the raw material and

samples after peeling operation, with levels of 18.2 and 15.4 mg -carotene eq.100 g-1

(p>0.05),

respectively. This result was already expected, since peels have a small contribute to carrots TCC

(8%), as previously shown (Part IV, section 1.1).

Figure 33 Minimal processing unit operation effects on total carotenoid content and respective



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After decontamination, carrot samples denoted a significant TCC decrease to 8.7 mg -carotene

eq.100 g-1

(Figure 33) equivalent to a 52% decline concerning the raw material TCC (18.2 mg -

carotene eq.100 g-1

) which may be attributed to carotenoid leaching and / or oxidation phenomena.

Conversely, Vandekinderen et al. (2008) found no significant losses in carotene content after

product decontamination with chlorine.

The subsequent rinsing operation did not introduced further losses in TCC since no significant

differences (p>0.05; Figure 33) are registered between decontaminated carrot samples and carrot

samples after packaging (11.5 mg -carotene eq.100 g-1

). As a result, by the end of the

technological processing of fresh-cut carrot, a 37% total decrease (p<0.05) in TCC was

determined regarding the raw material. Noteworthy is, despite the non-significant difference, the

carotenoid contents of samples after decontamination and packaging which, regarding the raw

material, registered losses of 59% and 37%. This difference could be related to oxidation

phenomenon attributed to chlorine. In fact, since analytical procedures were carried out 2 h after

minimal processing, the residual chlorine present in the collected samples after the

decontamination procedure may well have promoted additional compound oxidation while the

packaged samples had already gone through rinsing operation reducing residual chlorine to levels

that prevented the continuity of carotenoid oxidation by chlorine. Consequently it is found that post-

cut washing procedures could lead to deleterious effects over this composition especially taking

into account the chemical nature of the decontamination solution.

During the 7 days of storage (Figure 33), no significant changes in TCC were determined, varying

from 11.0 to 11.4 mg -carotene eq.100 g-1

, except on day 5 were a significant decrease was found

without any apparent justification (7.7 mg -carotene eq.100 g-1

, p<0.05). Nonetheless, from this

behavior, it is possible to state that considerable carotenoid losses occur during minimal

processing without additional changes during storage.


Shown in Figure 34 are the changes in antioxidant capacity as a result of minimal processing

operations and during storage. The minimal processing unit operation that mostly contributed to the

significant loss of the products antioxidant capacity (Figure 34) was the decontamination procedure

(66.7 and 50.8 mg TE.100 g-1

, p<0.05, for raw material and carrot samples after decontamination,

respectively). No further changes in AOx (p>0.05) were determined after sample packaging,

accounting for a total 22% significant decrease in AOx. These results are not unexpected

considering the significant decreases registered in phenolic and carotenoid contents which

compromise products antioxidant capacity.


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Figure 34 Minimal processing unit operation effects on antioxidant capacity and respective



During storage, a significant decrease in AOx was still found to day 3 (43.7 mg TE.100 g-1

, p<0.05)

which was maintained during the remaining shelf-life (p>0.05). The further decrease in AOx to day

3 could be attributed to the residual effects of chlorine as the oxidative character of chlorine might

induce consumption of antioxidant compounds (Figure 34).

1.5.1.5 Dietary fiber content

Changes in fiber content during storage of the fresh-cut product are shown in Table 17. The initial

raw material fiber content was of 27.4±4.4 g.100 g-1

(dry weight) and to this content the major

contribution is given by the insoluble fraction as shown in Table 17. Minimal processing operations

did not introduced significant changes over this composition (fresh-cut carrot at day 0), maintaining

the raw material fiber content, both insoluble and soluble fractions. Maintenance in fiber content

(total, insoluble and soluble) was also observed during the products shelf-life (Table 17).

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Table 17 Changes in fiber content (total, insoluble and soluble fractions) of carrot samples after

processing and at day 7 of low temperature storage (5 C).

Sample Storage time

(days)

Fiber content (g.100 g-1 dry weight)

I/S ratio Total* Insoluble fraction (I)

Soluble fraction (S)

Raw material - 27.4ns±4.4 22.1ns±2.4 9.2ns±2.1 2.4ab±0.3

Fresh-cut carrot 0 29.0ns±3.1 19.8ns±2.0 9.2ns±1.1 2.1a±0.1

7 33.0ns±6.3 24.3ns±4.6 8.7ns±1.7 2.8b±0.1

* Total fiber content is estimated as the sum of insoluble and soluble fiber content. Values represent the mean of three replicates±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test). ns – non-significant differences.

The relationship between the insoluble and soluble fiber fractions from carrot (raw material) could

be considered as well balanced (I/S ratio of 2.4) since, according to the recommendation of Spiller

(1986, cit in Grigelmo-Miguel and Martín-Belloso, 1999), the I/S ratio should be in the range of 1.0

to 2.3 in order to obtain the associated physiological effects. After processing, that ratio was

maintained (I/S ratio of 2.1, fresh-cut carrot at day 0; p>0.05) and during storage, although a

significant increase was determined (I/S ratio of 2.8 in fresh-cut carrot at day 7), no differences

were found regarding the raw material I/S ratio (p>0.05). As a result, there is an indication that fiber

content is relatively stable during low temperature storage and therefore, no changes in this

composition are expected during the products shelf-life.

1.5.1.6 pH

The influence of minimal processing operations over carrots pH is shown in Figure 35. Sample pH

varied between 6.4 (raw material) and 6.3 (carrot samples after packaging) from which it can be

concluded that no change in carrots’ pH arise from minimal processing. During storage, sample pH

was also unaffected (p>0.05), varying from 6.3 (at day 0) and 6.2 pH units (at day 7).


105

Figure 35 Minimal processing unit operation effects on pH and respective influence during product

storage (5 C, 7 days). Error bars represent the confidence interval at 95% considering the MP operation analysis

and Storage analysis independently.

1.5.1.7 Soluble solids content

On Figure 36, minimal processing operations influence regarding soluble solids content (SSC) is

shown. Peel removal did not introduce any significant change over carrots SCC (8.3 ºBrix, for both

raw material and carrot samples after peeling, p>0.05). However, after decontamination

procedures, a significant reduction in SSC was found in carrot samples to 6.2 Brix (p<0.05). This

behavior was already described by Alegria et al. (2009), in which significant decrease in SSC was

found in chlorinated samples and attributed to leaching phenomenon associated to

washing / decontamination procedures. By the end of processing, further significant losses in SSC

were found to 5.4 Brix (p<0.05) in carrot samples after packaging procedures from which it can be

deduced that the rinsing step further increased compound leaching.

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Figure 36 Minimal processing unit operation effects on soluble solids content (SSC) and respective



During storage (Figure 36), a significant increase in SSC was found to day 3 (from 5.3 to 5.9 Brix)

which could possibly be related to enzymatic hydrolysis of complex sugars to simple sugars. From

this date onwards a decrease tendency (p>0.05) back to initial content is highlighted which could

result from microbial development (sugar consumption). Despite the significant changes in SSC

during storage, the results suggest that the main significant losses in SSC of FC carrot occur as a

result of minimal processing operations (35%), which could have a significant impact over one of

carrots most appreciated sensorial traits, its respective sweetness.

1.5.1.8 Sensorial analysis

The mean scores for the sensorial attributes of color, fresh-like appearance and fresh-like aroma

and also the respective rejection index are shown in Table 18.

The color sensorial attribute (Table 18) was initially scored with 1.4±0.3 (day 0), which indicates a

small deviation to fresh carrot color (anchor 1). This change in orange color perception, which was

described by the panel member as “lighter orange”, could be due to the significant carotenoid

decrease (37%) as a result of compound leaching during washing / decontamination procedures,

has also already described by Sant’Ana et al. (1998). An increase tendency (p<0.05) in the color

scores was found during the product storage, reaching by the last day of storage (day 7) highest

mean scores, of 2.2±0.3. This tendency, meaning that panel members detected that the product

color had become lighter (anchor 2 corresponds to the perception of “slightly white” shreds) during

storage could be related to the known phenomenon of white-blush related with lignin deposition in

the cut surfaces (Cisneros-Zevallos et al., 1995; Howard et al., 1994; Howard and Griffin, 1993;

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Bolin and Huxsoll, 1991; Tatsumi et al., 1991) and / or to products superficial dehydration (Howard

et al., 1994; Bolin e Huxsoll, 1991; Barry-Ryan et al., 2000).

Similarly to color evaluation, fresh-like appearance scores also registered an increase tendency

during product storage (from 1.1±0.3 to 3.1±0.3, p<0.05). By the end of storage, mean scores of

3.1 were achieved deeming those samples at the saleability limit (Table 18).

Table 18 Sensorial scores and acceptance levels of fresh-cut carrot samples processed according to

the standard (industrial) practice during products storage (5 C, 7 days).

Storage time (days) Sensorial attributes

Rejection index Color Fresh-like appearance Fresh-like aroma

0 1.4a±0.3 1.1a±0.2 3.1b±0.5 1.6a±0.4

1 1.6a±0.4 1.3a±0.3 3.5bc±0.8 2.2ab±0.6

3 1.8ab±0.3 1.5a±0.5 4.1ac±0.6 2.4b±0.4

5 1.9ab±0.2 2.3b±0.3 4.5a±0.5 3.3c±0.3

7 2.2b±0.3 3.1c±0.3 4.7a±0.5 4.0d±0.5

Values represent the mean±SD of the scores given by 8 members of the sensorial panel. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

Color: 1 - Nonwhite; 2 - Slightly white; 3 - Moderate white; 4 - Severe white; 5 - Extreme white, corresponding carrot fresh color to anchor 1;

Fresh-like appearance: 1 - Excellent/fresh appearance; 2 - Moderate; 3 - Limit of saleability; 4 - Poor; and 5 - No fresh appearance, where anchor 1 matches to the perception of the fresh-cut untreated carrot immediately after cutting;

Fresh-like aroma: 1 - Very intense, 2 - Intense; 3 - Moderate; 4 - Low; and 5 - Absent, where anchor 1 corresponds to the perception of the carrots aroma immediately after cutting. Panelists are also asked to identify the presence of any off-odors in the comments section.

Rejection index: 1 – Excellent; 2 – Good; 3 - Limit of marketability; 4 - Poor and 5 - Unusable.

The sensorial attribute that underwent the most significant change during processing was the fresh-

like aroma (Table 18). Just after processing (day 0), mean aroma scores of 3.1±0.5 (Moderate)

were given revealing a significant deviation to carrots characteristic aroma. In fact, most members

of the panel commented that they could detect the presence of an off-odor, described as

“disinfectant”. During storage, progressive deterioration of samples fresh-like aroma attribute was

registered (scores ranging from 3.1±0.5 [Moderate; day 0] to 4.7±0.5 [Low to absent; day 7],

p<0.05) and by day 7, some members of the panel detected the presence of an off-odor described

as “fermented”. This comment might be an earlier indicator of excessive lactic acid bacteria growth

during product storage.

Sample rejection index during storage mirrors the overall sensorial quality of the product and, to

this acceptability judgment, fresh-like appearance evaluation was the sensorial attribute with

greater input as shown by the higher correlations found (rejection index [RI] vs. fresh-like

appearance; r2=0.8, p<0.05). In this sense, product rejection is considered when RI mean scores

above 3 (limit of marketability) are reached, since the product no longer meets the sensorial panel

quality standards. After processing (day 0) and at day 1, samples showed no significant differences

between respective RI scores (1.6±0.4 and 2.2±0.6, respectively, p>0.05), implying a good product

quality (Table 18). To day 3, a deviation from the initial sensorial quality (day 0) was detected

(p<0.05), but samples were scored below the limit of marketability (2.4±0.4). This limit was however


108

surpassed at day 5, were samples were rejected by panel members (scores of 3.3±0.3)

considering the color change (lighter samples), moderate fresh-like appearance and high deviation

to fresh-like aroma. According to this criterion, the usually given shelf-life to fresh-cut shredded

carrot (of 7 days) by the processors is over-estimated, since suitable product sensorial quality is

only assured during 3-4 days. The development of off-odors, loss in fresh-like appearance and

shred discoloration were the main sensorial quality aspects which limited the products shelf-life.

1.5.1.9 Microbiological responses

Initial mesophilic counts (TAPC) of 5.8±0.1 Log10 were found in the raw material. This

contamination level is well within the range of initial contamination of carrot found on literature

(Garg et al., 1990; Alegria et al., 2010; Carlin et al., 1989) and is usually related to the amount of

soil that is adhered to carrots surface. The effects of minimal processing unit operations and the

respective storage effects on the growth of this microbial group are shown in Figure 37. Significant

lower TAPC counts were determined in carrot samples after peeling regarding raw material,

(4.5±0.1 Log10 vs. 5.8±0.1 Log10, respectively). This result shows that peeling was responsible for a

significant 1.3 Log10 reduction in mesophilic contamination. Garg et al. (1990), while assessing

microbial counts during the different stages of a fresh-cut processing line, found that peeling

contributed to minimize carrots initial contamination by 2 Log10.

Figure 37 Minimal processing unit operation effects on total aerobic counts (TAPC) and respective

evolution during product storage (5 C, 7 days). Error bars represent the confidence interval at 95%


Just after the decontamination process (Figure 37), a further reduction (p<0.05) in TAPC counts

was determined to levels of 3.7±0.3 Log10, and the associated decontamination effect of the

chlorine solution (200 ppm/1 min; 5 C; HIPO) was of 0.8 Log10. No significant changes in

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mesophilic flora counts were found after product packaging as similar counts were found between

FC carrot samples after decontamination and packaging (p>0.05).

The decontamination effectiveness of HIPO has already been challenged by several authors

(Adams et al., 1989; Beuchat, 1999; Li et al., 2001; Nguyen-The and Carlin, 1994; Zhang and

Farber, 1996) and all report a low efficiency leading to the search of alternative and efficient

decontamination methodologies. However, it is a fact that most fresh-cut industries, particularly

fresh-cut carrot processors, use chlorine-based decontamination procedures (Rico et al., 2007;

Seymour, 1999).

The effect of chlorinated-water as a decontamination process reported in literature seems to be

somewhat inconsistent: Klaiber et al. (2005 a) found a 1.7 Log10 reduction using a 200 ppm

chlorine solution (120 s) on cv. Bangor carrot, while Sinigaglia et al. (1999) found no effect over

initial microbial load using a 100 ppm free chlorine solution. The determined 0.8 Log10

decontamination efficiency (Figure 37) is generally in agreement with the above mentioned data

and acknowledges chlorines’ reduced lethality in direct contact with carrot cut tissues. Contributing

to this result, failure to inactive microorganisms, could be the increase of organic load during

product immersion due the great surface area exposed by cutting operations and respective

compound release to the chlorine solution as also pointed by Gonzalez et al. (2004).

The microbial quality in fresh-cut products is compromised during storage due to excessive

microbial outgrowth and/or inadequate decontamination and the Health Protection Agency’s (HPA,

2009) issued generic guidelines for assessing the microbiological quality of ready-to-eat foods that,

even though not taking precedence over set microbiological criteria within European or national

legislation, set threshold limits to aerobic colony count levels and within this, limits to the

predominant microorganisms. To fresh-cut shredded carrot, included in food category 8 (extended

shelf-life food products requiring refrigeration) of the HPA guidelines, the threshold limit for aerobic

colony count levels is of 106-<10

8 cfu.g

-1 (>10

8 cfu.g

-1, the product is considered “Unsatisfactory”)

which is in agreement with the French legislation (counts of 5 x 106 cfu.g

-1; Ministere de l’Economie

des Finances et du Budget, 1988). In this study, a threshold limit of 7.5 Log10 was considered

which is within both HPA and French legislation proposed limit.

During storage, significant TAPC increases were registered (Figure 37), reaching counts of

7.2±0.4 Log10 by day 7 which can be considered as similar to the set threshold limit of 7.5 Log10.

This result shows that the usual expiry date of 7 days given by processors undertakes significance

and is well fitted considering that the mesophilic growth rate was almost linear and of about

0.5 Log10 per day.


110

Shown in Figure 38 are the results regarding lactic acid bacteria (LAB) and initial counts of

3.5±0.1 Log10 were found in the raw material.

Figure 38 Minimal processing unit operation effects on lactic acid bacteria counts (LAB) and

respective evolution during product storage (5 C, 7 days). Error bars represent the confidence interval at

95% considering the MP operation analysis and Storage analysis independently.

The initial contamination level was significantly reduced after peeling with counts of 2.7±0.1 Log10

implying a reduction effect of 0.8 Log10. Chlorine decontamination further significantly reduced the

LAB contamination level by another 0.8 Log10 which was again maintained till the end of processing

(FC carrot samples after packaging). The assigned decontamination efficiency of chlorine is

therefore of 0.8 Log10 regarding lactic flora, which once again shows that chlorine is far from being

a highly efficient decontamination treatment for fresh-cut carrot. As for TAPC, LAB counts also had

an almost linear growth rate, and significant increases were determined during the entire storage

period (Figure 38). The determined LAB counts at day 7, of 5.4±0.1 Log10, were below the HPA

guidelines threshold limit for the predominant microflora (LAB counts up to 108 cfu.g

-1, HPA, 2009).

Kakiomenou et al. (1996) and Carlin et al. (1989) reported that LAB group prevailed over

pseudamonads and yeasts in fresh-cut carrot with Leuconostoc mesenteroïdes mesenteroïdes

(heterofermentative microorganism) as the dominant microorganism within this microbial group.

The predominance of LAB in shredded carrot has marked consequences regarding the products

sensorial quality, namely off-odors development, mucilaginous (“slimy”) material accumulation on

product surface as a result of elevated lactic/acetic acids and ethanol concentrations (Sinigaglia et

al., 1999; Kakiomenou et al., 1996; Nguyen-The and Carlin, 1994).

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Given the increasing LAB contamination level during storage, one could assume that the

overwhelming CO2 increase to levels of 28% (Figure 31) was not only due to the products

respiration rate, but also to fermentation processes, namely those related to LAB metabolism.

Regarding the yeast and mold microbial group (Y&M), the effects of minimal processing operations

and respective evolution during product storage are shown in Figure 39.

Figure 39 Minimal processing unit operation effects on yeast and mold counts (Y&M) and

respective evolution during product storage (5 C, 7 days). Error bars represent the confidence interval at

95% considering the MP operation analysis and Storage analysis independently.

The raw material contamination level was of 3.2±0.2 Log10 and during the different stages of

minimal processing, no changes were found regarding Y&M counts, ranging from 3.2 to 3.1 Log10

(p>0.05). During the 7-day storage, no significant changes in Y&M counts were determined (counts

from 3.1 to 3.5 Log10, p>0.05) which might be related to the established atmosphere inside the

packages. Moreover, these results suggest that to fresh-cut carrot microbial quality this microbial

group is not “problematical”.

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2 HEAT SHOCK AND UV-C STRESS EFFECTS ON CARROT QUALITY: SINGLE

AND COMBINED APPLICATION

The studies under this research line include the evaluation of the single effects of heat shock and

UV-C treatment conditions (intensity and hierarchical stress order) regarding product bioactive

responses (studies: 2.1, 2.2 and 2.3). A further evaluation of the combination of treatments was

carried out to address the potential for additive or synergistic effects (study 2.4).

2.1 HEAT SHOCK AND UV-C STRESS EFFECTS ON FRESH-CUT CARROT QUALITY

This preliminary evaluation aimed to:

i. Evaluate the single effects of heat shock (100 C/45 s) and UV-C radiation (0.78 kJ.m-2

)

stress pre-treatments in whole carrots on the total phenolic and carotenoid contents, POD

activity and respiratory metabolism of fresh-cut carrot during storage (5 C, 10 days);

ii. Compare the effects of heat shock and UV-C radiation single stress pre-treatments as

alternatives to chlorine (200 ppm/1 min) regarding product decontamination and microbial

development during storage (5 C, 7 days).

A lot of 100 kg of cv. Nantes carrot was obtained and handled as described in Part III, section 1.

From the initial lot, a 10% sampling was used to conduct a physical-chemical and microbial

characterization of raw fresh carrot after peeling and the results are shown in Table App. 8

(appendix 2).

The remaining 90 kg of carrot were equally divided into three groups and used to prepare of fresh-

cut shredded carrot samples as shown in Figure 40. Carrots submitted to heat shock and UV-C

radiation (Part III, section 3.1) and further minimal processed (Part III, section 2.3) were identified

as HS and UV samples, respectively. Carrots only submitted to minimal processing according to an

industrial practice (chlorine decontamination, Part III, section 2.3), were identified as HIPO

samples. Samples were packaged in portions of 125 g using bags (200 x 110 mm) made from film

A (35 mm bioriented polypropylene with OTR and CTR of rates of 1100 cm3.m

-2.24 h

-1.atm

-1 and

3000 cm3.m

-2.24 h

-1.atm

-1 at 23 ºC, respectively). The bags were heat-sealed and stored at 5 C.

The abiotic stress pre-treatments (heat-shock and UV-C irradiation) were applied 24 h before

processing since, according to Lamikanra and Watson (2007), favorable changes in the metabolic

rates of quality-related enzymes (e.g. peroxidase, POD) are achieved during storage using this

hold-up period before processing. Evaluated responses during “MAP” storage (5 C, 10 days)

included total phenolic (TPC – protocol II) and carotenoid (TCC) content, peroxidase (POD),

HEAT SHOCK AND UV-C STRESS EFFECTS ON CARROT QUALITY: SINGLE AND COMBINED APPLICATION

114

CIELab color, headspace gas analysis, sensorial analysis (color, fresh-like appearance and aroma

and general acceptance) and total mesophilic aerobic counts (TAPC). Data from the trial was

subjected to analysis of variance (one-way ANOVA) and statistically significant differences

between samples were determined according to Tukey Honestly Significant Difference (HSD) test.

Figure 40 Flow diagram of minimal processing operations for shredded carrots according to each abiotic stress pre-treatment (heat shock and UV-C irradiation) and to the standard practice.


115



The headspace analysis (O2 and CO2 concentration, %) revealed that both pre-treated samples

had significantly reduced metabolic rates in comparison to HIPO samples (Figure 41). Both pre-

treatments were responsible for delaying (by one day at least) the inversion of the modified

atmosphere inside the packages to anaerobic conditions regarding the standard processing.

However, heat shock proved more favorable to the decrease in respiration metabolism since lower

O2/CO2 rates were registered during storage.

Figure 41 Changes in atmosphere composition (O2 and CO2 concentrations, %) of fresh-cut shredded carrot as affected by abiotic stress treatments during storage (5 °C, 10 days). Error bars

represent the 95% confidence interval. [HIPO: post-cut chlorine (200 ppm/1 min) decontamination; HS: heat shock

(100 C/45 s) applied 24 h before carrot shredding; UV: UV-C treatment (0.78 kJ.m-2) applied 24 h before carrot shredding].

Reduced respiration rates due to heat shock pre-treatments have already been reported in several

fruits and vegetables, namely shredded carrot, by different authors (Alegria et al., 2010; Rico et al.,

2008; Klaiber et al., 2005 a; Serrano et al., 2004). The same effect was also observed by Baka et

al. (1999) in UV-C treated strawberries during postharvest storage.

2.1.1.2 Total phenolic and carotenoid contents

At day 0, all samples showed a significant decrease (p<0.05) in total phenolic content (TPC –

protocol II) of ca. 37%, 24% and 17% for HIPO, UV and HS samples, respectively (Table 19) when

compared to the fresh content of 21.0±0.9 mg GAE.100 g-1

.

O2: HIPO HS UV

CO2: HIPO HS UV

0 3 5 7 10

Storage time (days)

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Table 19 Changes in total phenolic (TPC) and carotenoid content (TCC) for fresh-cut shredded

carrot samples during storage (5 C, 10 days).

Sample ID Storage time (days) TPC (mg GAE.100 g-1) TCC (mg -carotene eq.100 g-1)

Raw material - 21.0g±0.9 27.5b±0.4

HS

0 17.3def±2.3 19.9a±0.2 3 18.7fg±1.6 nd 5 18.9fg±2.1 25.0g±0.6 7 30.1h±1.1 13.6f±0.2

10 17.5def±1.1 nd

UV

0 16.0cde±1.1 10.0d±0.0 3 18.5efg±0.7 nd 5 18.8fg±1.1 20.2a±0.9 7 14.3abc±1.9 28.8h±0.0

10 17.0cdef±1.1 nd

HIPO

0 13.3ab±1.5 11.9e±0.0

3 15.6bcd±1.3 nd

5 16.3cdef±2.2 19.7a±0.3

7 12.3a±1.9 8.9c±0.0

10 16.9cdef±0.8 nd

mg GAE/100 g – mg gallic acid equivalents per 100 g fw; nd – not determined; Values are mean±SD; In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

Loss in phenolic content in FC carrot as a result of chlorinated-water treatment could be attributed

mainly to leaching phenomena, as previously discussed and also as reported by Vandekinderen et

al. (2008). Heat-induced losses of phenolic compounds could be attributed to phenolic thermal

degradation (autoxidation or breakdown). Also supporting this result is the work by Turkmen et al.

(2005) who found that blanching vegetables (boiling water) would cause phenolic losses up to

40%. It is known that oxidizing treatments such as UV-C light are likely to be involved in photo-

oxidation reactions in plants with production of free radicals (Maharaj et al., 1999), which can lead

to phenolic consumption as antioxidants. Losses in total phenolic content were also observed in

strawberries by Pan et al. (2004) 1 day after UV-C treatment (4.16 kJ.m-2

). However, also in

strawberry, Allende et al. (2007) observed maintenance in phenolic content just after UV-C

treatment (1 kJ.m-2

).

In general, HS and UV samples showed higher phenolic content during storage than HIPO

samples (Table 19) which maintained (p>0.05) initial post-processing levels throughout storage. HS

samples showed a significant (p<0.05) TPC increase at day 7 (30.2±0.8 mg GAE.100 g-1

, Table 19)

surpassing that of fresh peeled carrot (21.0±0.9 mg GAE.100 g-1

) followed by a significant

decrease to day 10 to about half the previous record. As for UV-C treated samples, higher TPC

levels were found regarding the HIPO sample, and a significant phenolic increase was observed

from day 0 to 5 (16.0±1.1 to 18.8±1.1 mg GAE.100 g-1

) barely reaching the raw material levels. To

day 7, a significant decrease in UV samples TPC was determined to levels of

14.0±1.9 mg GAE.100 g-1

, with no further significant changes to the end of storage. The level of

TPC changes during storage of UV samples comes as a surprise since significant phenolic

accumulation was expected: enhancement of bioactive compounds during storage of fresh and

processed fruits and vegetables using UV treatments has been reported by several authors (Du et


117

al., 2012; González-Aguilar et al., 2007 b; Surjadinata Cisneros-Zevallos, 2005; Baka et al., 1999)

attributing those increases to induced changes in several phytochemical pathways.

The changes in atmosphere composition (O2 decrease/CO2 increase) inside the packages of all

samples could also aid to justify the registered low to absent phenolic accumulation since

significant changes in the surrounding atmosphere occurred as a result of the respiration

metabolism (Figure 41) compromising suitable O2 levels for phenolic synthesis (Klaiber et al.,

2005 b; Babic et al., 1993). O2 concentration could therefore be regarded as a limiting factor to

phenolic synthesis.

Total carotenoid content (TCC) of all samples decreased considerably just after minimal processing

(Table 19). The highest loss (p<0.05) was registered in UV samples (64%) followed by HIPO (57%)

and HS (28%) samples when compared to fresh content (27.5±0.4 mg -carotene eq.100 g-1

).

Thereafter, carotenoid levels of all samples increased (p<0.05) at day 5 of storage followed by a

reduction (p<0.05) on the last day of analysis, except for UV samples. Moreover, UV samples

exhibited an interesting behavior during storage since a consistent increase was observed in TCC,

reaching levels three fold higher at day 7 than at day 0 (10.0±0.0 vs. 28.8±0.0 mg -

carotene eq.100 g-1

). In agreement are the findings of Liu et al. (2009), who reported that during

postharvest storage, carotenoid levels in UV treated tomato did not change during the early stages

of storage and started to increase thereafter. This behavior is of upmost importance since, in

human diet and from the nutritional standpoint, carrots are one of the major sources of carotenoids.

Nonetheless, this result could also be due to a higher extractability of these compounds since

increased cell wall depolymerization caused by this stress could also be in order (Bhat et al. 2007,

cit in Alothman et al. 2009).

2.1.1.3 POD activity

Peroxidase (POD) activity is stimulated by wounding, physiological stress and the readily

availability of phenolic substrates. Partial inhibitions in POD activity of 30% and 46% were found in

HS and UV samples respectively, when compared to the raw material (Figure 42). These levels of

inhibition were relatively maintained up to day 7 followed by an increase in activity (p<0.05) to day

10. In HIPO samples, maintenance in initial POD activity was also registered during 7 days but with

higher (p<0.05) levels when compared to pre-treated samples. Also, a significant increase was

found till day 10 surpassing by 20% the raw material level.


118

Figure 42 Effect of abiotic stress treatments on peroxidase (POD) activity of fresh-cut shredded carrot during storage at 5 °C. Error bars represent the 95% confidence interval. [HIPO: post-cut chlorine

(200 ppm/1 min) decontamination; HS: heat shock (100 C/45 s) applied 24 h before carrot shredding; UV: UV-C treatment (0.78 kJ.m-2) applied 24 h before carrot shredding].

The behavior between pre-treated and HIPO samples points out to the abiotic stress treatments

effects, particularly heat shock, over the enzyme activity level, which is in agreement with Alegria et

al. (2010). Moreover, Artés-Hernández et al. (2010) while studying the effects of UV-C treatments

on the quality of fresh-cut watermelon, found that treated samples showed an initial decrease in

enzyme activity (catalase) and suggested that the observed decrease could be evidence for the

protective role against active oxygen species induced by oxidative stress in which POD is also

involved (Apel and Hirt, 2004).

2.1.1.4 Color

Color changes, expressed as whiteness index, WI (Table 20), show an increase during storage in

all samples (p<0.05). HS samples had significantly lower WI mean scores in comparison to HIPO

and UV samples, until day 3. By the end of storage, all samples had identical WI values (p>0.05).

HIPO HS UV

0 3 5 7 10

Storage time (days)

25

30

35

40

45

50

55

60

65

70

75

80

85

PO

D (

U.g

-1)

Raw material


119

Table 20 Changes in color, expressed as whiteness index (WI), of FC carrot samples during storage

(5 C, 10 days).

Sample Storage time (days) Whiteness Index

HIPO samples 0 25.5b±2.1 3 27.3c±2.4 5 28.2cde±2.6 7 28.0cde±2.2 10 28.9e±2.7

HS samples 0 24.2a±2.2 3 25.9b±3.0 5 27.6cd±2.5 7 27.2c±2.7 10 28.2cde±2.7

UV samples 0 25.2ab±1.8 3 27.3c±2.0 5 28.2cde±2.2 7 28.7de±2.4 10 28.9e±2.3

Values are mean±SD; In the same column, different letters represent significant differences at P=0.05 (Tukey HSD test).

Increases in WI values during storage are usually correlated to surface whitening, a well-

documented defect of shredded carrot, and has been attributed to surface dehydration and/or lignin

synthesis (Barry-Ryan and O’Beirne, 1998). Whitening control in heat-treated shredded carrot has

been previously reported and correlated with peroxidase inhibition (Alegria et al., 2010; Howard et

al., 1994).


The evaluation of the sensorial attributes of color, fresh-like appearance, fresh-like aroma and

respective rejection index are shown in Figure 43 a and b, respectively.

No differences (p>0.05) in color (Figure 43 a) were detected after processing (day 0) between

samples. During storage, significant increases in the scores of sensorial color were registered in

HIPO and UV samples, from 1.4 (day 0) to 1.9 (day 10) in both samples (p>0.05), relating to a loss

in orange color intensity (lighter samples). In HS samples no significant color changes were

detected by the sensorial panel, registering lower scores during the entire storage period (scores

from 1.1 to 1.4, p>0.05). A significant and positive correlation (r2 = 0.7, p<0.05) was found between

color sensorial evaluation and the calculated color parameter WI. In fact, lower WI values were

determined in HS samples, matching the sensorial evaluation.

Fresh-like appearance registered an increase tendency all samples throughout storage (p>0.05;

Figure 43 a), relating to losses in this attribute. No differences between samples were found up to

day 5, with scores ranging from 1.0 to 1.5 (p>0.05). However, from this time onwards significant

differences were registered between samples and, once again, HS samples were classified with

lower (p<0.05) scores than HIPO and UV samples, with a final score (day 10) of 2.1 vs. 2.6 (HIPO

and UV samples, p>0.05). Nonetheless, by the end of storage, all samples were scored between

anchor 2 (moderate fresh-like appearance) and 3 (limit of saleability).


120

Figure 43 Effect of abiotic stress treatments on a) sensorial attributes of color, fresh-like appearance and aroma and b) acceptability levels (rejection index) of fresh-cut shredded carrot during storage at 5 °C. Error bars represent the 95% confidence interval. [HIPO: post-cut chlorine (200 ppm/1 min)

decontamination; HS: heat shock (100 C/45 s) applied 24 h before carrot shredding; UV: UV-C treatment (0.78 kJ.m-2) applied 24 h before carrot shredding].

As previously seen (Part IV, section 1.5), the fresh-like aroma (Figure 43 a) was negatively affected

by the standard industrial carrot processing since, at day 0, aroma scores of HIPO samples were of

3.0±0.5 (Moderate), and during storage further losses in the characteristic aroma was registered,

reaching scores of 4.4±0.3 by day 10 (low to absent). On the contrary, both HS and UV samples

denoted a good aroma retention after processing (mean scores of 1.3 and 1.2 at day 0, p>0.05,

respectively). Progressive losses in aroma were also registered in these samples during storage,

Storage time (days)

0 3 5 7 10

1

2

3

4

5

Sen

so

rial

sco

re

0 3 5 7 10 0 3 5 7 10

Color Fresh-like appearance Fresh-like aromaa)

HIPO HS UV

0 3 5 7 10

Storage time (days)

1

2

3

4

5

Re

jec

tio

n I

nd

ex

b)

Limit of marketability


121

but to a lower (p<0.05) extent than the one registered in HIPO samples. By the end of storage, HS

and UV samples had aroma scores of 2.8±0.3 and of 3.1±0.2 (p>0.05), respectively, meaning a

“moderate” fresh-like aroma. These results show that both abiotic stresses were favorable to the

retention of the carrots characteristic aroma during product storage.

By day 10, HIPO samples were rejected (rejection index scores above 3, limit of marketability) by

the sensorial panel as it can be seen from Figure 43 b, and barely accepted on day 7 (score of

2.8). On the contrary, HS and UV samples were always below this limit, indicating that during

storage, samples denoted a good overall sensorial quality, attesting favorable effects to the abiotic

stresses in regard to products sensorial quality. In this particular case, heat shock can be

considered as best suited for sensorial quality maintenance since fresh-like appearance was

achieved, aroma was best retained and no significant color changes were detected.


Decontamination effects were more expressive in HS samples, where a ≈ 2.5 Log10 reduction

(p<0.05) in the initial microbial load was found (Table App. 8, appendix 2), and only a 0.6 Log10

increase in TAPC was registered after 7 days of storage (Figure 44). This indicates the highly

significant effect of heat shock in controlling the microbial development, as also previously reported

by Alegria et al. (2010) and Klaiber et al. (2005 a).

Figure 44 Effect of abiotic stress treatments on mesophilic aerobic flora (TAPC) of fresh-cut shredded carrot at day 0 and 7 of storage at 5 °C. Means with the same letter are not significantly different

(p<0.05). Error bars represent the 95% confidence interval.

The significantly lower microbial development rates found in HS samples support that heat shock is

an effective decontamination alternative to microbial control in fresh-cut carrot. Nonetheless, further

studies are needed in order to understand this treatment effect over pathogenic flora. It is known

HIPO HS UV

0 7

Storage time (days)

0

1

2

3

4

5

6

7

8

TA

PC

(L

og

cfu

.g-1

)

Threshold limit

Initial contamination level (raw material)


122

that even though heat treatments may result in extended shelf-life, they may also provide

conditions for pathogens development and thereby increasing the risk for human health problems,

as demonstrated in lettuce (Li et al., 2002).

The use of heat in fresh and fresh-cut fruits and vegetables, for decontamination purposes, has

variable efficacies since it depends on the type of product and applied thermal intensity. Several

studies show that heat treatments have beneficial effects to achieve reduction in the deterioration

microflora (ranging from 1 Log10 to 3 Log10). As for pathogenic flora, the results are contradictory

since some studies demonstrate that the use of heat treatments may either increase the risk of

pathogen survival and growth, namely of E. coli and L. monocytogenes in iceberg lettuce (Delaquis

et al., 2002; Li et al., 2002), or favor pathogen elimination and/or inhibit its outgrowth during storage

as shown in cantaloupe melon (Annous et al., 2004; Solomon et al., 2006) and melon (Ukuku et al.,

2003). The use of temperature above 70 C, as is in the present study, is regarded as more

effective in the elimination of vegetative bacterial pathogens (Fan et al., 2009). Still, products’

safety issues derived from the elimination/reduction of the competition effects of the epiphytic

microflora as also the leakage of electrolytes after treatment (which provides favorable conditions

to bacteria growth) should, of course, be addressed.

UV-C stress treatment achieved a 1.7 Log10 reduction in the initial microbial load rendering it as

effective as chlorine decontamination (1.9 Log cycle). However, UV samples at the 7th day of

storage reached counts of 4 Log10 cfu.g-1

, with no differences (p>0.05) to HIPO samples.

Noteworthy is that all samples by day 7 had TAPC counts below the microbiological threshold limit

to define fresh-cut products shelf-life (7.5 Log10) (HPA, 2009).

2.2 UV-C SINGLE STRESS EFFECTS ON THE WOUND-INDUCED DYNAMICS

Considering that a hierarchical order between applied abiotic stresses (UV-C and shredding) could

influence the phenolic accumulation pattern of wounded tissues (Lamikanra and Watson, 2007;

Saltveit, 2000), it was aimed to study:

i. The UV-C dose (kJ.m-2

; =254 nm) that promotes higher phenolic synthesis without

impairing the carrot quality;

ii. The effects of UV-C treatment application in whole (pre-cut; 24 h before shredding) and in

shredded carrot (post-cut) on the phenolic synthesis dynamic.


123

2.2.1 RSM modeling of UV-C treatment

The effects of UV-C treatments (UV-C dose/application site) on shredded carrot bioactive and

sensorial quality during subsequent low temperature storage (5 C) were evaluated using the

response surface methodology (RSM) based on a two-variable central composite rotatable design

(CCRD) (Part III, section 5.2). The experiments were carried out following a CCRD as a function of

two factors: UV-C dose (UV, kJ.m-2

) and storage time (St, days; 5 C) on the total phenolic (TPC –

Protocol I) and carotenoid (TCC) contents and CIELab color (dependent variables) of shredded cv.

Nantes carrot. The ranges of interest of each independent variable were chosen in accordance with

the works of González-Aguilar et al. (2007 a,b), Erkan et al. (2008), Allende et al. (2006 b): UV-C

dose: 0.1-5 kJ.m-2

; Storage time: 0-8 days. Table 21 shows the decoded matrix of independent

variables.

Table 21 Decoded experimental design matrix.

Decoded factors

Run UV-Dose (kJ.m-2) / Treatment time (min:s) Storage time (days; 5 C)

5 0.1 / 0:27 4

3 4.2 / 18:26 1

12 (C) 2.5 / 10:58 4

6 5 / 18:26 4

13 (C) 2.5 / 10:58 4

11 (C) 2.5 / 10:58 4

2 0.7 / 3:05 7

9 (C) 2.5 / 10:58 4

1 0.7 / 3:05 1

4 0.7 / 3:05 7

8 2.5 / 10:58 8

7 2.5 / 10:58 0

14 (C) 2.5 / 10:58 4

10 (C) 2.5 / 10:58 4

The RSM conditions (Table 21) were implemented twice considering UV-C application in whole

carrot (pre-cut; 24 h prior to shredding) and in shredded carrot (post-cut) as schematically shown in

Figure 45. UV-C treatments were carried out as described in Part III, section 3.1.2 and samples set

as described in Part III, section 2.2 and “air” stored at 5 C. A control experiment was

simultaneously carried out (Part III, section 2.2), where shredded carrot was not subjected to any

UV-C treatment (Ctr samples). Control samples were stored in similar conditions and evaluated

using the same analytical protocol.


124

Figure 45 Schematic representation of the pursued strategy to evaluate UV-C treatment effects on wound-induced phenolic synthesis dynamic.

A lot of 20 kg of cv. Nantes carrot was obtained (Part III, section 1) and divided into two: 15 kg to

comply with the design requirements and remaining 5 kg stored at 5 C (10 days) for the UV-C

dose validation trial. From the initial lot, the raw material was characterized concerning total

phenolic and carotenoid content and PAL activity. The results are shown in Table App. 9 (appendix

2).

2.2.1.1 Results & Discussion

TOTAL PHENOLIC CONTENT

The mathematical model for total phenolic content (TPC) was developed by response surface

method (RSM) and its adequacy was checked by ANOVA analysis (Part III, section 5.2). To this

response, all effects except for the respective interaction were considered to model fitting and the

associated ANOVA analysis is shown in Table 22.

The ANOVA analysis of the regression model indicates that both TPC models were significant

(p<0.05), however exhibiting a significant lack of fit (p<0.05). The F-test for the lack of fit indicated

that the source of variance contained in the residuals could be explained by the experimental error,

at the confidence level of =0.05. Nonetheless, the regression coefficients (TPCPre-cut: r2=0.97 and

r2adj=0.96; TPCPost-cut: r

2=0.97 and r

2adj=0.97), explaining the variability of experimental data, were

good. The regression coefficients (r2) for TPCPre-cut and TPCPost-cut were close to the r

2adj (0.003 for

both models) showing that about 97% of the variation of the observations around the mean is

explained by the respective fitted regression model equations, remaining only 3% attributed to the

residuals. Also, it shows that the considered effects to the respective model equations are

adequate to the prediction quality of the evaluated response within the tested conditions.


125

Table 22 Analysis of variance of the second-order polynomial model for TPCPre-cut and TPCPost-cut of carrot subjected to UV-C treatments (dose/application site).

Model Source SS df MS F-ratio

(model significance) p*

TPCPre-cut Regression 32909.5 4 8227.4 264.6† 0.00

Residual 1150.5 37 31.1 Lack of fit 790.1 4 197.5 18.1‡ 0.000000 Pure Error 360.5 33 10.9 Total 34060.0 41

TPCPost-cut Regression 30844.6 4 7711.2 309.9† 0.00

Residual 920.7 37 24.9 Lack of fit 475.7 4 118.9 8.8‡ 0.000059 Pure Error 445.0 33 13.5 Total 31765.3 41

* F-test significance to p<0.05; † F-ratio (regression/residual); ‡ F-ratio (lack of fit/pure error); df degree of freedom; MS Mean squares; SS Sum of squares.

The response surfaces representing UV-C treatment effects on TPC of UV-C doses applied pre-cut

(TPCPre-cut) and applied post-cut (TPCPost-cut) are described by Eq. 7 and Eq. 8 (Table 23),

respectively. The effects parameter estimates, expressed in the model equations (Eq. 7 and Eq. 8)

and respective quadratic model probabilities are shown in Table App. 10 (appendix 2).

Table 23 Model equations for total phenolic content (TPC) changes as a function of UV-C dose (UV) and storage time (St) considering UV-C treatment application in whole peeled carrot (Pre-cut; 24 h before shredding) and in shredded carrot (Post-cut).

Model Equation

TPCPre-cut 𝑇𝑃𝐶 = 25.1 + 12.7 𝑈𝑉 − 2.6 𝑈𝑉2 + 5.2 𝑆𝑡 + 0.9 𝑆𝑡2 Eq. 7

TPCPost-cut 𝑇𝑃𝐶 = 26.6 + 10.6 𝑈𝑉 − 2.2 𝑈𝑉2 + 6.5 𝑆𝑡 + 0.7 𝑆𝑡2 Eq. 8

The model generated response surfaces for the TPCPre-cut and TPCPost-cut are shown in Figure 46 a)

and b), respectively. Both response surfaces suggest that the increase in UV-C dose has little input

to the phenolic accumulation level during storage as changes in phenolic content were more

dependent on storage time than on applied UV-C dose. Even so, the analysis of the response

surfaces allows to establish the most favorable UV-C doses that maximize phenolic increases

during storage. From that analysis, it is possible to verify that higher phenolic contents are

achieved earlier in time in the dose-range of 2 to 3 kJ.m-2

, with TPC levels ranging from 40 (day 0)

to 140 mg CAE.100 g-1

(day 8), independently of UV-C treatment application site. To this regard it

seems that, under the tested conditions, the UV-C treatment application site did not influenced the

phenolic increment during low temperature storage.


126

Figure 46 Total phenolic content response surface. a) TPCPre-cut, UV-C treatment application in whole peeled carrot (Pre-cut; 24 h before shredding); b) TPCPost-cut, UV-C treatment application in shredded carrot (Post-cut).

The numerical solution of the model TPCPre-cut (Eq. 7) and TPCPost-cut (Eq. 8) for a fixed 2.5 kJ.m-2

UV-C dose included within the range of interest, 2 to 3 kJ.m-2

where this response is maximized,

offers the estimated TPC evolution during storage. In this TPC estimate projection (Figure 47) it is

possible to observe that the TPC evolution in both pre- and post-cut UV-C treatments would result

in a TPC increase of 2.4 times the estimated initial content, a higher response than the one

determined in untreated Ctr samples (from 48.0 to 100 mg CAE.100 g-1

; 2 times initial content),

> 140

< 140

< 120

< 100

< 80

< 60

< 40

a)

> 140

< 140

< 120

< 100

< 80

< 60

< 40

< 20

b)


127

showing the increased effect of the UV-C treatment on the wound-induced phenolic synthesis as a

response to shredding (Ctr samples). Also, this UV-C dose would be sufficient to meet the raw

material TPC level (66.3±5.8 mg CAE.100 g-1

) one day earlier than in untreated Ctr samples

(Figure 47). By day 7, when significant phenolic accumulation is usually registered considering the

previous studies, UV-C treated samples according to the model projection, would have surpassed

the raw material level by 80% while untreated (Ctr) samples surpassed (p<0.05) that level by

40% (as previously characterized, Part IV, section 1.4) indicating that UV-C treatments influence

wound-induced phenolic synthesis.

Figure 47 Estimated TPC changes at low temperature storage (5 C) of pre- and post-cut UV-C (2.5 kJ.m-2) treated carrot and changes in phenolic content of untreated shredded carrot samples

during low temperature storage (5 C, 8 days). Error bars represent the confidence interval at 95%.

[ 2.5 kJ.m-2 Pre-cut: whole peeled carrot if submitted to a 2.5 kJ.m-2 UV-C dose 24 h prior to shredding

2.5 kJ.m-2 Post-cut: shredded carrot if submitted to a 2.5 kJ.m-2 Ctr: untreated shredded carrot].

The comparison of UV-C treatments effects on the antioxidant component, namely in the increase

of fruits and vegetables phenolic content, does not come easily since the application conditions of

the UV-C treatments vary significantly (e.g. product distance from the source of radiation; the

environmental temperature during treatment application; lamp power) and influence the applied

dose. Accordingly to the diverse results reported, the effects of UV-C on phenolics content have

not been yet completely elucidated and further research is needed. Still, several studies report that

the increases in phenolic content induced by UV treatments are a result of the treatments

interference in the plants defense mechanism, particularly the phenylpropanoid pathway, which

induces the production of secondary metabolites such as phenolic compounds. As examples, in

shredded carrot, Surjadinata (2006) demonstrated that the use of UV-C (60 W x 50 cm distance)

for 15 min increased by ~15% the wound-induced phenolic accumulation after storing carrots for 4

2.5 kJ.m -2 Pre-cut 2.5 kJ.m -2 Post-cut Ctr

0 1 2 3 4 5 6 7 8

Storage time (days)

20

40

60

80

100

120

140

160

TP

C (

mg

CA

E.1

00

g-1

)

Raw material


128

days at 15 C. In blueberry fruit, Erkan et al. (2013) showed that doses of 2.03 and 4.12 kJ.m-2

induced higher phenolic contents and antioxidant capacity during berry storage (5 C, 20 days)

while preventing respective decay compared to untreated berries. Also on blueberries, Perkins-

Veazie et al. (2008) reported a UV-induced phenolic response after a 1 kJ.m-2

UV-C treatment in

cv. Collins berries stored after 7 days at 5 C plus 2 days at 20 C regarding untreated berries while

in cv. Bluecrop berries no effects over the phenolic content were observed. In strawberry,

increased phenolic content was found in fruits treated with UV-C doses of 2.15 and 4.30 kJ.m-2

regarding untreated fruits after 15 days of storage at 10 C (Erkan et al., 2008). In tomato, Bravo et

al. (2013) also registered significant increases in phenolic content of UV-C treated tomatoes (1.0,

3.0 and 12.2 kJ.m-2

), just after two days (room temperature) of treatment application. On the

contrary, Artés-Hernández et al. (2010) reported a significant decrease in phenolic levels during

storage (5 C, 11 days; passive MAP) of watermelon cubes submitted to UV-C doses ranging from

1.6 to 7.2 kJ.m-2

however unrelated to the significant increase in antioxidant activity attributed to the

combined effect of the stress induced by the UV-C treatment and storage.

TOTAL CAROTENOID CONTENT

The results for total carotenoid content (TCC), of carrot samples subjected to UV-C treatments

were not adjustable to quadratic models (Table App. 11, appendix 2). The low regression

coefficients (<0.4) allied to the F-test for the lack of fit (p<0.05) confirmed the inadequacy of the

models to evaluate the considered effects. However, TCC was not affected by the independent

variables (UV and St) (Table App. 14, appendix 2) since the changes in TCC (ranging from 9.8 to

12.3 mg -carotene eq.100 g-1

; p>0.05) were fitted within the range of the TCC changes

determined in untreated (Ctr) samples during storage (Table 24).

Table 24 Changes in total carotenoid content (TCC) of untreated shredded carrot (Ctr samples)

during low temperature storage (5 C; Ctr samples).

Storage time (days) 0 1 4 7 8

TCC (mg -carotene eq.100 g-1) 10,6b±0,3 9,6a±0,6 11,3bc±0,3 12,1c±0,1 9,3a±0,1

Values represent the mean of three replicates±SD. In the same line, different letters represent significant differences at p=0.05 (Tukey HSD test).

Despite the differences between UV-C stress application in both studies (e.g. treatment

temperature; lamp number and distribution along the illumination area), carrot samples submitted

to a 0.78 kJ.m-2

UV-C dose 24 h prior to shredding (Part IV, section 2.1) lead to a significant TCC

decrease attributed to a possible photobleaching phenomenon (Bravo et al., 2013) and a significant

increase was found during storage believed to be supported by an increase in compound

extractability due to increased cell wall depolymerization (Bhat et al. 2007, cit in Alothman et al.

2009). This behavior was not found in this subsequent study either in pre-cut (24 h before

shredding) or post-cut UV-C treated carrot samples, irrespective of applied UV-C dose (0.1 to

5 kJ.m-2

), assigning to the UV-C treatments little to no effect over TCC changes during storage. It is


129

clear that the effects of UV-C treatments over the carotenoid content are far from been elucidated

and in need to be further explored namely its effects on the enzymes involved on the carotenoid

biosynthetic pathway.

COLOR

Color data, namely hue parameter and calculated whiteness index (WI) of carrot samples

subjected to UV-C treatments were not adjustable to quadratic models as confirmed by the

unacceptable regression coefficients (<0.4) and lack of fit (Table App. 12 and Table App. 13,

respectively, appendix 2). The inadequacy of the second-order polynomial models to describe the

changes in hue and WI changes could be related to the lack of treatment effects or storage time in

these responses since the found range of variation during storage for hue was between 63º-64º

and 29-33 WI units, irrespective of pre-cut or post-cut UV-C treatments (Table App. 14, appendix

2). Moreover, the observed maintenance of these color parameters was within the variation of

untreated (Ctr) carrot samples during storage, as shown in Table 25, suggesting that the UV-C

dose did not interfered with carrots’ characteristic orange color. Despite the significant increase in

WI of untreated (Ctr) samples, from 28 to 32 WI units (p>0.05; Table 25), the final mean WI value

was below the 38.4 ± 1.3 WI interval that relates to the perception of a “slight white surface of the

shreds” as proposed by Cisneros-Zevallos et al. (1995).

Table 25 Changes in color (expressed as hue angle and whiteness index, WI) of untreated shredded

carrot (Ctr samples) during low temperature storage (5 C, 8 days).

Storage time (days) 0 1 4 7 8

Color parameter

Hue 63.7ns±0.8 63.2ns±0.6 63.1ns±0.8 63.5ns±0.9 63.1ns±1.3

WI 28.0a±1.4 28.8a±2.3 30.1ab±2.4 31.4b±2.8 32.1b±3.2

Values represent the mean of 15 measurements ± SD. In the same line, different letters represent significant differences at p=0.05 (Tukey HSD test).

2.2.2 UV-C dose validation

Considering the RSM modeling, an UV-C dose within the range of 2 to 3 kJ.m-2

would anticipate

phenolic accumulation during low temperature storage (5 C) of shredded carrot, without

compromising carotenoid content or perceived color, independently of application site. In order to

validate these effects on shredded carrot total phenolic content and related induced phenylalanine

ammonia lyase (PAL) activity, an UV-C dose of 2.5 kJ.m-2

was selected. The stored (5 C, 10 days)

5 kg carrot sub-lot from the previous study was used to constitute three sample types (in triplicate):

two UV-treated samples (sample id.: 2.5 kJ.m-2

Pre-cut and 2.5 kJ.m-2

Post-cut UV-C application)

and one untreated sample (sample id.: Ctr). The application of 2.5 kJ.m-2

UV-C dose in whole (pre-

cut) and in shredded (post-cut) carrot was carried out as described in Part III, section 3.1.2 and


130

samples (UV-treated and untreated) prepared as described in Part III, section 2.2 and “air” stored

at 5 C. The total phenolic content (TPC – protocol I) and PAL activity levels were determined at

days 0 and 7 of storage (5 C) and treatments influence on evaluated responses during storage

was established from sample mean comparison (ANOVA, Tukey HSD test, p=0.05).



The effects of a 2.5 kJ.m-2

UV-C dose applied in whole carrot (24 h before shredding; pre-cut) and

in shredded carrot samples (post-cut) on the phenolic content during storage are shown in Figure

48.

Figure 48 Effect of a 2.5 kJ.m-2 UV-C dose, applied pre- and post-cut, on the total phenolic content

of shredded carrot at low temperature storage (5 C, 7 days). Error bars represent the confidence interval at

95%. [ 2.5 kJ.m-2 Pre-cut: whole peeled carrot submitted to a 2.5 kJ.m-2 UV-C dose 24 h prior to shredding;

2.5 kJ.m-2 Post-cut: shredded carrot submitted to a 2.5 kJ.m-2; Ctr: untreated shredded carrot].

The phenolic accumulation registered during low temperature storage was significantly higher in

UV-C treated samples than in untreated (Ctr) samples (Figure 48) and corroborates the RSM

estimate projection. After 7 days of storage, all samples had already surpassed the raw material

phenolic level (66.3±5.9 mg CAE.100 g-1

) by 124 to 145% and 89% (UV-treated and untreated

samples, respectively).

The determined increases in UV-C treated samples were higher than those predicted by the

models but then again, so were the increases found in untreated (Ctr) samples when compared to

the previous study (of 40%, Part IV, section 1.4), maintaining the relativity (20-30%) in increased


0 7

Storage time (days)

0

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

g-1

)

Raw material


131

wound-induced response as a result of UV-C treatments. Despite the higher (p<0.05) TPC level

determined in post-cut UV-C treated samples after 7 days of storage regarding pre-cut UV-C

treated samples, the registered difference between sample TPC levels was of around

14 mg CAE.100 g-1

(148.2±5.5 vs. 162.4±3.0 mg CAE.100 g-1

, respectively), which is below the

estimated TPC variation band found to be significant in the RSM models, of 20 mg CAE.100 g-1

.

PAL ACTIVITY

Changes in PAL activity as influenced by the 2.5 kJ.m-2

UV-C treatments during storage are shown

in Figure 49. The UV-C treatment induces prompt changes (day 0) in the basal PAL activity levels

as shown by the significantly higher PAL activity levels of both UV-C treated samples (28.9 in pre-

cut UV-C treated samples and 37.4 U.100 g-1

in post-cut treated samples, p>0.05) regarding the

untreated (Ctr) sample (10.6 U.100 g-1

) or the raw material (8.1 ± 2.6 U.100 g-1

). Nigro et al. (2000)

have also shown PAL activity was stimulated after UV-C treatments and, 12 hours after treatment

application, significantly higher PAL activity levels were determined in UV-C treated strawberries

(0.5-2.5 kJ.m-2

) regarding untreated samples.

Figure 49 Effect of a 2.5 kJ.m-2 UV-C dose, applied pre- and post-cut, on PAL activity of shredded

carrot at low temperature storage (5 C, 7 days). Error bars represent the confidence interval at 95%.

[ 2.5 kJ.m-2 Pre-cut: whole peeled carrot submitted to a 2.5 kJ.m-2 UV-C dose 24 h prior to shredding;

2.5 kJ.m-2 Post-cut: shredded carrot submitted to a 2.5 kJ.m-2; Ctr: untreated shredded carrot].

Further increases (p<0.05) were observed in PAL activity levels after 7 days of storage (Figure 49)

and again higher (p<0.05) activity levels were determined in UV-C treated samples than in

untreated (Ctr) samples: 138.8, 165.8 and 246.4 U.100 g-1

, for Ctr, pre-cut and post-cut UV-C

treated samples, respectively. Despite the significant difference in PAL activity registered between

UV-C treated samples, the registered phenolic accumulation in both samples was similar not


0 7

Storage time (days)

0

50

100

150

200

250

300

PA

L (

U.1

00

g-1

)

Raw material


132

allowing to establish if the UV-C enhanced wound-induced response is regulated by a hierarchical

order between stresses (UV-C treatment and shredding). The found differences between UV-C

treated and untreated samples PAL activity levels suggest that enhanced PAL activity is

attributable to UV-C treatment effects on the de novo synthesis of the enzyme. As Reyes et al.

(2007) pointed out, the mechanism by which the combination of wounding and other abiotic

stresses induces a synergistic response is not clear but it is possible that all these stresses share a

common signaling molecule that may amplify the desirable response.

2.3 HEAT SHOCK [100 C/45 S] SINGLE STRESS EFFECTS ON THE WOUND-INDUCED

DYNAMICS

Given that peels have higher ability to phenolic synthesis (Part IV, section 1.3) and that peel

removal is advantageous for fresh-cut microbial stability (Part IV, section 1.5) this study aimed to

evaluate the effects of a 100 C/45 s heat shock, applied before or after peeling, on the phenolic

synthesis mechanism of shredded carrot during low temperature storage (5 C; 10 days).

2.3.1 Phenolic synthesis as affected by heat shock application before or after peel removal

To assess the 100 C/45 s heat shock effects on shredded carrot phenolic content considering heat

shock application prior to and after peeling (Figure 50) as well as the respective accumulation

during low temperature storage (5 C, 10 days), a full factorial design was established with

“Peeling” and ”Heat shock” as independent variables. The application of heat shock in shredded

carrot was not considered since, in previous studies (Alegria et al., 2009; Alegria, 2007), it was

demonstrated that post-cut application of heat shocks (100 C/45 s; 70 C/30 s; 50 C/60 s)

significantly compromised the quality of FC carrot due to significant SSC losses, color changes,

impairment of fresh-like aroma perception and significant changes in product firmness.


133

Figure 50 Schematic representation of the pursued strategy to evaluate the 100 C/45 s heat shock treatment effects on wound-induced phenolic synthesis dynamic.

From a lot of 10 kg of cv. Nantes carrot (Part III, section 1), 4 sample types (in triplicate; 125 g per

sample replicate) were set and identified as shown Table 26. Heat shock treatments were carried

out as described in Part III, section 3.1.1 and heat-treated and untreated (with or without peel)

samples prepared as described in Part III, section 2.2 and “air” stored at 5 C. The total phenolic

content (TPC - protocol I) was determined during storage at days 0, 3, 7 and 10. The evaluation of

the raw material phenolic content was also considered as to estimate the stresses initial impact

(day 0) over this bioactive response and assessment of phenolic recovery during storage as a

result of induced phenolic synthesis. The influence of “Peeling” and “Heat shock” on phenolic

accumulation were established from sample mean comparison (ANOVA, Tukey HSD test, p=0.05).

Table 26 Experimental design matrix and sample ID.

Factors / Independent variables

Sample ID

A B Peeling Heat shock (100 C / 45 s)

-1 -1

No peeling

No heat shock NP

-1 1 Heat shock NP_HS

1 -1

Peeling

No heat shock P

1 1 Heat shock P_HS


134



The initial impact of the considered independent variables, peeling and 100 C/45 s heat shock, on

shredded carrot TPC levels evaluated at day 0 were compared with the raw material levels and the

results are shown in Table 27.

Despite of the known phenolic sensitivity to thermal degradation (Xu et al., 2007), no significant

differences were found between TPC levels of heat-treated (NP_HS and P_HS) and untreated

samples (NP and P) indicating that the 100 C/45 s heat shock stress does not introduce additional

TPC changes than those caused by peeling and shredding. In fact, as it can be observed in Table

27, the losses of 48-49% regarding the raw material content were similar (p>0.05) in both

unpeeled samples (NP_HS and NP), endorsing the already discussed increase of inner to peel

tissue ratio after carrot shredding (Part IV, section 1.4), and the losses of 62-68% were also

similar (p>0.05) in both peeled samples (P_HS and P) and attributed, of course, to peel removal.

Table 27 Peeling and heat shock effects over the total phenolic content (TPC) of cv. Nantes shredded carrot at day 0.

Independent variables Bioactive response

Sample Id. Peeling Heat shock

(100 C / 45 s)

TPC (mg CAE.100 g-1)

Stress impact* (%)

RM - - 74.6c±1.8 -

NP No peeling

No heat shock 38.8b±4.8 48.0a±6.4

NP_HS Heat shock 38.3b±3.5 48.7a±4.7

P Peeling

No heat shock 28.1a±0.5 62.3b±0.7

P_HS Heat shock 23.7a±0.2 68.2b±0.3

*Stresses impact expressed as % of TPC decrease regarding raw material (RM sample). Values represent the mean of three replicates±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

Changes in phenolic content of the different shredded carrot samples during low temperature

storage are shown in Figure 51 (univariate table of effects given in Table App. 15, appendix 2). All

samples showed significant increases in TPC during low temperature storage and it is interesting to

notice that the first significant TPC increase, registered from day 3 to 7, was not influenced by the

application of the 100 C/45 s heat shock.


135

Figure 51 Peeling and heat shock (100 C / 45 s) effects on the total phenolic content (TPC) of

shredded carrot during low temperature storage (5 C, 10 days). Error bars represent the 95% confidence

interval. [ NP: unpeeled; NP_HS: unpeeled heat-treated (100 C/45 s); P: peeled; P_HS: peeled

heat-treated (100 C/45 s)].

As it can be observed in Figure 51, lower TPC levels were registered in both unpeeled and peeled

heat-treated samples (NP_HS and P_HS samples, respectively) regarding untreated samples. Peel

removal prior to heat treatment did not influenced the registered phenolic accumulation during

storage since no differences were found between TPC levels of unpeeled and peeled heat-treated

samples (p>0.05). Consequently, no advantages are foreseen in the maintenance of peels in

shredded carrot to the phenolic accumulation pattern in heat-treated samples. Still, and despite the

significant phenolic accumulation registered, of 1.7 and 2.6 times the initial contents (NP_HS and

P_HS samples, respectively), by the end of storage (10 days) none of the heat-treated samples

had reached the raw material TPC level (74.6 mg CAE.100 g-1

). In contrast, both untreated

samples (NP and P) had similar (p>0.05) TPC increases during storage and the achieved phenolic

accumulation was sufficient to match the raw material level after 7 days of storage, but fell short

from the accumulation levels observed in previous studies in which similar samples had exceeded

the raw material by 30-40%.

2.3.2 Heat sock effects on wound-induced phenolic synthesis dynamic

In order to better understand the induced changes by the 100 C/45 s heat shock on the phenolic

synthesis dynamics, namely the heat shock effects on the biochemical dynamics of PAL activity

during low temperature storage (5 C; 10 days) of peeled shredded carrot, two sample types

(triplicates; 125 g per sample replicate), peeled heat-treated (sample id.: HS) and peeled untreated

samples (sample id.: Ctr) were set from a lot of 10 kg of cv. Nantes carrot (Part III, section 1). Heat

shock treatments were carried out as described in Part III, section 3.1.1 and heat-treated (HS) and

NP NP_HS P P_HS

0 3 7 10

Storage time (days)

0

10

20

30

40

50

60

70

80

90

100

110

TP

C (

mg

CA

E.1

00

g-1

) Raw material


136

untreated (Ctr) samples prepared as described in Part III, section 2.2 and “air” stored at 5 C. Total

phenolic (TPC – protocol I) and carotenoid (TCC) content, PAL and POD activity levels were

evaluated at days 0, 3, 5, 7 and 10 of storage (5 C). The same responses were also determined in

the raw material. The influence of heat shock on evaluated responses during storage was

established from sample mean comparison (ANOVA, Tukey HSD test, p=0.05).


TOTAL PHENOLIC CONTENT & PAL ACTIVITY

The effects of the 100 C /45 s heat shock on the total phenolic content and PAL activity are shown

in Figure 52 and Figure 53, respectively.

The initial phenolic losses registered (at day 0) in heat-treated (HS) and untreated (Ctr) samples

are similar (p>0.05) and of 30% regarding the raw material content (67.1±1.8 mg CAE.100 g-1

),

confirming once more that the 100 C /45 s heat shock does not involve additional losses to the

ones promoted by peeling and shredding.

During storage, significant phenolic accumulation was found in heat-treated (HS) samples

accounting for a 2.3 times increase in the initial content (from 47.5 to 107.6 mg CAE.100 g-1

,

Figure 52). After 7 days of storage, the registered TPC accumulation in heat-treated samples

(74.0±1.8 mg CAE.100 g-1

) was sufficient to match the raw material content which was not

observed in the previous study. After 7 days of storage, the phenolic accumulation registered in

untreated (Ctr) samples had exceeded the raw material levels in 63% (confirming the former

characterizations) and the same outcome in heat-treated (HS) samples was just observed after 10

days of storage suggesting that the heat shock treatment introduced a delay to the wound-induced

phenolic synthesis.


137

Figure 52 Heat shock effects on total phenolic content of peeled shredded carrot during low

temperature storage (5 C, 10 days). Error bars represent the 95% confidence interval. [ Ctr: untreated;

HS: heat-treated (100 C/45 s)].

Figure 53 Heat shock effects on PAL activity levels of peeled shredded carrot during low



Ctr HS

0 3 5 7 10

Storage time (days)

30

40

50

60

70

80

90

100

110

120

130

TP

C (

mg

CA

E.1

00

g-1

)

Raw material

Ctr HS

0 3 5 7 10

Storage time (days)

0

50

100

150

200

250

300

350

400

450

PA

L (

U.1

00

g-1

)

Raw material


138

During storage, heat-treated (HS) samples had always PAL activity levels below untreated (Ctr)

samples. As shown in Figure 53, both sample types register a significant increase in PAL activity in

the first tested time interval, from day 0 to 3, however PAL activity levels of heat-treated (HS)

samples were lower (p<0.05) than that of untreated (Ctr) samples. This difference in activity levels

could justify the maintenance in TPC levels in heat-treated (HS) samples while a significant TPC

increase was registered in untreated (Ctr) samples from day 0 to 3 (Figure 52).

The positive and significant correlation found between the changes in TPC and PAL activity during

storage (r=0.9, p<0.05) show a direct dependency between the respective levels and determined

increments. The lower increments in PAL activity of heat-treated (HS) samples could be justified by

the 100 C /45 s heat shock effect over PAL de novo synthesis, leading to a metabolic shift to

synthetize heat shock proteins (Campos-Vargas et al., 2005; Saltveit, 2000). Choi et al. (2005 b)

suggested that stress treatments could either decrease PAL synthesis or increase respective

destruction. From our results, it is therefore a possibility that heat shock might not have directly

reduced the activity of PAL, but could have led to delay the tissues capacity to synthetize PAL

enzyme as a response to shredding by at least two days. Moreover, according to the findings of

Campos-Vargas et al. (2005), heat shocks limit the wound-induced PAL response by inhibiting the

accumulation of PAL proteins either by preventing the translation of PAL mRNA or accelerating the

turnover of PAL proteins leading to a relatively low enzyme activity.

The significant decrease in PAL activity registered between day 7 and 10 of storage in untreated

(Ctr) samples (Figure 53) appears to show that PAL activity levels are set by phenolic

concentration supporting the PAL activity retro-inhibition hypothesis (Part IV, section 1.3).

Moreover, the found maintenance in TPC levels in the mentioned time period (from day 7 to 10;

Figure 52) reinforces this hypothesis and suggests that there might be a limit to phenolic

accumulation during storage.


139

TOTAL CAROTENOID CONTENT

The effects of the 100 C /45 s heat shock on the changes in total carotenoid content (TCC) are

shown in Figure 54.

Figure 54 Heat shock effects on total carotenoid content of peeled shredded carrot during low



In heat-treated (HS) and untreated (Ctr) samples no significant differences are found to the raw

material TCC (15.2±0.4 mg eq. -carotene.100 g-1

) and maintenance in TCC levels is found during

storage (p>0.05), except at day 5 in heat-treated (HS) samples which, without apparent reason, a

significant increase was determined and could be assigned as an experimental error. The found

maintenance in TCC levels confirms the stability of these compounds during low temperature

storage and that the 100 C /45 s heat shock does not further increase the change pattern of this

bioactive marker. These results are however conflicting with the previously characterized

carotenoid losses (p<0.05) at day 0 as a result of heat shock treatment (of 28%, Part IV, section

2.1).

POD ACTIVITY

Changes in POD activity levels as affected by the 100 C /45 s heat shock during low temperature

storage is shown in Figure 55. The comparison between POD activity levels of heat-treated (HS)

and untreated (Ctr) samples at day 0 suggest that the lower (p<0.05) activity levels found in heat-

treated (HS) samples were consequent of a partial thermal inactivation of the enzyme, which had

already been observed in previous studies (Alegria et al., 2010; Alegria, 2007).

Ctr HS

0 3 5 7 10

Storage time (days)

12

14

16

18

20T

CC

(m

g E

q.

b-c

aro

ten

e.1

00

g-1

)

Raw material


140

POD activity increases as a result of fresh-cut processing (wounding operations) and storage could

be considered as a manifestation of the oxidative stress level. As Lamikanra and Watson (2001)

pointed, POD under such stress conditions would act to reduce potential oxidative damage to the

vegetable (Lamikanra and Watson, 2001) as it provides protection against atmospheric oxygen

trough POD involvement in the lignification process (Howard et al., 1994) and thereby reducing

oxidative degradation rate during storage until no further POD is needed.

Figure 55 Heat shock effects on peroxidase (POD) activity of peeled shredded carrot during low



The maintenance of low enzymatic activity levels registered in heat-treated (HS) samples during

storage in opposition to the increase in activity (p>0.05) in untreated (Ctr) samples could be related

to the general diversion of protein synthesis induced by the 100 C/45 s heat shock to the

production of heat shock proteins (Salveit, 1998). The reduced POD activity levels found during

storage of heat-treated (HS) samples could therefore either result from direct enzyme thermal

inactivation or from reduced POD synthesis during storage. Reduced POD synthesis, as Martín-

Diana et al. (2005) reported, could be due either to an indirect effect caused by feedback inhibition

from the lack of phenolic compounds (substrate) or to a direct effect on an unknown receptor

implicated in the synthesis of POD. From our results and concerning the reduced POD synthesis

hypothesis, it seems more likely that the 100 C/45 s heat shock had a direct effect on reported

unknown receptor involved in POD synthesis (Martín-Diana et al., 2005) since significant phenolic

accumulation was achieved in heat-treated samples during storage.

Ctr HS

0 3 5 7 10

Storage time (days)

2

4

6

8

10

12

14

16

18

20

22

24

PO

D

(U.g

-1)

Raw material


141

Overall and considering all bioactive responses, the application of the 100 C/45 s heat shock in

whole carrot does not change the initial phenolic levels of shredded carrot but it changes the

phenolic synthesis dynamic. The possible effects of the heat stress in this dynamic are

schematically shown in Figure 56.

Figure 56 Effect of heat shock on wound-induced phenolic synthesis dynamic in shredded carrot. (Scheme adapted from Salveit, 2000).

The PAL-induced phenolic synthesis mechanism was altered by the heat shock and a two-day

delay in phenolic increase (p<0.05) was observed during storage regarding the absence of

treatment. The lower PAL activity levels in heat-treated shredded carrot may result from two

effects: PAL enzyme thermal inhibition or due to a metabolic shift induced by the treatment.

Similarly, the reduced POD activity levels (p<0.05) achieved and maintained during storage could

also be related to heat inactivation or to lower POD synthesis as a result of the diversion of

metabolic pathways. This effect could have a significant influence on the phenolic degradation

pattern during storage of fresh-cut carrot since POD is related to phenolic oxidation.


142

2.4 HEAT SHOCK AND UV-C COMBINED STRESS EFFECTS ON THE WOUND-INDUCED

DYNAMICS

It was aimed to evaluate the effectiveness of abiotic stress treatment combination (heat shock

[100 C/45 s] and UV-C [2.5 kJ.m-2

]) to improve shredded carrot bioactive quality during storage

(5 C) regarding the standard (industrial) decontamination treatment (chlorine, 200 ppm x 1min).

Multitarget preservation is here considered, seeing as it introduces a series of disturbances

(simultaneous or sequential) that act on different targets, changing products physiology. From this

concept, it is thought-out that abiotic stress treatments, heat shock and UV-C applied in

combination, offers better technological approaches to the standard industrial processing practice

(use of post-cut chlorine decontamination solution) of fresh-cut carrot regarding product

decontamination and maintenance / enhancement of bioactive phytochemicals. Considering this

approach, schematically shown in Figure 57, it is expected that, apart from the decontamination

efficiency, the partial inhibition of stress-related enzymes (PAL and POD) as a result of heat shock

will be overcome by the UV-C treatment effects on the wound-induced phenolic synthesis

dynamics, especially if this latter stress is applied after shredding.

Figure 57 Multitarget approach: combination of heat shock and UV-C stresses.


143

The experiment considered the evaluation of the effects of heat-shock (100 C/45 s) applied in

whole peeled carrot and of UV-C (2.5 kJ.m-2

; =254 nm) treatment applied after shredding, as

single or combined treatments on the quality of shredded carrot during low temperature storage

(5 C, 7 days).

To comply with trial requirements a lot of 10 kg of cv. Nantes carrot was obtained (Part III, section

1) and a 10% sampling was collected to characterize the raw material (Table App. 17, appendix 2).

Remaining carrots were equally divided into groups to constitute 4 sample types: whole peeled

carrots submitted to heat shock treatment (100 C/45 s; sample id.: HS) and then shredded;

shredded carrot submitted to UV-C treatment (2.5 kJ.m-2

; sample id.: UV); sequential combination

of heat shock and UV-C stress treatments (sample id. HS x UV) and samples submitted to the

standard minimal processing post-cut chlorine decontamination procedure (200 ppm / 1 min;

sample id.: HIPO). Three independent replicates per treatment were carried out and samples

processed as shown in Figure 58.

Heat shock, UV-C and combined stress treatments were carried out as described in Part III, section

3.1 (General methods), and chlorine treatments as described in Part III, section 2.3 (excluding

packaging operation). Samples were then set as described in Part III, section 2.2 and “air” stored at

5 C. Evaluated responses included total phenolic (Protocol I) and carotenoid content, antioxidant

capacity (AOx – Protocol II), PAL and POD activity, CIELab color, pH, soluble solids content and

dry matter. Analytical procedures were carried out just after processing (day 0) and after 7 days of

storage at 5 C. The influence of single and combined stress effects on evaluated responses during

storage was established from sample mean comparison (ANOVA, Tukey HSD test, p=0.05).


144

Figure 58 Flow diagram of minimal processing operations for shredded carrots according to the multitarget strategy (single and combined heat shock and UV-C stress treatments) and to the standard practice.



In Figure 59 the total phenolic content as affected by stress treatments during storage is shown. At

day 0, all treatments induced a significant decrease in initial TPC content (raw material;

54.6±2.2 mg CAE.100 g-1

), ranging from 23% to 32% with no differences determined between

treatments. The found decrease could once more be related to the peeling effect, attesting that the

100 C/45 s heat shock and 2.5 kJ.m-2

UV-C stress treatments, in single or combined application,

do not further increase phenolic losses attributed to processing operations. Unexpectedly and

despite the higher TPC decrease found after chlorine treatment (HIPO samples; 32%), the

accounted phenolic loss was similar (p>0.05) to the other treatments, contrasting with the further

significant TPC loss registered after the decontamination procedure when evaluating minimal

processing operations effects over this bioactive marker (Part IV, section 1.5).


145

Figure 59 Changes in total phenolic content as affected by selected abiotic stress treatments during

storage (7 days, 5 C). Error bars represent the confidence interval at 95%. [ HS: heat-treated (100 C/45 s);

UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated ([100 C/45 s]x[2.5 kJ.m-2]); HIPO: chlorine-treated (200 ppm/1 min)].

During storage all samples showed significant TPC increases (Figure 59), more expressive

(p<0.05) in samples submitted to heat shock and UV-C stress treatments, single (HS and UV

samples) or combined (HS x UV samples) application, regarding chlorine-treated (HIPO) samples.

After 7 days of storage, samples treated with UV-C after shredding, UV and HS x UV samples

(p>0.05), registered the highest TPC increases followed by heat-treated (HS) samples (p<0.05).

The induced phenolic accumulation in these samples was sufficient to reach and surpass the raw

material TPC level by 60% (HS x UV samples), 70% (UV samples) and 38% (HS samples) after

a 7-day storage period (Figure 59). However, despite the significant phenolic increase registered in

chlorine-treated (HIPO) samples to day 7, the induced phenolic accumulation was still insufficient

to match the raw material TPC level and a 14% difference was still registered. In spite of the

difference regarding storage conditions (“air” vs. “MAP” storage), this result supports the already

described chlorine-induced phenolic oxidation during storage, preventing the accumulation levels

necessary to reach the raw material TPC level.

The phenolic accumulation levels determined after 7 days of storage in heat-treated (HS) and UV-

treated (UV) samples, single application (38% and 70% above the raw material TPC levels,

respectively), subscribe the already discussed effects of the 2.5 kJ.m-2

UV-C and 100 C/45 s heat

shock stress treatments over the wound-induced phenolic synthesis (Part IV, sections 2.2.2 and

2.3.2, respectively). The registered phenolic increase (p<0.05) found in samples submitted to the

combined application of stresses (HS x UV samples) was similar (p>0.05) to that of UV-C single

stress application (UV samples) and higher (p<0.05) than the one achieved by heat shock single

HS UV HSxUV HIPO

0 7

Storage time (days)

20

30

40

50

60

70

80

90

100

TP

C (

mg

CA

E.1

00

g-1

)

Raw material


146

stress application (HS samples) suggesting that an additive effect might be in order rather than a

synergic one (Figure 59). Moreover, these results point out that the introduced delay in phenolic

synthesis by the heat shock treatment might be overcome by the UV-C effects over the

phenylpropanoid mechanism, supporting the hypothesis that the multitarget preservation strategy

(sequential combination of abiotic stresses) is more favorable to the wound-induced phenolic

synthesis during low temperature storage of shredded carrot.

2.4.1.2 Total carotenoid content

Changes in total carotenoid content as influenced by heat shock, UV-C and chlorine treatments

during storage of shredded carrot are shown in Figure 60. No significant differences were detected

between TCC levels of single heat- or UV-treated samples with the raw material (16.2±0.1 mg -

carotene eq.100 g-1

) which agrees with previous findings considering the same storage conditions

(“air” storage, Part IV, sections 2.2.1 and 2.3.2). Unexpectedly, the TCC content of samples

submitted to treatment combination (HS x UV samples) was higher (p<0.05) than the raw material

without apparent reason. On the contrary, the registered 38% decrease (p<0.05) in TCC of

chlorine-treated (HIPO) samples regarding the raw material was to be expected has significant

compound leaching to washing solutions (decontamination procedure as also rinsing operation)

was already characterized (Part IV, section 1.5) and of the same order.

Figure 60 Changes in total carotenoid content as affected by selected abiotic stress treatments

during storage (7 days, 5 C). Error bars represent the confidence interval at 95%. [ HS: heat-treated

(100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated ([100 C/45 s]x[2.5 kJ.m-2]); HIPO: chlorine-treated (200 ppm/1 min)].

After 7 days of storage (Figure 60), the initial TCC levels of heat-treated (HS), UV-treated (UV) and

chlorine-treated (HIPO) samples was maintained (p>0.05) supporting the previously described

stability in carotenoid content during low temperature storage and confirming that heat shock and

HS UV HSxUV HIPO

0 7

Storage time (days)

8

10

12

14

16

18

20

22

24

TC

C (

mg

-c

aro

ten

e e

q.1

00

g-1

)

Raw material


147

UV-C treatments do not further increase the changes in this composition under “air” storage

conditions. Despite the significant decrease registered in samples submitted to treatment

combination (HS x UV samples) from day 0 to 7, the TCC levels of these samples registered no

differences (p>0.05) with the heat-treated (HS) or UV-treated (UV) samples and were similar to that

determined in the raw material. In this sense, it appears that stress combination should be able to

at least maintain the raw material carotenoid level during low temperature storage.


A high correlation between TPC and determined antioxidant capacity (hydrophilic component) was

again found (r = 0.85; p<0.05), and the same behaviors described for TPC also apply to AOx

(Table 28). After 7 days of storage, AOx levels of heat and UV-treated samples, single or combined

application, were higher (p<0.05) than those initially registered and chlorine-treated (HIPO)

samples registered a significant decrease in AOx, possibly attributed to the induced oxidation of

phenolics during storage.

Table 28 Changes in antioxidant capacity as affected by selected abiotic stress treatments during

storage (5 C, 7 days).

Sample Id. Storage time (days) AOx (µg TE.100 g-1)

Raw material - 471.7a±4.3

HS 0 478.1a±33.6

7 746.7b±44.8

UV 0 487.5a±21.3

7 855.4c±30.9

HSxUV 0 438.7a±16.6

7 794.7bc±16.7

HIPO 0 470.1a±39.5

7 343.9d±4.3

Values represent the mean of three replicates±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).


Changes in PAL activity as affected by abiotic stress treatments during low temperature storage

are shown in Figure 61. At day 0, all samples exhibit higher PAL activity levels, ranging from 9.4 to

24.9 U.100 g-1

(p>0.05), than the ones determined in the raw material (4.0±0.8 U.100 g-1

) and

significant differences were found between samples (Figure 61): UV-treated samples, single (UV

sample) or combined application (HS x UV samples), had the highest (p<0.05) PAL activity level

(19.8 and 24.9 U.100 g-1

, p>0.05, respectively), while heat- (HS) and chlorine-treated (HIPO)

samples had lower PAL activities with no differences between them (11.6 and 9.4 U.100 g-1

,


148

p>0.05, respectively). The higher initial increases in PAL activity registered in UV-C treated

samples could be related to the previously described effect of UV-C treatments regarding further

stimulation of the wound-induced de novo synthesis of PAL and the registered difference (p<0.05)

between heat-treated (HS) and stress combination samples (HS x UV) at day 0 might be an early

indicator of the minimization of the introduced delay in PAL synthesis by heat shock, as formerly

proposed.

Figure 61 Changes in phenylalanine ammonia lyase (PAL) activity as affected by selected abiotic

stress treatments during storage (7 days, 5 C). Error bars represent the confidence interval at 95%. [ HS:

heat-treated (100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated ([100 C/45 s]x[2.5 kJ.m-

2]); HIPO: chlorine-treated (200 ppm/1 min)].

From day 0 to 7, increases (p<0.05) in PAL activity were detected in all samples (Figure 61) and

highest PAL activity was determined in UV-treated (UV) samples, single application (85.1 U.100 g-1),

supporting the UV-C treatment effect on increasing the wound-induced synthesis of this enzyme

leading to the higher phenolic accumulation registered in these samples (Figure 59). In contrast,

despite the significant increase in PAL activity levels registered to day 7 in chlorine-treated (HIPO)

samples, these samples registered the lower (p<0.05) activity level (47.5 U.100 g-1

, Figure 61)

among samples. This result puts forward the possibility that chlorine could have delayed onset of

PAL activity, suggesting that there are inhibitory effects on plant metabolism imposed by chlorine

which is in accordance with the findings of Klaiber et al. (2005 b). This effect has marked

consequences on the wound-induced phenolic accumulation (Figure 59), preventing the necessary

phenolic synthesis to reach the raw material levels.

The comparison between PAL activity levels at day 7 of heat- and UV-C treated samples, single

(HS and UV) and combined (HS x UV) application (Figure 61), of 61.5, 85.1 and 76.8 U.100 g-1

(p<0.05), respectively, agrees with the possibility that an additive effect is achieved by the stress

HS UV HSxUV HIPO

0 7

Storage time (days)

0

10

20

30

40

50

60

70

80

90

100

PA

L (

U.1

00

g-1

)

Raw material


149

combination. The estimated two-day delay in the wound-induced PAL synthesis introduced by heat

shock could have been lessened by the application of the UV-C stress treatment after shredding

allowing for a significantly higher phenolic accumulation than the one registered in heat-treated

(HS) samples and with no differences to UV-C treated (UV) samples (Figure 59). These findings

reinforce the combined sequential application of abiotic stresses strategy as to achieve higher

phenolic accumulation during storage.


Regarding POD activity (Figure 62), lower activity levels were determined at day 0 in heat-treated

samples, single (HS) and combined (HS x UV) application (p<0.05), comprising a 54 to 64%

activity inhibition regarding the raw material (Table App. 17, appendix 2) with no further significant

changes (p>0.05) during storage. Earlier it was suggested that the 100 C/45 s heat shock could

have directly inactivated the enzyme or induced a reduction in POD synthesis. Within the latter

hypothesis, two distinct effects were highlighted regarding the low POD activity levels during

storage: feedback inhibition due to the lack of phenolics (substrates) or due to heat shock effect on

an unknown receptor implicated in the synthesis of POD (Martín-Diana et al., 2005). Since higher

phenolic levels (Figure 59) were determined in samples submitted to the combined application of

heat shock and UV-C stresses (HS x UV samples) than in samples submitted to the single

application of heat shock (HS samples), it seems that feedback inhibition from the lack of

substrates can be excluded, narrowing the heat shock effects to a direct thermal inhibition or to a

direct effect on the receptors signaling to POD synthesis.

Figure 62 Changes in peroxidase (POD) activity as affected by selected abiotic stress treatments

during storage (7 days, 5 C). Error bars represent the confidence interval at 95%. [ HS: heat-treated

(100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated ([100 C/45 s]x[2.5 kJ.m-2]); HIPO: chlorine-treated (200 ppm/1 min)].

HS UV HSxUV HIPO

0 7

Storage time (days)

0

5

10

15

20

25

30

35

40

PO

D (

U.g

-1)

Raw material


150

The remaining samples also denoted reduced POD activity levels at day 0 (39 to 50%, HIPO and

UV samples, p<0.05, respectively) but, during storage significant increases were found in these

samples, but below the raw material level. This behavior was already described under “MAP”

storage (Part IV, section 2.1), but higher POD increases were found regarding chlorine-treated

(HIPO) samples which can be related to the oxidative stress level during storage as pointed by

Lamikanra and Watson (2001) and could also justify the lower phenolic accumulation level found

(Figure 59). The found differences between sample behaviors indicates that the multitarget strategy

(heat shock and UV-C stress treatments combination) is more beneficial regarding the

achievement of low POD levels during “air” storage preventing related phenolic degradation.

2.4.1.6 pH, SSC, water content and CIELab color

Regarding pH (Table 29), no significant changes were determined either as an immediate

consequence of applied treatments or during storage. As for soluble solids content (SSC), chlorine-

treated (HIPO) samples registered a significant decrease just after treatment (day 0, Table 29)

which was maintained during storage, while no significant changes in this quality parameter were

detected in the remaining samples. The reduced SSC as a result of chlorine treatment was already

described (Part IV, section 1.5) and attributed to compound leaching to the treatment solution

during the decontamination procedure and advises against post-cut immersion treatments as

suggested by Alegria et al. (2009). No significant color changes, expressed as whiteness index

(WI, Table 29), were found in treated samples at day 0 or after 7 days of storage as also no

changes were found regarding the water content parameter (Table 29).

Table 29 Changes in pH, SSC, water content and color (expressed as whiteness index, WI) as

affected by selected abiotic stress treatments during storage (5 C, 7 days).

Sample Id. Storage time (days) pH SSC

(ºBrix)

Water content

(%) WI

Raw material - 6.2ns±0.0 9.4abc±0.4 88.1ns±4.4 *

HS 0 6.3ns±0.0 9.4ac±0.2 88.6ns±5.3 27.5b±2.5

7 6.2ns±0.0 9.5a±0.4 88.8ns±5.3 29ab±2.4

UV 0 6.4ns±0.0 9.7a±0.1 88ns±5.3 25.9a±1.7

7 6.3ns±0.0 9.7a±0.1 88ns±5.3 29.9a±2.9

HSxUV 0 6.3ns±0.0 9bc±0.0 88.7ns±4.4 26.1b±2.2

7 6.3ns±0.0 8.8b±0.0 89ns±4.5 28.8a±2.4

HIPO 0 6.1ns±0.0 7.0e±0.0 91.1ns±4.6 27.9ab±1.9

7 6.0ns±0.0 6.3d±0.1 90.6ns±4.5 29.4a±2.5

* Not comparable; Values represent the mean of three replicates±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).


151

Maintenance of these physical-chemical responses after heat shock or UV-C stress treatments,

single or combined application, during storage, support that these treatments are more

advantageous over the standard industrial practice (post-cut chlorine decontamination) since the

raw material characteristics are better retained during products storage and, indirectly, suggests

that changes in FC carrot sensorial characteristics (sweetness and color) are lessened by these

treatments.


152

2.5 CLOSING REMARKS

In this chapter, insights regarding the effects of heat shock and UV-C abiotic stress treatments on the shredded carrot quality are highlighted in Table 30,

considering the single and combined stress application.

Table 30 Abiotic stress single and combined application effects on shredded carrot quality.

Abiotic stress treatment

UV-C Heat shock Stress combination

Bio

acti

ve q

ual

ity

“Air”

sto

rage

- Phenolic content: accumulation during storage shows to be dependent on UV-C dose; o A 2.5 kJ.m-2 dose (pre- and post-cut

application) promotes PAL activity leading to the maximization of phenolic accumulation regarding the absence of treatment; Surpasses the fresh content by 70-145%

(7 days), regardless of pre- or post-cut application.

- Carotenoid content: maintenance during storage.

- Phenolic content: accumulation during storage is

achieved with 100◦C/45 s stress condition;

o Introduces a two-day delay to the phenolic synthesis dynamic when compared to the absence of treatment;

Surpasses the fresh content by 38% (7 days).


- Phenolic content: accumulation during storage is only enhanced when compared with heat shock single effect; o UV-C allows accumulation to occur early

during storage by amplifying the initial stress signals promoting de novo synthesis of PAL;

Surpasses the fresh content by 60% (7 days).


“MA

P”

stor

age

- Phenolic content: no significant accumulation during storage was verified at 0.78 kJ.m-2 dose (pre-cut application) (10 days); o Anoxic conditions inside the packages (7

days) could be partially responsible for this result.

- Carotenoid content: enhanced content during

storage; o Carotenoid extractability could have been

affected.

- Phenolic content: accumulation during storage is achieved; o The delay in reaching anoxic conditions (10

days) could be partially responsible for this result; Surpasses the fresh content by 30% (7

days).

- Carotenoid content: inconsistent changes during

storage

Under consideration in the next chapter

(continues)


153

Table 30 (cont.) Abiotic stress single and combined application effects on shredded carrot quality.

Abiotic stress treatment

UV-C Heat shock Stress combination

Sen

sori

al q

ual

ity

“Air”

sto

rage

- Does not affect characteristic carrot color, regardless of applied dose or application site (pre- or post-cut).

- Does not affect characteristic carrot color and sweetness (7 days).

- Does not affect characteristic carrot color and sweetness (indirectly shown by SSC maintenance) during storage (7 days).

“MA

P”

stor

age

- Acceptable sensory properties (0.78 kJ.m-2 dose; 10 days)

- Higher sensory scores comparing to UV-C treatment (10 days).


Mic

rob

iolo

gic

al q

ual

ity

“Air”

sto

rage

- Not applicable - Not applicable - Not applicable

“MA

P”

stor

age

- Decontamination efficiency of 1.7 Log10 similar to that of chlorine (0.78 kJ.m-2 dose).

- Decontamination efficiency of 2.5 Log10 significantly higher than UV-C treatment and with effect in controlling microbial development (10 days).


Other effects

- Lower POD activity levels regarding raw material could be supported by the stress-induced diversion of metabolic pathways; o Activity regeneration levels in MAP storage

conditions still needs confirmation for 2.5 kJ.m-2 dose.

- Lower POD activity levels regarding raw material could be supported either by the stress-induced diversion of metabolic pathways or by enzyme thermal inactivation; o Activity regeneration levels in MAP storage

conditions still needs confirmation considering the effective control under air storage conditions;

- Treatment effects on O2/CO2 changes inside the packages are justified by lower carrot respiration rate as previously confirmed (Alegria, 2007);

- Lower POD activity levels regarding raw material could be supported either by the stress-induced diversion of metabolic pathways or by enzyme thermal inactivation; o Activity regeneration levels in MAP storage

conditions needs to be evaluated; - Treatment effects on O2/CO2 changes inside the

packages need to be tested.

155

3 TECHNOLOGICAL PROPOSAL: HURDLE CONCEPT INTO ACTION

The hurdle concept is explored to the fullest within this research line through the integration of

selected abiotic stress treatments, in single or in combination, with two distinct MAP solutions (OPP

vs. micro-perforated OPP films) (study 3.1). Finally, the best technological solution found was

compared with the standard (industrial) minimal processing performance and product shelf-life

estimated according to the fulfillment of three quality criteria: microbiological, sensorial and

bioactive (study 3.2).

3.1 SELECTED HEAT SHOCK AND UV-C STRESS TREATMENTS AND MAP EFFECTS ON

FRESH-CUT CARROT QUALITY

The aim of this study was to evaluate the following on the overall quality of fresh-cut carrot during

low temperature storage (5 C, 14 days):

i. The effects of selected abiotic stress treatments (heat shock: 100 C/45 s; UV-C: 2.5 kJ.m-2),

applied as single or in combination;

ii. The effects of two MAP conditions set by used packaging film (non-perforated vs. micro-

perforated).

The treatments under evaluation were (3 independent replicates): heat shock (100 C/45 s; sample

id. HS; treated as described in Part III, section 3.1.1), UV-C radiation (2.5 kJ.m-2

; sample id. UV;

treated as described in Part III, section 3.1.2) and the combination of heat shock and UV-C

radiation (sample id. HSxUV; treated as described in Part III, section 3.2). A control experiment

was set considering untreated shredded carrot (Ctr) samples. For subsequent evaluation, fresh-cut

carrot samples prepared according to the industrial standard which uses 200 ppm chlorine

solutions as a post-cut decontamination treatment (sample id. HIPO; treated as described in Part

III, section 2.3) were set in parallel.

Two MAP storage conditions were evaluated and established by the use of two types of oriented

polypropylene (OPP) films (Amcor Flexibles Neocel – Embalagens Lda., Lisboa, Portugal) of 35 µm

with OTR and CTR of 1100 ml.m-2

.d-1

.atm-1

and 3000 ml.m-2

.d-1

.atm-1

, respectively. The difference

between films is related to the lack of, film A, or presence of laser established micro-perforations at

120 µm intervals, film B (PPlus - 35PA120), which ensures significant changes in the film

permeability.

A lot of 100 kg of cv. Nantes carrot was obtained (Part III, section 1) and a 10% sampling was used

to conduct the raw material characterization (Table App. 18, appendix 3). The remaining 90 kg of

carrot were equally divided into five groups and used to prepare treated (HS, UV, HSxUV

samples), untreated (Ctr) and the chlorinated (HIPO) samples as shown in Figure 63. Samples

TECHNOLOGICAL PROPOSAL: HURDLE CONCEPT INTO ACTION

156

were packaged in both packaging conditions in 125 g portions and the bags (200 x 110 mm) heat-

sealed and stored at 5 C until analysis (days 0, 3, 5, 7, 10, 12 and 14).

Figure 63 Flow diagram of minimal processing operations for preparing FC carrot packaged under two MAP conditions according to the selected abiotic stress treatments and to the standard practice.

The evaluated responses during storage (5 C, 14 days) included the determination of the

headspace gas analysis, of the bioactive responses (TPC – protocol I, chlorogenic acid [CA], TCC,

-carotene, AOx [both in hydrophilic and lipophilic extracts; protocol I] and stress related enzymes

activities [PAL and POD]), physical-chemical responses (pH, SSC, color), sensorial analysis and

microbiological responses (TAPC, LAB and Y&M).

In order to assess the single or combined effects of the stress treatments on evaluated responses,

treated samples (HS, UV and HSxUV) were compared to the untreated (Ctr) samples (ANOVA,

Tukey HSD test, p=0.05) and hierarchical cluster and principal component (PCA) analyses were

carried out as to establish the relations between stresses, MAP storage and the quality variables

assessed in this study. The best technological alternative regarding bioactive, sensorial and

microbiological quality was then selected to be compared (ANOVA, Tukey HSD test, p=0.05) with


157

the industrial reference (HIPO samples) in order to estimate the predicted shelf-life for the

technological proposal (following section 3.2).



The O2 and CO2 (%) evolution inside the sample packages during storage are shown in Figure 64.

Figure 64 Changes in atmosphere composition ( O2 and CO2 concentrations, %) of shredded carrot samples packaged with a) film A and b) film B as affected by abiotic stress

treatments during low temperature storage (5 C, 14 days). Vertical bars denote the confidence interval at

95%. [Ctr: untreated; HS: heat-treated (100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated

(100 C/45 s x 2.5 kJ.m-2)].

a)

a)

Storage time (days) O2 CO2

0 3 5 7 1012140

5

10

15

20

25

30

35

40

45

50

Ga

s c

on

ce

ntr

ati

on

(%

)

0 3 5 7 101214 0 3 5 7 101214 0 3 5 7 101214

Ctr samples HS samples UV samples HSxUV samples

a)

a)

a)


0 3 5 7 1012140

5

10

15

20

25

30

35

40

45

50

Ga

s c

on

ce

ntr

ati

on

(%

)

0 3 5 7 101214 0 3 5 7 101214 0 3 5 7 101214

Ctr samples HS samples UV samples HSxUV samples

b)


158

Stress treatments, single and combined application, and packaging film had significant effects over

the gaseous composition of the packages achieved passively by the product respiration and/or

microbial development, with packaging film being more influential to those changes as expressed

by the highest F-value (Table App. 19, appendix 3).

During storage, anoxic conditions (O2 levels <0.5%) were reached in the early stages of storage

when samples were packaged with film A (Figure 64 a), condition that never occurred when

samples were packaged with film B (Figure 64 b) and could be related to the existing micro-

perforations in film B that convey adequate ventilation/gas exchanges.

In samples packaged with film A, distinctive responses were found between applied stress

treatments (Figure 64 a): heat- (HS) and combined-treated (HSxUV) samples showed lower O2

decreases reaching anoxic conditions (O2 levels <0.5%) at least 7 days after UV-treated samples

(UV) and untreated (Ctr) samples (HS samples – day 12; HSxUV samples – day 10; UV samples –

day 5; Ctr samples – day 3). The same behavior was found in samples packaged with film B

(Figure 64 b), and again heat- (HS), combined-treated (HSxUV) samples had lower O2 decreases

(p<0.05) regarding UV-treated (UV, single application) and untreated (Ctr) samples. With this

packaging solution, the lowest O2 level registered was of 1.5% in untreated (Ctr) samples after 12

days of storage which was much lower (p<0.05) than the O2 levels registered in treated samples, of

about >10% in O2.

Concerning CO2 levels, during storage, samples packaged with film A (Figure 64 a) reached

surprisingly high CO2 levels, up to 43.1% at the end of considered storage. This CO2 level was

registered in UV-treated (UV, single application) samples with no differences (p>0.05) with the

untreated (Ctr) samples, suggesting that the applied UV-C dose had no effect on products

respiration rate. Heat- (HS) and combined-treated (HSxUV) samples had lower (p<0.05) CO2

increases, with maximums of 25.3 and 24.7% (p>0.05), respectively, after 14 days of storage.

These significant lower CO2 levels support the heat shock treatment effect in lowering the

respiration rate of shredded carrot as demonstrated in previous works (Alegria, 2007).

The CO2 evolution in samples packaged with film B (Figure 64 b) followed a similar pattern, with

UV-treated (UV, single application) and untreated (Ctr) samples registering the highest (p<0.05)

increases (18.8 and 14.8%, respectively) after the 14 days of storage while heat- (HS) and

combined-treated (HSxUV) samples (p>0.05) had CO2 levels of 7%.

Between the applied treatments, the 100 C/45 s heat shock, applied as single or in combination,

showed to be the most favorable stress treatment to effectively lower the product respiration

metabolism (reduced O2 consumption/CO2 production rates) during storage. Reduced respiration

rates as a result of heat treatments have also been reported by other authors in shredded carrot

(100 C/45 s; 50 C/120 s) (Alegria et al., 2009; Alegria, 2007; Klaiber et al., 2005 a), plums

(45 C/10 min) (Serrano et al., 2004), shredded iceberg lettuce (47 °C/30–180 s) (Odumeru et al.,

2003), and fresh-cut lettuce (steamed, 100 °C/10 s) (Rico et al., 2008).


159


Changes in total phenolic content (TPC) as influenced by stress treatments and MAP storage are

shown in Figure 65. The TPC levels registered in all treated (HS, UV, HSxUV) and untreated (Ctr)

samples at day 0 (ranging from 50.4 - 57.7 mg CAE.100 g-1

, p>0.05) were significantly lower than

the ones determined in the raw material (85.3 mg CAE.100 g-1

; Table App. 18, appendix 3). The

registered loss (36%) was attributed to the already quantified effects of minimal processing

operations (Part IV, section 1.5), confirming once more that the abiotic stresses, as single or

combined treatments, do not further increase to processing losses.

During storage, the used packaging film was the most influential factor to the TPC changes of FC

carrot as shown by the higher F-value (Table App. 20, appendix 3). In samples packaged with film

A (where there is a rapid change of gaseous levels as previously characterized – “low” permeability

film), there was a significant phenolic increase after 7 days of storage (Figure 65), regardless of

applied treatment. The phenolic increments to day 7 were not influenced by treatment nature (heat

shock or UV-C) but by treatment combination (p<0.05). Thus the accounted increases of 1.8 and

1.5 times regarding initial contents in heat (HS) and UV-treated (UV) samples (single application)

allowed to reach the levels present in the raw material (Table App. 18, appendix 3), and the

2.3 times increase registered in combined-treated (HSxUV) samples exceeded by 36% the levels

of the raw material.

Figure 65 Changes in total phenolic content (TPC) of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low temperature storage

(5 C, 14 days). Vertical bars denote the confidence interval at 95%. [ Ctr: untreated; HS: heat-treated

(100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated (100 C/45 s x 2.5 kJ.m-2)].

Storage time (days) Ctr HS UV HSxUV

0 3 5 7 10 12 140

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

g-1

)

0 3 5 7 10 12 14

Film BFilm A

Raw material


160

For all samples there was a decrease (p<0.05) from day 7 to 10 back to the respective initial

contents (day 0, p>0.05): TPC levels of heat- (HS) and UV-treated (UV) samples decreased to

about half the previous record and combined-treated (HSxUV) samples decreased about 2.5-fold.

No further significant TPC changes were determined till the end of considered storage period (days

10 to 14, Figure 65).

Concerning samples packaged with film B (“high” permeability film), there was also a significant

phenolic increase after 7 days of storage, regardless of applied treatment and higher (p<0.05) than

those observed in film A (Figure 65). However, the phenolic increments to day 7 were influenced

(p<0.05) by treatment nature (heat shock and UV-C) and by treatment combination since significant

differences were registered between treated (HS, UV and HSxUV) and untreated (Ctr) samples. At

day 7, the phenolic increments found in heat- (HS) and combined-treated (HSxUV) samples

(p>0.05, 2.4 and 3 times, respectively), exceeded the raw material TPC by 36% while in UV-treated

(UV) samples the increase was lower (p<0.05) but sufficient to match the raw material (Figure 65).

The registered phenolic accumulation at day 7 in heat-treated (HS) and combined-treated (HSxUV)

samples was significantly higher than the one registered in untreated (Ctr) samples and,

considering the similar (p>0.05) behavior of both samples, it can be suggested that the 100 C/45 s

stress treatment allied to perforation mediated (PM)-MAP storage favored the wound-induced

phenolic synthesis mechanism.

To day 10 still a significant TPC increase was registered in all treated samples (p>0.05) reaching

maximum TPC levels three days after the maximum found in untreated (Ctr) samples. This

behavior indicates that all stress treatments, single and combined application, allied to the

established atmosphere inside the packages provided by the micro-perforated film, induced

favorable changes to the phenylpropanoid metabolism. From that point on, decreases were also

registered but without compromising the maintenance of the raw material TPC level until the end of

considered storage period (day 14).

The found differences between treated (HS, UV and HSxUV) and untreated (Ctr) samples reveal

that the effect of the UV-C treatment fell short from the expected (further stimulation of the wound-

induced phenolic synthesis) but these results could be justified by differences in the established

modified atmosphere inside sample packages which are significantly different from “air” storage

conditions. This result also suggests that there might be a critical O2 level to sustain phenolic

synthesis.

It would be expected that the abiotic stresses and MAP conditions effects on the chlorogenic acid

(CA) response were to be similar to the ones found regarding TPC since chlorogenic acid is the

main synthetized phenolic in FC carrot (Part IV, section 1.4). The projection of CA changes during

storage as a result of abiotic stress treatments and gaseous composition imposed by packaging

conditions is shown in Figure 66.


161

Figure 66 Changes in chlorogenic acid content of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low temperature storage



At day 0 and regarding the raw material (12.30±1.17 mg.100 g-1

; Table App. 18, appendix 3), a

significant decrease in CA content was registered in all samples (60%-80%), irrespective of the

used film for packaging. The similarity (p>0.05) in CA contents of heat- (HS) and UV-treated (UV)

samples and the untreated (Ctr) samples suggests that these treatments, applied as single, do not

add to CA losses attributed to peeling operation (by thermal degradation or oxidation) but

respective treatment combination (HSxUV samples) might minimize this loss as higher contents

(p<0.05) were determined.

During storage, samples packaged with film A showed a relative maintenance (p>0.05) in CA

content except for combined-treated samples, in which a significant increase was found to day 10

followed by a decrease (p<0.05) back to initial levels (Figure 66), and the depicted behavior can be

supported by the found differences regarding TPC changes.

When samples were packaged with film B, a significant CA increase was registered in all samples

to day 7, however insufficient to reach the raw material level (Table App. 18, appendix 3)

regardless of applied treatment. Still significant increases were found in CA to day 10 which were

dependent on applied treatment: heat-treated (HS) and combined-treated (HSxUV) samples

(p>0.05) had already reached the raw material CA content while UV-treated (UV) samples were still

below that level. This difference could find support in the heat shock effects of in lowering the

product respiration rate as previously seen, which can be seen as beneficial to phenolic synthesis,

namely of chlorogenic acid. Further increases were determined to day 12 in all treated samples

whereas untreated (Ctr) samples maintained the previous record till the end of storage. It of interest


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2

4

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8

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CA

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162

to notice that heat-treated samples, single (HS) application, registered significant CA increments

from day 5 until the end of storage (day 14), sufficient to reach and surpass the raw material

content, but combined-treated (HSxUV) and UV-treated (UV, single application) samples registered

significant decreases from day 12 to 14, which could be attributed to differences in the phenolic

degradation pattern.

3.1.1.3 Total carotenoid and -carotene content

Changes in total carotenoid (TCC) and -carotene contents of fresh-cut carrot are shown in Figure

67 and Figure 68, respectively.

At day 0, all samples registered similar TCC levels (Figure 67) regardless treatment or packaging

film, with levels close to the ones determined in the raw material (18.2±0.3 mg -carotene eq.100 g-1;

Table App. 18, appendix 3).

Figure 67 Changes in total carotenoid content (TCC) of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low temperature storage



The comparison between TCC changes of treated and untreated samples suggest that the abiotic

stress treatments did not significantly influence this bioactive composition during storage, as only

promptly significant differences are found. The established atmosphere inside the packages as set

by the film used for packaging also did not significantly influenced TCC changes during storage

(Figure 67). These results support the found relative stability of this composition during storage

(Part IV, sections 2.2, 2.3 and 2.4).


0 3 5 7 10 12 140

5

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163

Concerning -carotene content (Figure 68), at day 0 and in general, samples had higher contents

than the raw material (16.43±0.85 mg.100 g-1

; Table App. 18, appendix 3) without apparent reason.

Consequently, it can be assumed that the application of the abiotic stress treatments did not

introduced any immediate change to this composition.

Figure 68 Changes in -carotene content of fresh-cut carrot as affected by abiotic stress treatments

and MAP conditions (set by the films used for packaging) during low temperature storage (5 C, 14

days). Vertical bars denote the confidence interval at 95%. [ Ctr: untreated; HS: heat-treated (100 C/45 s);

UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated (100 C/45 s x 2.5 kJ.m-2)].

During storage, a decrease (p<0.05) is found in -carotene content to day 3 (Figure 68),

irrespective of applied abiotic stress or MAP condition, which was relatively maintained for the

remaining storage period. From sample comparison and despite occasional significant differences

with the untreated control, it can be speculated that the applied stress treatments or the established

atmosphere inside the packages resulting from the film used for packaging do not alter the -

carotene content change pattern during storage. Overall, it can be assumed that FC carrot -

carotene content is fairly stable during storage and comparable to that of the raw material content.


The abiotic stress treatment and packaging film effects on the hydrophilic (AOxH) and lipophilic

(AOxL) antioxidant capacity of fresh-cut carrot during storage are shown in Figure 69 a and b,

respectively. At day 0 and irrespective of considered packaging film, no differences (p>0.05) were

found in AOxH (Figure 69 a) and AOxL (Figure 69 b) between treated (HS, UV, HSxUV) and

untreated (Ctr) samples, with AOx levels similar to those determined in the raw material (Table

App. 18, appendix 3), despite the significant decrease in TPC levels (Figure 65).


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-C

aro

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.10

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164

Figure 69 Changes in hydrophilic (AOxH, a) and lipophilic (AOxL, b) antioxidant capacity of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for

packaging) during low temperature storage (5 C, 14 days). Vertical bars denote the confidence interval at

95%. [ Ctr: untreated; HS: heat-treated (100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV:

combined-treated (100 C/45 s x 2.5 kJ.m-2)].

As for TPC, during storage, the gaseous composition inside the packages was most influential to

the AOxH changes (Figure 69 a) as demonstrated by the highest F-value (Table App. 22, appendix

3). Likewise, in samples packaged with film A significant increases were found to day 7 and

maximum AOxH levels were registered, however dependent on applied treatment: heat-treated

(HS) samples registered the highest AOxH increase (50% above the raw material level), followed

by combined-treated (HSxUV) samples (27% above the raw material level) and UV-treated (UV)


0 3 5 7 10 12 1420

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Film A Film B

Raw material

a)


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35

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AO

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b)


165

samples with similar levels to that of raw material. To day 10, a decrease (p<0.05) was registered

in heat-treated samples (single and combined application) to AOxH levels just below the raw

material with no further changes during the remaining storage period.

A similar behavior was found in samples packaged with film B (Figure 69 a), however the AOxH

increment to day 7 was significantly higher than the one registered in samples packaged with film

A. No differences were found between heat-treated (HS) and combined-treated (HSxUV) samples

which had the highest (p>0.05) AOxH increase (61% above the raw material) or between UV-

treated (UV) and untreated (Ctr) samples. Moreover, despite the found decrease (p<0.05) in AOxH

to day 10, the raw material AOxH levels were assured till the end of storage (day 14), regardless of

applied treatment.

Concerning changes in AOxL during storage (Figure 69 b), no effect from established MAP

conditions or applied treatment stands out. A significant decrease to day 3 to levels below the raw

material was detected with no further changes (p>0.05) during storage, irrespective of treatment or

the film used for packaging, agreeing with the relative maintenance in total carotenoid and -

carotene contents.

To the total antioxidant capacity (Table App. 23, appendix 3), little input is given by the AOxL

fraction (r2=0.08, p>0.05). On the contrary, the significant correlation found with total AOxH

(r2=0.99, p<0.05), major contributor to carrots total antioxidant capacity, corroborates that the film

used for packaging was more influent to the found changes in total AOx of FC carrot during

storage. As a result, the taken considerations about treatment and packaging film effects on AOxH

during storage stand as well for total AOx.


Changes in PAL activity during storage as affected by abiotic stress treatments and gaseous

composition imposed by the films used for packaging are shown in Figure 70. Independently of

packaging film, at day 0 no significant changes in PAL activity were detected between treated and

untreated samples (p>0.05). Nonetheless, despite the non-significant difference, the highest PAL

activity levels determined in UV- (UV) and combined-treated (HSxUV) samples suggest that the

UV-C treatment, as single hurdle or in combination, further stimulated the wound-induced de novo

synthesis of PAL as previously seen (Part IV, sections 2.2 and 2.4). Moreover, the absolute

difference in PAL activity levels between heat- (HS) and combined-treated (HSxUV) samples found

at day 0 could support the registered difference in phenolic accumulation during storage.


166

Figure 70 Changes on phenylalanine ammonia lyase activity (PAL) of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low

temperature storage (5 C, 14 days). Vertical bars denote the confidence interval at 95%. [ Ctr: untreated;

HS: heat-treated (100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated

(100 C/45 s x 2.5 kJ.m-2)].

In samples packaged with film A, a significant increase in PAL activity was found in all samples to

day 3 as a result of wounding, and the activity increments were not, however, influenced by

treatment nature or by treatment combination (Figure 70): treated samples (HS, UV and HSxUV)

had similar (p>0.05) PAL activity levels regarding the untreated (Ctr) samples, but highest levels

were determined in combined-treated (HSxUV) samples which support the higher TPC and CA

increases to day 7 registered in these samples in comparison with the single application of either

abiotic stress. These results are in accordance with Klaiber et al. (2005 b) who found that

anaerobic atmospheres resulting from the use of low oxygen permeability films as packaging

solution for shredded carrot, such as film A, resulted in a maximum activity peak between storage

days 2 and 4 with PAL activity declining back to negligible values thereafter. Nevertheless, despite

the significant decrease in PAL activity of all samples to day 5, the registered activity levels allowed

for the continuity of phenolic synthesis to day 7 (Figure 65), supporting the previous findings that

show that even though PAL synthesis is suppressed, the existing enzyme level supports phenolic

synthesis.

Conversely, samples packaged with film B show a general increase tendency in PAL activity levels

during storage (Figure 70). The first significant increase in PAL activity was, similarly to film A,

registered to day 3 but the respective activity increments were much higher (p<0.05) than the ones

found in samples packaged with film A. In this situation (samples packaged with film B) the PAL

activity increase to day 3 was only influenced by the 100 C/45 s heat shock single application (HS

samples), condition that lead to the lower (p<0.05) activity increment regarding UV-C treatment,


0 3 5 7 10 12 140

50

100

150

200

250

300

350

PA

L (

U.1

00

g-1

)

0 3 5 7 10 12 14

Film A Film B

Raw material


167

treatment combination or even the absence of treatment (UV, HSxUV and Ctr samples,

respectively; p>0.05). This result supports once more that heat shock introduces a delay to PAL

synthesis either due to decreased PAL synthesis as a result of the induced metabolic shift or due to

increased thermal destruction as previously suggested (Part IV, section 2.3). Moreover, the

significant difference found between PAL activity levels of heat-treated (HS) and combined-treated

(HSxUV) samples confirms that the heat-induced delay on de novo synthesis of PAL can be

overcome by UV-C application allowing to at least match the wound-induced PAL synthesis (Ctr

samples). However from the comparison between UV-treated (UV) and untreated (Ctr) samples

activity levels (p>0.05) at day 3, it is possible to observe that the UV-C treatment did not further

stimulate the wound-induced de novo synthesis of PAL contrary to previous findings (Part IV,

section 2.2).

From day 3 to 5, all samples denoted a significant decrease in PAL activity levels maintaining the

previous relations between samples and, from day 5 to 10 consistent significant increases were

registered in all samples (Figure 70). From that point on, treated samples registered maintenance

(p>0.05) of the achieved activity levels to the end of storage (previously characterized behavior)

except for heat-treated samples, single application (HS samples), that still registered significant

activity increments to day 12, corroborating the heat-induced delay in PAL synthesis. In spite of the

lower activity levels registered in these later samples, higher TPC and CA levels were found

regarding untreated (Ctr) samples which could be attributed to differences in the degradation rates

of phenolics (consumed as antioxidants or enzymatic substrates) between samples.

Overall, the differences between samples packaged with low (film A) and high (film B) permeability

films or even “air” storage (2), suggest that the desirable abiotic stress treatments effects over the

phenolic synthesis dynamic can only be achieved if adequate O2 levels are maintained. The

characterized differences between sample O2/CO2 levels support the found differences regarding

phenolic synthesis (TPC and PAL levels): phenolic accumulation was completely suppressed under

ultralow oxygen conditions (<0.5%; film A), while maintenance of O2 levels above 2% were a

necessary condition to phenolic synthesis during storage. Using a micro-perforated film (film B),

enables higher O2 levels during storage (averaged in 9% from day 3 to 14) resulting in a longer

TPC synthesis period which benefits the product bioactive composition, irrespective of applied

stress treatment. Generally speaking, the use of the micro-perforated film enabled, in the early

stages of storage, the achievement of a steady-state compatible with phenolic synthesis in

opposition to the non-perforated film where reaching those steady-state conditions are challenging

to put it mildly. These findings support that the phenolic synthesis dynamics in shredded carrot is

firstly determined by established modified atmosphere inside the packages then by applied

treatment.


168


Shown in Figure 71 are the changes in POD activity during storage as affected by abiotic stress

treatments and gaseous composition imposed by the film used for packaging. Raw material POD

activity level was unusually high considering previous studies (97.1±1.8 U.g-1

; Table App. 18,

appendix 3) and, after processing, all samples exhibit significantly lower activity levels (from 10 to

50 U.g-1

).

Figure 71 Changes on peroxidase activity (POD) of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low temperature storage



Irrespective of packaging film, at day 0, POD activity was significantly influenced by treatment

nature and by treatment combination (Figure 71): heat- (HS) and combined-treated (HSxUV)

samples reveal lowest (p<0.05) activity levels considering UV-treated (UV) or untreated (Ctr)

samples, representing a partial inhibition of about 65% regarding the absence of treatment (Ctr

samples).

During storage, and regardless of packaging solution, heat- (HS) and combined-treated (HSxUV)

samples maintained (p>0.05) the initial low activity levels confirming once more the 100 C/45 s

heat shock effects over this oxidative enzyme (Part IV, sections 2.3 and 2.4), acting either on its

thermal inhibition or by compromising its synthesis during storage. No effect is attributed to UV-C

treatment on POD activity changes during storage since no differences (p>0.05) were found

regarding untreated (Ctr) samples. However, the effects of the established gaseous composition

inside the packages of UV-treated (UV) and untreated (Ctr) samples significantly influenced POD

changes from day 7 onward. To this regard, samples packaged with film A denoted a decrease


0 3 5 7 10 12 140

20

40

60

80

100

120

PO

D (

U.g

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0 3 5 7 10 12 14

Film A Film B

Raw material


169

tendency during the remaining storage period while samples packaged with film B denoted an

opposite behavior, with increases (p<0.05) in the activity levels (Figure 71). The difference in

behaviors to the last 7 days of storage could be related with the higher oxygen levels registered in

samples packaged with film B.

3.1.1.7 Phenolic synthesis dynamic

Phenolic (TPC) changes during storage are a result of synthesis and degradation reactions

involving both PAL and POD enzymes. The two mechanisms can be described by fitting the TPC

data to the equation proposed by Amodio et al. (2014; Eq. 6) and the projections are shown in

Figure 72 (model parameters reported in Table App. 25 and regression analysis in Table App. 26,

appendix 3.).

The phenolic synthesis registered until day 7 was influenced by the application of heat shock either

as single or in combination (Figure 72 b and d, respectively) but no influence of UV-C treatment is

observed (Figure 72 c), regardless of the film used for packaging. Similarly, the phenolic

degradation phenomenon was also affected by heat shock since the respective effect over POD

(partial inhibition) lessens phenolic oxidation during storage which favors to increment the products

bioactivity. Moreover, to the phenolic degradation behavior, the gaseous composition established

inside the packages was extremely influential: while decreases in phenolic content are well defined

in samples packaged with film A irrespective of treatment nature or combination, when packaged

with film B the behavior is countered and further phenolic synthesis is achieved during storage

minimizing also respective degradation. This shift to the degradation behavior highlights the

extreme importance of ensuring adequate oxygen levels for the continued synthesis of phenolic

compounds, namely chlorogenic acid, where stress nature is indirectly influential to the response.

These findings are in good agreement with the studies of Babic et al. (1993) and Klaiber et al.

(2005 b): while first authors found that high phenolic accumulation was achieved when storing

shredded carrots in air, the second authors found that carrot stick samples packaged with films of

distinct (“low” and “high” permeability films) had distinguishable phenolic accumulation behaviors

much alike the ones observed herein.


170

Figure 72 Changes in TPC and model fitting to the proposed equation by Amodio et al. (2014) applied to fresh-cut shredded carrot samples packaged with film

A and B stored at 5 C (14 days) and according to abiotic stress treatment: a) control (Ctr, Untreated), heat-treated (HS, 100 C/45 s), UV-treated (UV, 2.5 kJ.m-2) and combination of heat and UV-treated (HSxUV, [100 C/45 s] x [2.5 kJ.m-2]).

Ctr samples

Film A Film B

0 3 5 7 10 12 14

Storage time (days)

0

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

g-1

)

Raw material

a) HS samples

Film A Film B

0 3 5 7 10 12 14

Storage time (days)

0

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

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)

Raw material

b)

UV samples

Film A Film B

0 3 5 7 10 12 14

Storage time (days)

0

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

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)

Raw material

c) HSxUV samples

Film A Film B

0 3 5 7 10 12 14

Storage time (days)

0

20

40

60

80

100

120

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160

180

TP

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d)


171

3.1.1.8 pH and soluble solids content (SSC)

As it can be observed in Figure 73, no immediate change in pH was registered as a result of abiotic

stress treatments just after processing (pH of 6.2 in all samples vs. 6.4 in raw material; Table App.

18, appendix 3), irrespective of the film used for packaging. During storage, maintenance (p>0.05)

in sample pH is observed until day 7 and no differences are found between treated samples nor

between MAP conditions. From day 7 onwards, a decrease (p<0.05) tendency is observed in all

samples which was influenced by treatment nature, treatment combination and also by MAP

conditions. From day 7 to 10, UV-treated (UV) samples packaged with film A, registered a

significant pH drop, from 6.3 to 5.7 pH units, surpassed (p<0.05) only by untreated (Ctr) samples

(to 5.3 pH units), and further continuous decreases (p<0.05) were found to day 14 (4.3 pH units,

Figure 73). When packaged with film B, UV-treated (UV) samples also registered a similar drop to

day 10, but pH decrease (p<0.05) to the last day of storage (day 14) was lower (p<0.05) and to

about 5.0 pH units (Figure 73).

Unlike UV-treated samples, from day 7 to 10 maintenance (p>0.05) in pH values were found in

heat- (HS) and combined-treated (HSxUV) samples irrespective of the film used for packaging

(Figure 73). From that point on, also a significant drop in pH was registered and maintained

(p>0.05) to day 14 (final pH values of 5.5 and 5.2 units for HS and HSxUV samples, respectively).

Figure 73 Changes in pH of fresh-cut carrot as affected by abiotic stress treatments and MAP

conditions (set by the films used for packaging) during low temperature storage (5 C, 14 days).

Vertical bars denote the confidence interval at 95%. [ Ctr: untreated; HS: heat-treated (100 C/45 s); UV:

UV-treated (2.5 kJ.m-2); HSxUV: combined-treated (100 C/45 s x 2.5 kJ.m-2)].

The higher pH decreases found in UV-treated (UV) samples, similar to those observed in untreated

(Ctr) samples, and particularly when packaged with film A, could be related to an insufficient


0 3 5 7 10 12 144,0

4,5

5,0

5,5

6,0

6,5

7,0

pH

0 3 5 7 10 12 14

Film A Film B

Raw material


172

decontamination efficiency, leading to the outgrowth of microbial population, namely of LAB group.

Accordingly, FC carrot acidification is closely related to the accumulation of fermentation by-

products such as lactic and acetic acids (Kakiomenou et al., 1996; Carlin et al., 1990) resulting

from LAB growth/metabolism. The found pH differences between heat- (HS), combined-treated

(HSxUV) samples and UV-treated (single application, UV) or untreated (Ctr) samples suggest that

the 100 C/45 s heat shock was responsible for controlling the development of this contaminating

group, preventing accelerated product acidification.

Concerning soluble solids content (SSC), no effect was attributed to stress application, as single or

in combination, as no significant changes were found regarding the raw material (8.3±0.1 Brix;

Table App. 18, appendix 3). During storage, no significant changes (p>0.05) in SSC were found

either regarding applied stress treatments or the film used for packaging: SSC varied between 8.1-

8.7 Brix (day 0) and 7.3-9.1 (day 14; Table App. 28, appendix 3).

The found maintenance in SSC values could be justified by treatment (heat shock) application

before shredding and by the absence of post-cut immersion treatments (UV-C treatment) therefore

preventing losses to this quality parameter so closely related to perceived carrot sweetness

(Seljåsen et al., 2001).

3.1.1.9 Color

Changes in CIELab color parameters and whiteness index (WI) of fresh-cut carrot are shown in

Table App. 30 (appendix 3) and Figure 74, respectively.

No effect is attributed to stresses application on CIELab color changes during storage as no

differences (p>0.05) are found regarding the untreated (Ctr) samples during storage, and only

promptly differences were found concerning MAP conditions (Table App. 30, appendix 3).

As for sample whiteness index (WI, Figure 74), similar behavior is found, and no significant

changes in WI were found relating to applied stress treatment or packaging film, with WI values

ranging from 26.0-28.8 (day 0) to 27.1-30.5 WI units (day 14). These WI values are below the

38.4 ± 1.3 WI interval considered to be sensorially perceived as a “slight” whitening of the shred

(Cisneros-Zevallos et al., 1995).


173

Figure 74 Changes in color of fresh-cut carrot, expressed as whiteness index (WI), as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low

temperature storage (5 C, 14 days). Vertical bars denote the confidence interval at 95%. [ Ctr: untreated;

HS: heat-treated (100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV: combined-treated

(100 C/45 s x 2.5 kJ.m-2)].


Changes in the sensorial attributes scores of FC carrot during storage as influenced by abiotic

stress treatments and MAP conditions are shown in Figure 75 and respective rejection index is

shown in Figure 76.

Focusing on color changes during storage (Figure 75 a), treatment nature and treatment

combination significantly influenced the perceived sample color however unrelated with objective

color measurements (sensorial color scores vs. CIELab parameters or whiteness index, r2<0.04;

p>0.05). The unrelated objective color measurements and sensorial color perception subscribes

the difficulty in accurately and objectively measure shredded carrot color due to the product

topographic heterogeneity (uneven surfaces), easily creating shadows, compromising the objective

reading.

UV-treated (UV) samples had increasingly higher (p<0.05) color scores during storage, from 1

(Nonwhite) to 3 (Moderate white; day 14), than heat- (HS) and combined-treated (HSxUV)

samples, always scored between anchors 1 (Nonwhite) and 2 (Slightly white; day 14), irrespective

of packaging film. These results indicate that the application of the 100 C/45 s heat shock, single

or combined, favors color maintenance during storage. Color maintenance in fresh-cut products as

a result of heat treatments has also been described by other authors, namely in carrot

[100 C/45 s; 50 C/120 s] (Alegria et al., 2010; Klaiber et al., 2005 a), pear [35 C/20 min] (Abreu

et al., 2011) and lettuce [steam - 100 °C/10 s] (Rico et al, 2008).


0 3 5 7 10 12 1421

22

23

24

25

26

27

28

29

30

31

32

33

WI

0 3 5 7 10 12 14

Film A Film B


174

Figure 75 Changes in the sensorial scores of a) color, b) appearance and c) aroma of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for


95%. [ Ctr: untreated; HS: heat-treated (100 C/45 s); UV: UV-treated (2.5 kJ.m-2); HSxUV:

combined-treated (100 C/45 s x 2.5 kJ.m-2)].


Scale: 1 - Nonwhite (Fresh); 2 - Slightly white; 3 - Moderate white; 4 - Severe white; 5 - Extreme

white

0 3 7 10 14

1

2

3

4

5

Co

lor

sc

ore

0 3 7 10 14

Film A Film Ba)


Scale: 1 - Excellent/fresh appearance; 2 - Moderate; 3 - Limit of saleability; 4 - Poor; 5 - No fresh

appearance

0 3 7 10 14

1

2

3

4

5

Fre

sh

-lik

e a

pp

ea

ran

ce

sc

ore

0 3 7 10 14

Film A Film B

Limit of saleability

b)


Scale: 1 - Very intense (Fresh), 2 - Intense; 3 - Moderate; 4 - Low; 5 - Absent

0 3 7 10 14

1

2

3

4

5

Fre

sh

-lik

e a

rom

a s

co

re

0 3 7 10 14

Film A Film Bc)


175

Regarding fresh-like appearance (Figure 75 b), increasing scores were given to UV-treated

samples, single application (UV samples), with no differences regarding the absence of treatment

(Ctr samples). Despite the higher (p>0.05) scores found in these samples when packaged with film

A regarding film B, at day 7 scores around 3 (Limit of saleability) were attributed and by the end of

the considered storage period (day 14), scores as high as 5 (No fresh appearance) were attributed

to UV-treated (UV) samples surpassing (p<0.05) the Poor classification of untreated (Ctr) samples.

From day 7 onward, the panel members included an important comment describing UV-treated

(UV) and untreated (Ctr) samples as “slimy looking”, irrespective of MAP conditions. The presence

of a “slimy viscous substance” on the shreds surface has already been described in FC carrot and

attributed to the development of lactic acid bacteria (Carlin et al., 1989), particularly Leuconostoc

mesenteroïdes sp. as reported by Kakiomenou et al. (1996).

Both heat- (HS) and combined-treated (HSxUV) samples were also increasingly scored regarding

fresh-like appearance without however reaching an unacceptable level during storage, with

maximum scores of 2.3 (HS samples) and 2.8 (HSxUV samples), irrespective of packaging

solution.

Loss in aroma intensity was detected by panel members during storage (Figure 75 c) and the

deviation from fresh-like aroma was influenced by applied treatment and the established

atmosphere inside the packages as influenced by the film used for packaging. The effect of the

100 C/45 s heat shock, as single or combined, was significant to best retain this sensorial attribute

during storage regarding other samples with aroma scores always below anchor 3 (Moderate

perception). The influence of the established atmosphere inside the packages of heat- (HS) and

combined-treated (HSxUV) samples was only observed from day 10 onwards, from which samples

packaged with film B had lower (p<0.05) scores than homologous packaged with film A, signifying

a higher fresh-like aroma perception (Figure 75 c).

During storage, significantly higher (p<0.05) aroma scores were always attributed to UV-treated

(UV) samples (with no differences with the untreated control; p>0.05) in regard to heat- (HS) and

combined-treated (HSxUV) samples. At day 7, UV-treated (UV) samples were scored around

anchor 4 (Poor perception of fresh-like aroma), irrespective of MAP condition. By day 10, all panel

members had identified an off-odor described as “fermented” in UV-treated samples packaged with

film A, attributing scores of 5 (Absent), which contrasts with homologous samples packaged with

film B, with lower (p<0.05) scores and no mention to the off-odor. By the end of storage (day 14),

the “fermented” comment was used to describe UV-treated (UV) samples irrespective of packaging

solution. The aroma depreciation found in these samples is consistent with the growth of lactic acid

bacteria (LAB) due to respective fermentative metabolism by-products and also agrees with the

fresh-like appearance depreciation registered in these samples from day 7 onwards. Moreover, the

found difference in these samples aroma classification and identification of the “fermented” off-odor

also subscribe the influence of the established atmosphere composition inside the packages as

anoxic conditions propitious to LAB fermentative metabolism were avoided by the use of a micro-

perforated film (film B).


176

The increase in sample rejection during storage (Figure 76) was correlated (p<0.05) with all

sensorial attributes, and correlation values of 0.86, 0.92 and 0.94 were determined between the

rejection index and color, fresh-like appearance and aroma, respectively. From these correlations it

is suggested that changes in sample appearance and aroma are more determinant to sample

rejection than changes in color (which scores were always below anchor 3 - Moderate white).

According to the panel members, UV-treated (UV) samples were quite acceptable within the first 3

days of storage but, at day 7, these samples were clearly rejected irrespective of MAP conditions.

The increasing rejection level of UV-treated (UV) samples during storage (with no differences with

the untreated control) could be related to the already described depreciation in appearance and

aroma suggesting high counts of the LAB microbial group.

Figure 76 Acceptance level of fresh-cut carrot as affected by abiotic stress treatments and MAP

conditions (set by the films used for packaging) during low temperature storage (5 C, 14 days).

Vertical bars denote the confidence interval at 95%. [ Ctr: untreated; HS: heat-treated (100 C/45 s); UV:

UV-treated (2.5 kJ.m-2); HSxUV: combined-treated (100 C/45 s x 2.5 kJ.m-2); Rejection index scale: 1 – Excellent; 2 – Good; 3 - Limit of marketability; 4 - Poor and 5 - Unusable].

In contrast, heat- (HS) and combined-treated (HSxUV) samples (p>0.05) were always preferred by

the panel members irrespective of MAP conditions, and only samples packaged with film A

reached the marketability limit by day 14 (mean scores of 2.9 and 3.1, respectively; p>0.05),

situation that never occurred when packaged with film B (mean scores of 2.5 and 2.6, respectively;

p>0.05). From these results, it is clear that higher FC carrot sensorial quality is achieved by the use

of the 100 C/45 s heat shock treatment due to the lower product respiration rate and possible

effects in controlling LAB development, regardless the film used for packaging.


0 3 7 10 14

1

2

3

4

5

Re

jec

tio

n I

nd

ex

0 3 7 10 14


Film A Film B


177


The decontamination efficiency of the abiotic stress treatments, estimated by taking into account

the respective counts at day 0, is summarized in Table 31. Initial mesophilic counts (TAPC) of 5.8

Log10 were found in the raw material (Table App. 18, appendix 3) and after processing (day 0), an

expressive averaged reduction on mesophilic counts was found in untreated (Ctr) samples

(reduction of 1.6 Log10, Table 31) which ascribes to the peel removal operation a significant effect

to reduce initial microbial contamination as previously demonstrated (Part IV, section 1.5).

Table 31 Decontamination efficiency of abiotic stress treatments (heat shock, UV-C and respective combination).

Abiotic stress treatment* Initial counts (day 0; Log10 cfu.g-1) Decontamination efficiency** (Log10 cfu.g-1)

TAPC LAB Y&M TAPC LAB Y&M

Ctr 4.2d±0.2 2.6c±0.1 3.3c±0.2 1.6±0.2 0.9±0.1 0.1±0.1

HS

[100 C/45 s] 2.5b±0.2 0.0a±0.0 0.0a±0.0 1.7c±0.2 2.6c±0.1 3.3b±0.2

UV

[2.5 kJ.m-2] 3.8c±±0.2 2.1b±0.2 3.1b±0.2 0.5b±0.3 0.5b±0.2 0.2a±0.2

HSxUV

[100 C/45 s] x [2.5 kJ.m-2] 1.8a0.4 0.0a±0.0 0.0a±0.0 2.4d±0.3 2.6c±0.1 3.3b±0.2

*Reference to the control (Ctr) sample is made in order to quantify the peel removal effect on the reduction of contaminating flora, regarding raw material counts (Table App. 18, appendix 3); **Decontamination efficiency was estimated taking into consideration the peeling effect (reference taken as Ctr sample). Values represent the mean±SD of three replicates. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

UV-C stress treatment was considered in this study as a phenolic synthesis promoter but still a

decontamination effect was thought to be associated with this treatment application as referenced

by other authors (Allende et al., 2006 b). In spite of described UV-C treatments effects on reducing

mesophilic counts (Allende and Artés, 2003 a,b), no significant decontamination effect was found in

UV-treated (UV) samples registering only a 0.5 Log10 reduction in TAPC regarding the absence of

treatment (Ctr sample). The low decontamination efficiency could be explained by the uneven

surface topography (shreds) of the product, shielding microorganisms from incident UV.

The 100 C/45 s heat shock aptitude to effectively reduce the initial TAPC load is again

demonstrated by the attained decrease (p<0.05) found in heat-treated samples: taking into account

the initial reduction provided by peel removal (Ctr samples), the heat shock single application (HS

samples) was still able to further reduce the contamination levels by 2 Log10. An additive effect

(p<0.05) was found when heat shock was combined with UV-C treatment and a 2.4 Log10 TAPC

reduction over the peeling effect (Table 31) was found in combined-treated (HSxUV) samples.

In raw material (Table App. 18, appendix 3), mean LAB counts of 3.5 Log10 were determined and,

just after minimal processing (day 0), lactic acid bacteria count (LAB) in untreated (Ctr) samples

recorded a 0.9 Log10 reduction showing again that peeling operation provides a 1 Log10 reduction

in contaminating lactic flora. UV-C treated (UV) samples registered a similar LAB reduction


178

efficiency to that of TAPC, of 0.5 Log10 (Table 31), pointing to the low efficiency of the UV-C

treatment as a single hurdle towards fresh-cut carrot decontamination. In contrast, LAB counts in

heat- (HS) and combined-treated (HSxUV) samples were below the detection limit (<101 cfu.g

-1)

expressing the sensitivity of this microbial group to heat (Breidt and Costilow, 2004).

Initial mean Y&M counts were of 3.2 Log10 (raw material, Table App. 18, appendix 3) and after

processing (day 0), only untreated (Ctr) and single UV-treated (UV) samples, registered Y&M

counts which were of the same order than that of the raw material (3 Log10, Table 31). Heat- (HS)

and combined-treated (HSxUV) samples had counts below the quantification limit (<101 cfu.g

-1),

demonstrating the heat-sensitivity of this microbial group and the generally recognized destruction

of Y&M at treatment temperatures above 60 C-70 C (Breidt and Costilow, 2004).

The development TAPC, LAB and Y&M during storage as influenced by stress treatments and

established gaseous compositions inside the packages as set by used films are shown in Figure

77 a, b and c, respectively.

Increases in TAPC counts were found during storage in all evaluated samples (Figure 77 a) which

were significantly influenced by applied stress treatment. TAPC growth was unaffected by the

single application of the 2.5 kJ.m-2

UV-C treatment (UV samples) (no differences [p>0.05] with the

untreated control) and after 7 days of storage UV-treated samples had already reached the critical

microbial criterion (TAPC mean counts of 7.5 Log10 cfu.g-1

), irrespective of MAP conditions

(p>0.05). The rapid TAPC outgrowth registered in UV-treated samples demonstrates that the use

of the selected dose (2.5 kJ.m-2

) to promote phenolic synthesis is inadequate to effectively control

microbial development during storage of shredded carrot.

The initially found additive effect of heat shock and UV-C treatment combination was not

maintained during storage (Figure 77 a) and no differences (p>0.05) were found between heat-

(HS) and combined-treated (HSxUV) samples, irrespective of packaging film. Nevertheless, both

sample types registered 2.0-2.5 Log10 lower (p<0.05) TAPC counts regarding UV-treated samples

maintaining that difference throughout storage. Heat- (HS) and combined-treated (HSxUV)

samples never reached the critical microbial criterion (maximum counts of 7 Log10 cfu.g-1

)

endorsing the 100 C/45 s heat-shock as a highly effective treatment to reduce and control

microbial development in fresh-cut shredded carrot, assuring products microbial quality for a 14-

day period.

The outgrowth of LAB during storage was observed in all sample types, but the increase rate was

significantly influenced by MAP conditions and applied treatment. The significant influence of the

film used for packaging in LAB counts is clearly demonstrated in untreated (Ctr) samples, were

lower counts (in about 1 Log10 cfu.g-1

) are found in samples packaged with film B (Figure 77 b) from

day 7 onwards regarding untreated samples packaged with film A. This inhibitory effect attributed

to the used packaging film relates to the existence of micro-perforations in film B preventing CO2

accumulation to levels favorable to the anaerobic metabolism of LAB which had a significant impact

regarding sample sensorial evaluation.


179

Figure 77 Microbial development of total aerobic plate counts (TAPC, a), lactic acid bacteria counts (LAB, b) and yeast and molds counts (Y&M, c) of fresh-cut carrot as affected by abiotic stress treatments and MAP conditions (set by the films used for packaging) during low temperature storage




0 3 7 10 140

1

2

3

4

5

6

7

8

9

10

11

TA

PC

(L

og

10 c

fu.g

-1)

0 3 7 10 14

Threshold limit

Film A Film Ba)


0 3 7 10 140

1

2

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4

5

6

7

8

9

10

11

LA

B (

Lo

g1

0 c

fu.g

-1)

0 3 7 10 14

Film A Film B

<10 cfu.g-1 HS & HSxUV

b)

Threshold limit

<10 cfu.g-1 HS & HSxUV


0 3 7 10 140

1

2

3

4

5

6

7

Y&

M (

Lo

g1

0 c

fu.g

-1)

0 3 7 10 14

Film A Film B

<10 cfu.g-1 HS & HSxUV <10 cfu.g-1 HS & HSxUV

c)


180

LAB growth was unaltered by the single application of the 2.5 kJ.m-2

UV-C treatment (UV samples;

Figure 77 b) and similar (p>0.05) counts regarding the untreated (Ctr) samples are found, except

on day 7 regarding samples packaged with film B.

Although the single application of heat shock or even the combination with UV-C treatment resulted

in LAB counts to levels below the quantification limit after processing (day 0), respective outgrowth

during storage was registered (Figure 77 b), higher (p<0.05) in samples packaged with film A

(maximum counts of 5.3 and 6.4 Log10 cfu.g-1

for HS and HSxUV samples, respectively, p>0.05)

than in samples packaged with film B (maximum counts of 3.4 and 5.8 Log10 cfu.g-1

for HS and

HSxUV samples, respectively, p>0.05). Despite onset development, considerable lower counts (up

to 3 Log10 cfu.g-1

) were determined after 14 days of storage in heat-treated and combined-treated

(HS and HSxUV) samples in comparison with UV-treated (UV) or untreated (Ctr) samples,

contributing to the relevant effects of heat shock as to prevent accelerated microbiological growth.

During storage, no differences (p>0.05) were found between Y&M counts of untreated (Ctr)

samples packaged with film A or B (Figure 77 c), demonstrating a lack of effect of established

gaseous composition inside the packages. Applied treatment had however a significant effect

regarding Y&M development: heat- (HS) and combined-treated (HSxUV) samples had during the

entire storage period Y&M counts below the quantification limit (<101 cfu.g

-1), demonstrating the

effectiveness of the 100 C/45 s heat shock to the elimination of this heat-sensitive microbial group,

supporting previous findings (Alegria et al., 2010; Alegria, 2007). No significant effect to control the

development of this microbial group was found regarding the application of the 2.5 kJ.m-2

UV-C

treatment (UV samples) and counts up to 5 Log10 cfu.g-1

(with no differences [p>0.05] with the

untreated control sample) were found after 14 days of storage, irrespective of MAP conditions. The

increase of Y&M population in these samples could also have a significant impact to the

established modified atmosphere inside the packages, contributing to the increase of CO2

concentrations and, in turn, favoring LAB development which has sensorial implications.

Overall, it is possible to state that the 100 C/45 s heat shock allied to the use of a micro-perforated

film is an effective hurdle combination as to prevent microbiological spoilage during storage of FC

carrot.

3.1.1.12 Hierarchical cluster and principal component analyses

Hierarchical cluster and principal component analyses were used to establish a relationship

between abiotic stresses, MAP conditions (as set by film used for packaging) and FC carrot quality

evolution during storage (0-14 days). The data set included eight categories of variables

identifying untreated (control; C), heat-treated (H), UV-treated (U) and combined-treated (HU)

samples packaged in two film types (A and B). Quantitative variables corresponded to gas

composition (O2 and CO2), microbiological data (TAPC, LAB and Y&M), pH, total phenolic content

(TPC), PAL activity (PAL), and sensorial rejection scores (Rejection). The resulting data matrix

contained 56 samples and 9 variables codified as shown in Table App. 33 (appendix 3).


181

Principal components analysis allowed to explain 76.72% of the original data variability in the first

two dimensions (principal components, PC, 1 and 2) which is considered adequate to define a

good model for qualitative purposes since a significant percentage of original information (>70%)

was accumulated within the first two PC’s (Larrigaudière et al., 2004).

The PC1 accounted for the highest proportion of the variation (55%) in the data and was most

heavily loaded with microbiological counts, pH, sensorial rejection and gaseous composition inside

the packages. As it can be seen in Figure 78 a, the variables vector projection on PC1 indicates

that sensorial rejection was significantly dependent on microbial development (TAPC, LAB and

Y&M), all positively loaded with PC1. Within the studied microbial groups it can be stated that LAB

accounted the most for TAPC counts, as shown by the respective vector projection juxtaposition on

PC1. The development of this microbial group contributed heavily to deteriorative processes,

relevant FC carrot quality changes, leading to product rejection as illustrated by the almost

overlapping vector angles of LAB and rejection variables. Additionally, from the observed variable

correlations with PC1, it was found that LAB growth was favored by the establishment of anaerobic

conditions as illustrated by the proximity between the LAB and CO2 vectors. This relation had

significant implications on product acidification as shown by the negatively loaded pH vector on

PC1.

The phenolic synthesis dynamic (represented by TPC and PAL variables) is described by PC2

(accounting for 22% of data variability) and the weak TPC and PAL vector projection on PC1

indicates that changes in these variables do not have any sensorial implications. The oxygen-

dependence of phenolic synthesis mechanism is suggested by the location of both TPC and PAL

vectors in regard to the O2 vector (Figure 78 a) on the 2nd

quadrant (Figure 78 a).

Thus, a consistent trend in the loading plot is shown. Variable loadings on PC1 and PC2,

respectively, measure independent mechanisms responsible for FC carrot quality changes during

storage: PC1 refers to sensorial quality loss promoted by microbial growth and PC2 describes the

phenolic synthesis dynamic. It is interesting to notice that both mechanisms are significantly

influenced by O2 and CO2 concentrations and therefore influenced by the film used for packaging.

Despite the interdependence of O2/CO2 concentrations, microbiological growth is directly

influenced by CO2 concentration while changes in O2 concentration are critical to the phenolic

synthesis dynamic. Higher CO2 concentrations lead to higher LAB counts, sensorial rejection and

limit phenolic synthesis during storage suggesting that a compromise solution could be found

regarding FC carrot microbiological, sensorial and bioactive quality, addressing the importance of

knowing O2/CO2 thresholds levels which limit/promote those phenomena.


182

Figure 78 Principal component analysis of fresh-cut carrot as affected by abiotic stress treatments

and packaging film during low temperature storage (5 C, 14 days) for a) the quality parameters (loading plot) and b) samples in study (score plot). [ CAi: untreated samples packaged with film A; CBi:

untreated samples packaged with film B; HAi: heat-treated (100 C/45 s) samples packaged with film A; HBi: heat-

treated (100 C/45 s) samples packaged with film B; UAi: UV-treated (2.5 kJ.m-2) samples packaged with film A; UBi:

UV-treated (2.5 kJ.m-2) samples packaged with film B; HUAi: combined-treated (100 C/45 s x 2.5 kJ.m-2) samples

packaged with film A; HUBi: combined-treated (100 C/45 s x 2.5 kJ.m-2) samples packaged with film B; i stands for storage day].

TPC

PAL

O2

CO2

TAPC

LAB

Y&M

pH

Rejection

-1 0 1

PC1: 55.21%

-1

0

1

PC

2:

21

.51

%

TPC

PAL

O2

CO2

TAPC

LAB

Y&M

pH

Rejection

a)

CA0CA3

CA5

CA7

CA10

CA12 CA14

CB0

CB3CB5

CB7

CB10

CB12

CB14

HA0 HA3

HA5 HA7

HA10

HA12HA14

HB0

HB3 HB5

HB7

HB10

HB12

HB14

UA0

UA3

UA5UA7

UA10UA12

UA14

UB0

UB3

UB5 UB7

UB10

UB12UB14

HUA0

HUA3

HUA5

HUA7

HUA10HUA12

HUA14

HUB0

HUB3HUB5

HUB7

HUB10HUB12

HUB14

-5 -4 -3 -2 -1 0 1 2 3 4 5 6

PC1: 55.21%

-3

-2

-1

0

1

2

3

4

PC

2:

21

.51

%

CA0CA3

CA5

CA7

CA10

CA12 CA14

CB0

CB3CB5

CB7

CB10

CB12

CB14

HA0 HA3

HA5 HA7

HA10

HA12HA14

HB0

HB3 HB5

HB7

HB10

HB12

HB14

UA0

UA3

UA5UA7

UA10UA12

UA14

UB0

UB3

UB5 UB7

UB10

UB12UB14

HUA0

HUA3

HUA5

HUA7

HUA10HUA12

HUA14

HUB0

HUB3HUB5

HUB7

HUB10HUB12

HUB14

b)

Group B

B1

Group A

A1

A2

B2


183

The projection of analyzed samples in the space of PC1 and PC2 is shown in Figure 78 b and the

distinction between the samples was clearly visible as the points were well-spread within the

scatter plot. Irrespective of treatment or used packaging film, all samples moved in a similar pattern

during storage indicating progressive quality loss and increased rejection, as samples from day 0

are located to the far left of the PC1 and PC2 space and subsequent dates spread to the right

(Figure 78 b). Within the score plot, sample grouping as indicated by the hierarchical cluster

analysis dendrogram (Figure App. 11, appendix 3) is also shown. According to this sample

grouping, the first encountered division, groups A and B, is made over PC1 as is related with the

first identified mechanism. This division suggests that FC carrot overall quality during storage is

firstly impaired by changes in sensorial. With the second encountered division (groups A1, A2, B1

and B2) the treatments and packaging film effects over the two independent mechanisms

responsible for FC carrot quality changes during storage are highlighted. The sample grouping

provided by this analysis indicates heat shock combined with film B as the technological choices

that simultaneously achieve the objectives of promoting product bioactivity and best preserve FC

carrot sensorial quality.

Exploring sample placing in the space of PC1 and PC2 (Figure 78 b), UV-treated (UV) and

untreated (Ctr) samples lie very close to each other considering sampling dates (from days 0 to

14), signifying a lack of effect from the UV treatment to prevent microbiological development.

Conversely, on the negative part of the axis defined by PC1, the relative positioning of heat- and

combined-treated samples, up to day 10 (film A) or 14 (film B), and of UV-treated samples up to

day 3 (film A) or 5 (film B), shows the significant effects attributed to heat shock in controlling the

onset microbial development. Heat shock effects regarding initial decontamination are also

demonstrated by the relative positioning of heat-treated samples towards untreated and UV-treated

samples at day 0.

Sample positioning in regard to the axis defined by PC2 (Figure 78 b) shows the distinction

between samples packaged with film B regarding homologous samples packaged with film A

(positive and negative sections of the axis, respectively). This sample placement confirms the

importance of oxygen levels to phenolic accumulation during storage, irrespective of applied

treatment.

The multivariate approach confirms that the 100 C/45 s heat shock treatment has a multi-target

effect of reducing the incidence of bacterial proliferation, preserving the sensorial characteristics

and improving the bioactive quality. These effects are further enhanced when combined with the

use of a micro-perforated packaging film (film B).


184

3.2 PROPOSED TECHNOLOGICAL ALTERNATIVE: EFFECTS ON FRESH-CUT CARROT QUALITY

AND SHELF-LIFE

It was aimed to evaluate the effects of the 100 C/45 s heat shock in comparison to the standard

200 ppm x 1 min chlorine decontamination procedure regarding product quality during two MAP

storage conditions. This assessment was achieved by comparing heat-treated (HS) and chlorine-

treated (HIPO) samples packaged with film A and B (refer to the previous section) and product

shelf-life associated with the best technological alternative was estimated.



Changes in atmosphere composition (O2 and CO2 concentrations, %) achieved passively during

storage under the influence of the film used for packaging of heat- and chlorine-treated samples

are shown in Figure 79.

The atmosphere composition established inside the packages during storage of heat-treated (HS)

samples were more favorable (p<0.05) to quality maintenance (higher O2/lower CO2) than that

registered in chlorine-treated (HIPO) samples, regardless the film used for packaging. Steady-state

conditions were not attained (chlorine-treated samples) or were achieved late during storage ( 10

days; heat-treated samples) when samples were packaged using film A (Figure 79 a). On the other

hand, when samples were packaged with film B ((Figure 79 b), steady-state conditions were

established earlier in time ( 3 days) in both sample types, but with higher/lower O2/CO2 levels

being registered in heat-treated (HS) samples in comparison with chlorine-treated (HIPO) samples.

The existing micro-perforations increased the film permeability allowing to reach equilibrium

between the flux of O2 and CO2 due to product respiration and the flux due to film permeation.

Moreover, the heat shock treatment reduced the product respiration rate and, as pointed out by the

PCA analysis (Part IV, section 3.1.1.12), that effect significantly contributes to the products’ quality

maintenance during storage, particularly when combined with the use of the micro-perforated film

which increases gas transfer through the package and establishes a favorable gaseous

composition (O210% and CO210%) inside the packages.


185

Figure 79 Changes in atmosphere composition ( O2 and CO2 concentrations, %) of shredded carrot samples packaged with a) film A and b) film B as affected by heat shock stress

treatment and chlorine (industrial standard) during low temperature storage (5 C, 14 days). Vertical

bars denote the confidence interval at 95%. [HS: heat-treated (100 C/45 s); HIPO: chlorine-treated (200 ppm/1 min)].


In Figure 80, the changes in total phenolic content (TPC) induced by heat shock treatment (HS

samples), chlorine decontamination treatment (HIPO samples) and packaging film (film A vs. film

B) are shown. Just after processing (day 0), the effect of heat shock over this response is clear in

regard to a higher retention of phenolic compounds, independently of packaging film (54 vs.

35 mg CAE.100 g-1

, HS vs. HIPO samples, respectively, p>0.05). Whilst non-significant, and

Film A


0 3 5 7 10 12 140

5

10

15

20

25

30

35

40

45

50

55

Ga

s c

on

ce

ntr

ati

on

(%

)

0 3 5 7 10 12 14

a)

HS samples HIPO samples

Film B


0 3 5 7 10 12 140

5

10

15

20

25

30

35

40

45

50

55

Ga

s c

on

ce

ntr

ati

on

(%

)

0 3 5 7 10 12 14


b)


186

besides the already discussed peeling effect, the lower TPC levels of HIPO samples could be

mainly attributed to leaching phenomena as samples are immersed in chlorinated water after

shredding which favors the phenomena (Part IV, section 1.5).

Figure 80 Changes in total phenolic content (TPC) of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films used for


95%. [ HS: heat-treated (100 C/45 s); HIPO: chlorine-treated (200 ppm/1 min)].

During storage and in general, heat-treated (HS) samples had always higher TPC levels than

chlorine-treated (HIPO) samples. Taking into consideration the used packaging film during storage,

and as mentioned, samples packaged with film B (PM-MAP) showed to have a higher phenolic

accumulation than that registered in samples packaged with film A (conventional MAP).

Irrespective of packaging film, heat-treated (HS) samples at day 7 had already matched the raw

material TPC content (85.3 mg CAE.100 g-1

, Table App. 18, appendix 3) while in chlorine-treated

(HIPO) samples that situation was only registered when samples were packaged with film B.

Furthermore, only heat-treated (HS) samples packaged with film B were able to maintain TPC

levels compatible with the raw material from day 7 onwards. In contrast, chlorine-treated (HIPO)

samples registered a significant decrease after day 7 compromising the products’ value regarding

this bioactive marker, regardless packaging film.

These results support the importance of established atmosphere conditions inside the packages

over phenolic synthesis to which, despite the introduced delay to the synthesis dynamic (refer to

the previous section) the 100 C/45 s heat shock also contributes to maintain adequate O2/CO2

concentrations to promote phenolic accumulation during longer storage periods and to minimize

respective degradation/consumption.

Storage time (days) HS HIPO (industrial reference)

0 3 5 7 10 12 140

20

40

60

80

100

120

140

160

180T

PC

(m

g C

AE

.10

0 g

-1)

0 3 5 7 10 12 14

Film A Film B

Raw material


187

Changes in chlorogenic acid (CA) as a result of heat shock treatment, chlorine decontamination

treatment and MAP/PM-MAP are shown in Figure 81. At day 0, no differences were found between

CA content of heat- (HS) or chlorine-treated (HIPO) samples (p>0.05), irrespective of packaging

film and a significant difference is registered to the raw material (Table App. 18, appendix 3). The

registered initial losses in CA were of the same order as the one previously registered in untreated

samples (up to 80%, Part IV, section 3.1.1.2) indicating that losses in this content (due to thermal

degradation or oxidation) were not further increased by the application of either treatment and

could be attributed to the peeling operation effect. However, changes in CA contents during

storage were dependent on the established atmosphere inside sample packages and higher CA

levels were determined in samples packaged in film B than in film A, which is consistent with TPC

variations and by the low O2 levels registered in samples packaged with film A.

Figure 81 Changes in chlorogenic acid (CA) of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films used for packaging)

during low temperature storage (5 C, 14 days). Vertical bars denote the confidence interval at 95%. [ HS:

heat-treated (100 C/45 s); HIPO: chlorine-treated (200 ppm/1 min)].

During storage, no changes in sample initial CA levels were determined under the influence of film

A: CA content of heat-treated (HS) samples varied between 2.70 and 2.89 mg.100 g-1

(p>0.05) and

in chlorine-treated (HIPO) samples from 2.29 to 2.87 mg.100 g-1

(p>0.05). However, under the

influence of film B, significant increases in CA were registered in both sample types during storage.

When packaged in film B, heat-treated (HS) samples started to significantly accumulate CA from

day 3 till the end of storage, where increases of 5.6 times the initial content were registered (from

3.77 to 21.16 mg.100 g-1

). Chlorine-treated (HIPO) samples also registered significant increases

from day 3 (3.99 mg.100 g-1

) to 10 (7.60 mg.100 g-1

) of 1.9 times, maintaining those levels to the

last day of storage (8.15 mg.100 g-1

, p>0.05). The raw material CA content (12.30 mg.100 g-1

) was


0 3 5 7 10 12 140

2

4

6

8

10

12

14

16

18

20

22

24

CA

(m

g.1

00

g-1

)

0 3 5 7 10 12 14

Film A Film B

Raw material


188

only matched by heat-treated (HS) samples at day 10, and the further CA synthesis increased this

content by 42% (at day 14). This result supports the already discussed O2 influence over induced

phenolic synthesis and also suggests that, although CA is the main synthetized phenolic, the found

TPC decreases at the later storage period (irrespective of sample type) are not of this particular

phenolic compound.

3.2.1.3 Total carotenoid and -carotene content

Regarding the changes in total carotenoid content, Figure 82, both treatments induced a decrease

in raw material TCC (18.2±0.3 mg -carotene eq.100 g-1

) just after processing (day 0), however

dependent on treatment nature: heat-shock treatment promoted a 15% decrease regarding the

raw material carotenoid content while chlorine treatment more than doubled that reduction (37%).

The higher losses (p<0.05) registered in chlorine-treated (HIPO) samples could be related to the

treatment procedure itself (post-cut decontamination with an oxidizing agent) which promotes

leaching and oxidation phenomena as previously characterized (Part IV, section 1.5). It is therefore

reasonable to assume that the heat shock treatment, as a pre-cut decontamination solution, best

preserves this composition.

Figure 82 Changes in total carotenoid content (TCC) of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films used for



The modified atmosphere developed inside the packages established by used packaging film had a

non-significant effect over TCC levels since no differences were found between samples packaged

with film A or film B during storage. The observed changes were dependent on applied treatment

and, in general, heat-treated (HS) samples show higher mean TCC levels than chlorine-treated


0 3 5 7 10 12 140

5

10

15

20

25

TC

C (

mg

-c

aro

ten

e e

q.1

00

g-1

)

0 3 5 7 10 12 14

Film A Film B

Raw material


189

(HIPO) samples, but only occasionally with statistical significance. At day 14, heat-treated (HS)

samples had significantly higher TCC levels (1.5 times) than chlorine-treated (HIPO) samples,

irrespective of packaging film. To this bioactive response, besides the already established relative

stability during storage, heat shock treatment was more suitable to minimize losses attributed to

minimal processing as well as to ensure maintenance of higher carotenoid levels during storage.

To -carotene content (Figure 83), the raw material level (16.43±0.85 mg.100 g-1

) was not affected

either by treatment or packaging film after processing (day 0). This result seems strange since

decreases in total carotenoid content were found after processing, higher in chlorine-treated

samples, but no significant correlation (<0.1) was determined between TCC and -carotene

content.

Figure 83 Changes in -carotene content of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films used for packaging)



Irrespective of treatment and packaging film, the determined concentrations described an

inconsistent behavior during storage, with -carotene levels ranging from 11 to 20 mg.100 g-1

(regardless significance). Consequently, to -carotene changes in fresh-cut carrot during storage,

no differences are found regarding treatment or developed modified atmosphere inside the

packages.


The antioxidant capacity of fresh-cut carrot show significant differences as a result of applied

treatment and modified atmosphere imposed by packaging films during storage (Figure 84).


0 3 5 7 10 12 140

5

10

15

20

25

30

-c

aro

ten

e (

mg

.10

0 g

-1)

0 3 5 7 10 12 14

Film A Film B

Raw material


190

Despite the reduced TPC and TCC contents of both treated samples at day 0, AOxH and AOxL

levels after processing (day 0) were similar to the ones determined in raw material (66.7±6.2 and

28.8±2.8 mg TE.100 g-1

, respectively) without apparent reason.

Figure 84 Changes in a) hydrophilic and b) lipophilic antioxidant capacity of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by

the films used for packaging) during low temperature storage (5 C, 14 days). Vertical bars denote the

confidence interval at 95%. [ HS: heat-treated (100 C/45 s); HIPO: chlorine-treated (200 ppm/1 min)].

As for the AOx hydrophilic component (Figure 84 a), respective changes during storage were

dependent on both treatment type and developed modified atmosphere as influenced by packaging

film. Regarding packaging solution, the registered changes in AOxH during storage were


0 3 5 7 10 12 1420

40

60

80

100

120

140

160

180

200A

Ox

H (

mg

TE

.10

0 g

-1)

0 3 5 7 10 12 14

Film A Film Ba)

Raw material


0 3 5 7 10 12 140

5

10

15

20

25

30

35

40

AO

xL

(m

g T

E.1

00

g-1

)

0 3 5 7 10 12 14

Film A Film Bb)

Raw material


191

significantly higher in samples packaged with film B regarding samples packed with film A,

agreeing with the described behavior of phenolic accumulation (TPC and CA) in FC carrot during

storage.

As for treatments influence on AOxH, a peak in respective levels was registered between days 5 to

10 in heat-treated (HS) samples, irrespective of packaging film, while in chlorine-treated (HIPO)

samples that behavior was only matched in samples packaged with film B. Moreover, during the full

storage period and irrespective of packaging film, the raw material AOxH levels were kept (and

surpassed (p<0.05) in the 5-10 day time-frame) in heat-treated (HS) samples, condition that was

only observed in chlorine-treated (HIPO) samples packaged with film B.

A relative stability in AOxL (Figure 84 b) was to be expected during storage as no noteworthy

changes in TCC or -carotene content were found. In fact, apart from the significant decrease in

AOxL from day 0 to 3 regardless treatment (p>0.05) or packaging film (p>0.05), maintenance in

this component was found during the remaining storage period (15-19 mg TE.100 g-1

in HS

samples and 13-15 mg TE.100 g-1

in HIPO samples).

From the results, and seeing as for total antioxidant capacity the hydrophilic component is the

major contributor, the 100 C/45 s heat shock combined with film B as packaging solution assures

a better maintenance of total AOx during storage.


The effects of heat shock and chlorine treatments combined with MAP and PM-MAP on stress

related enzymes activities, phenylalanine ammonia lyase (PAL) of fresh-cut carrot are shown in

Figure 85.

The dependency of PAL activity changes on the established modified atmosphere conditions

imposed by packaging film was already discussed and is again evident in Figure 85: samples

packaged with film B (PM-MAP) show higher PAL activity increases (p<0.05) to day 3 than that

observed in samples packaged in film A (conventional MAP). During storage, the changes in PAL

activity were also significantly influenced by the modified atmosphere conditions and again the

distinct change patterns previously characterized were observed: after day 3, PAL activity declines

to initial levels in samples packaged with film A and further activity increases were determined in

samples packaged with film B reaching maximums from day 10 onwards. The fast establishment of

anoxic conditions when using film A and in opposition to the maintenance of higher O2

concentrations facilitated by the existing micro-perforations in film B (as previously demonstrated;

Part IV, section 3.1.1.1), inhibit the de novo synthesis of PAL which impaired phenolic synthesis

during storage.


192

Figure 85 Changes in phenylalanine ammonia lyase (PAL) activity of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films

used for packaging) during low temperature storage (5 C, 14 days). Vertical bars denote the confidence

interval at 95%. [ HS: heat-treated (100 C/45 s); HIPO: chlorine-treated (200 ppm/1 min)].

Treatment effects are also observable (Figure 85) and heat-treated (HS) samples had almost

double the PAL activity level found on chlorine-treated (HIPO) samples at day 3 in both packaging

conditions, however only with statistical meaning in samples packaged with film B. The higher

activity increase registered in heat-treated samples could be assigned to the heat shock effects in

lowering the product respiration rate which benefits the de novo synthesis of PAL, irrespective of

used packaging film.

Focusing on samples packaged with film B and even though heat-treated samples had always

higher (p<0.05) PAL activity levels (with one exception at day 5) than chlorine-treated samples, the

increase behavior was similar in both samples up to day 10. From this sampling date, heat-treated

samples registered still significant activity increases to day 12 while chlorine-treated maintained the

previous lower (p<0.05) record till the end of storage.

As discussed, the higher PAL activity levels registered in heat-treated samples supports the higher

TPC and CA accumulation in these samples during storage and, when compared with chlorine-

treated samples supports the beneficial effects of heat shock to maintain O2 levels compatible with

phenolic synthesis as a result of PAL activity and/or by inhibiting enzymes that modify or degrade

accumulated phenolic compounds. Moreover, the behavior found in PAL activity levels of chlorine-

treated samples upkeep the raised hypothesis (Part IV, section 2.4) that chlorine could have an

inhibitory effect on plants defense metabolism delaying the de novo synthesis of PAL and

compromising phenolic accumulation.


0 3 5 7 10 12 140

50

100

150

200

250

300

PA

L (

U.1

00

g-1

)

0 3 5 7 10 12 14

Film A Film B

Raw material


193


Figure 86 shows the changes in POD activity during storage as influenced by heat shock and

chlorine treatments combined with MAP and PM-MAP. The significant POD reduction (65%

regarding the untreated control sample) attained by the application of the 100 C/45 s heat shock

was already established in the previous section. When comparing the initial POD activity level of

heat-treated (HS) and chlorine-treated (HIPO) samples also a significant 65% difference is

registered, irrespective of used packaging film. The found difference indicates that the post-cut

chlorine decontamination treatment does not introduce any changes in activity level of this

oxidative enzyme whereas the heat shock treatment promoted a significant partial inhibition.

Figure 86 Changes in peroxidase (POD) activity of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films used for packaging)



During storage and regardless of used packaging film, the initial reduction in POD activity

registered in heat-treated samples was maintained (p>0.05), with one exception at day 10,

supporting once more that the applied heat shock has significant effects over POD synthesis during

storage or by inducing respective thermal inactivation. Concerning changes in POD activity during

storage of chlorine-treated (HIPO) samples, also independent (p>0.05) of used packaging film, a

distinct behavior was found describing an initial activity decrease (p<0.05) to day 5 and then an

increase (p<0.05) to day 7 starting again to decrease to initial levels.

In view of the results, advantages are foreseeable regarding the products phenolic composition

with the use of the heat shock treatment since maintenance of low POD activity levels throughout

storage are achieved possibly preventing phenolic enzymatic degradation.


0 3 5 7 10 12 140

20

40

60

80

100

120

PO

D (

U.g

-1)

0 3 5 7 10 12 14

Film A Film B

Raw material


194

3.2.1.7 pH and soluble solids content (SSC)

The comparison between pH and soluble solids evolution during storage of heat shock and chlorine

samples under the influence of distinct packaging solutions is shown in Figure 87 a and b,

respectively.

Figure 87 Changes in a) pH and b) soluble solids content (SSC) of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films




0 3 5 7 10 12 144

5

6

7

pH

0 3 5 7 10 12 14

Film A Film Ba)

Raw material


0 3 5 7 10 12 144

5

6

7

8

9

10

11

SS

C (

ºBri

x)

0 3 5 7 10 12 14

Film A Film Bb)

Raw material


195

No significant change in pH was found as a result of applied treatment (p>0.05), and similar pH

values were determined up to day 10 (6 pH units) in both samples packaged with film A (Figure

87 a). From day 10 to 12, significant decreases were registered in sample pH, where chlorine-

treated (HIPO) had lower (p<0.05) pH than heat-treated (HS) samples (from 4.9 vs. 5.4 pH units,

respectively). In samples packaged with film B, similar behavior was found, only heat-treated (HS)

samples maintain the respective pH until day 12, decreasing (p<0.05) to day 14 (from 6.0 to 5.5 pH

units), while chlorine-treated (HIPO) samples register this decrease earlier, from day 10 onwards

(from 6.0 to 5.1 pH units, p<0.05).

Product acidification was already related to the LAB outgrowth and is commonly attributed to the

accumulation of respective fermentation by-products (Kakiomenou et al., 1996) and it could be

argued that chlorine decontamination was unable to successfully control LAB development.

Significant differences were registered in sample soluble solids content (SSC) just after processing,

regardless of packaging solution (Figure 87 b). Heat-treated (HS) samples had similar SSC

(8.1±0.1 ºBrix) to that determined in the raw material (8.3±0.1 ºBrix, Table App. 18, appendix 3)

while significant lower SSC was found in chlorine-treated (HIPO) samples (5.0±0.2 ºBrix). Despite

this difference, during storage relative maintenance in respective initials SSC was found in both

sample types, independently of the distinct modified atmosphere conditions. The maintenance in

SSC initial values of heat-treated samples in both MAP conditions could be explained by treatment

application prior to shredding, preventing the known and significant compound leaching effect

resulting from post-cut decontamination processes (up to up to 60% in SSC, Alegria et al., 2009).

3.2.1.8 Color

In Figure 88, the whiteness index (WI) values of fresh-cut carrot as affected by the heat shock and

chlorine treatments and packaging films during storage are shown. Just after processing (day 0),

no differences in WI (p>0.05) were detected between sample types.


196

Figure 88 Changes in color, expressed as whitening index (WI), of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference) and MAP conditions (set by the films



No significant changes in WI values during storage (mean values between 27.2 and 28.0 WI units,

p>0.05) were registered in heat-treated (HS) samples packaged with film A. Although chlorine-

treated samples packaged with the same film had higher WI mean values than heat-treated

samples, significant differences were only detected at the last days of storage (from day 10

onwards), reaching WI values of 31 units. The increase (p<0.05) behavior found in chlorine-

treated samples is consistent with a reversible color change associated to shred surface

dehydration (physical response to stress) since no significant phenolic accumulation was registered

in these samples (Cisneros-Zevallos et al., 1995; Emmambux and Minaar, 2003). Nonetheless,

considering the whiteness index scale proposed by Cisneros-Zevallos et al. (1995), the determined

WI values in chlorine-treated samples packaged with film A, are well fitted into the defined interval

correspondent to the perception of a nonwhite surface.

While packaged with film B, an increase tendency in WI values is perceived (Figure 88), probably

due to a higher surface dehydration, but again, without statistical significance, independently of

sample type. The highest reached WI value, of 31 units, was still well within the above mentioned

interval where samples are considered to have a nonwhite surface.


Changes in the sensorial scores of color, fresh-like appearance and fresh-like aroma of fresh-cut

carrot submitted to heat shock and chlorine decontamination treatments and packaged with two

distinct films are shown in Figure 89 and respective sample rejection index is shown in Figure 90.


0 3 5 7 10 12 1425

26

27

28

29

30

31

32

33

WI

0 3 5 7 10 12 14

Film A Film B


197

Increasing (p<0.05) color scores were registered in both samples during storage, irrespective of

packaging film. Chlorine-treated (HIPO) samples (Figure 89) showed the highest color scores and

by the 14th day scores of 3 (moderate white) were attributed, relating to white perception on the

shreds surface. Since phenolic levels in chlorine-treated samples were significantly lower than

those registered in heat-treated samples, the white perception could therefore be related to a more

pronounced superficial dehydration in chlorine-treated samples than in heat-treated samples

(Figure 89), irrespective of packaging film.

Figure 89 Sensorial attributes scores of shredded carrot as affected by heat shock stress treatment

(100 C/45 s) and chlorine (industrial standard; 200 ppm/1 min) and MAP conditions (set by the films

used for packaging – a) Film A; b) Film B) during low temperature storage (5 C, 14 days). Vertical

bars denote the confidence interval at 95%.[ Color: 1 - Nonwhite; 2 - Slightly white; 3 - Moderate white; 4 - Severe

white; 5 - Extreme white, corresponding carrot fresh color to anchor 1; Fresh-like appearance: 1 - Excellent/fresh appearance; 2 - Moderate; 3 - Limit of saleability; 4 - Poor; and 5 - No fresh appearance, where anchor 1 matches to the

perception of the fresh-cut untreated carrot immediately after cutting; Fresh-like aroma: 1 - Very intense, 2 - Intense; 3 - Moderate; 4 - Low; and 5 - Absent, where anchor 1 corresponds to the perception of the carrots aroma immediately after cutting].

Film A

Storage time (days) Color Fresh-like appearance Fresh-like aroma

0 3 7 10 14

1

2

3

4

5

Se

ns

ori

al

sc

ore

0 3 7 10 14

HIPO samplesHS samples

a)

Film B

Storage time (days) Color Fresh-like appearance Fresh-like aroma

0 3 7 10 14

1

2

3

4

5

Se

ns

ori

al

sc

ore

0 3 7 10 14


b)


198

Regarding fresh-like appearance, during storage samples also denoted losses in this attribute,

more pronounced in chlorine-treated samples. By day 7, chlorine-treated samples had already

reached a score correspondent to “Limit of saleability” (Figure 89 a) and panel comments indicated

the presence of a “slimy substance” on chlorine-treated samples packaged with film A whereas

heat-treated samples only surpassed anchor 2 (moderate fresh-like appearance) by the 14th day

even when packaged with film A (Figure 89 a). Chlorine-treated samples continued to receive

increasingly higher scores during the remaining storage period, reaching mean scores close to 5

(No fresh appearance), especially when packaged with film A (Figure 89 a). As previously

discussed, this considerable loss in fresh-like appearance attributed to a “slimy substance” is

related to the outgrowth of LAB bacteria.

The aroma attribute was also increasingly lost during storage (Figure 89). Heat-treated samples

showed however a better aroma retention, reaching scores between 2.9 and 2.5 for samples

packaged with film A (Figure 89 a) and B (Figure 89 b) at day 14, respectively. Just after

processing (day 0) chlorine-treated samples had a significant deviation from the characteristic

carrot aroma with samples being scored close to 3 (Moderate) and with the detection of an off-odor

by panel members, identified as “disinfectant”. Increasing depreciation in aroma was registered

during storage and, by day 7, chlorine-treated samples packaged with film A were negatively

appreciated due to the identified “fermented” off-odor (Figure 89 a). Considering that this off-odor

detection matches the recognition of a “slimy substance” on the shreds, reinforces the deleterious

effects of LAB growth to shredded carrot sensorial characteristics. By the end of the considered

storage period, chlorine-treated samples had an “absent” classification of fresh-like aroma and all

members were consensual identifying the “fermented” scent, irrespective of used packaging film

(Figure 89).

From sensorial attributes depreciation during storage increase in samples rejection index is

foreseeable (Figure 90). Chlorine-treated samples were undoubtedly rejected by day 7, irrespective

of packaging solution whilst heat-treated samples were scored below this marketability limit during

the entire storage period. Nonetheless, heat-treated samples packaged with the micro-perforated

film had a higher acceptance level when compared to homologous samples packaged with the

non-perforated film.


199

Figure 90 Rejection index scores of shredded carrot as affected by heat shock stress treatment and chlorine (industrial standard) and MAP conditions (set by the films used for packaging) during low

temperature storage (5 C, 14 days). Vertical bars denote the confidence interval at 95%. [ HS: heat-treated

(100 C/45 s); HIPO: chlorine-treated (200 ppm/1 min); Rejection index scale: 1 – Excellent; 2 – Good; 3 - Limit of marketability; 4 - Poor and 5 - Unusable].

The achieved benefits regarding color maintenance, fresh-like appearance and aroma retention

during the 14 days of storage, assigns to heat-shock beneficial effects for fresh-cut carrot sensorial

quality maintenance during storage, irrespective of packaging film. Concerning the respective cut-

off limit of products acceptability, chlorine-treated samples provide acceptable sensorial quality for

a period of time under 7 days, while heat-treated samples were always accepted during the

considered 14 days.


As previously discussed, peel removal significantly contributes to reduce the existing contaminating

microflora (Part IV, section 3.1.1.11; Ctr samples; Table 31). For that reason, treatments

decontamination efficiency was therefore estimated after peel removal (counts of 4.2±0.2, 2.6±0.1

and 3.3±0.2 Log10 cfu.g-1

, for TAPC, LAB and Y&M, respectively) and is shown in Table 32.

Table 32 Decontamination efficiency of heat shock (HS) and chlorine (HIPO) treatments.

Initial counts (day 0; Log10 cfu.g-1) Decontamination efficiency (after peeling;

Log10 cfu.g-1)

Treatment TAPC LAB Y&M TAPC LAB Y&M

HS

[100 C/45 s] 2.5a±0.2 0.0a±0.0* 0.0a±0.0* 1.7b±0.2 2.6b±0.1 3.3b±0.2

HIPO [200 ppm/1 min]

3.7b±0.2 1.9b±0.2 3.1b±0.3 0.5a±0.3 0.7a±0.1 0.2a±0.2

*Counts below the quantification limit of 10 cfu.g-1. In the same column different letters represent significant differences at p=0.05 (Tukey HSD test)


0 3 7 10 14

1

2

3

4

5

Re

jec

tio

n I

nd

ex

0 3 7 10 14


Film A Film B


200

To heat shock a significant decontamination efficiency of 2 Log10 was estimated while chlorine

post-cut decontamination was only responsible for a 0.5 Log10 reduction of initial mesophilic

contaminating flora (TAPC; p<0.05). As for lactic acid bacteria (LAB) and yeast and mold (Y&M)

contamination, heat shock provided significant reduction of these microbial groups since counts

below 101 cfu.g

-1 were assessed after processing, demonstrating these microbial groups

susceptibility to heat. On the contrary, chlorine decontamination came far behind this efficiency

achieving a 0.7 Log10 reduction concerning LAB flora and no significant effect was registered

regarding Y&M (0.2 Log10). These results per se indicate that post-cut chlorine decontamination

was inadequate to achieve a desirable microbial reduction (to levels of about 103 cfu.g

-1 according

to Guerzoni et al., 1996) in shredded carrot and also suggest that the heat shock treatment was

more suitable for this purpose.

The effects of treatment and packaging film on the microbiological development during storage of

fresh-cut carrot are shown in Figure 91. Regarding TAPC growth during storage (Figure 91 a), no

significant differences were found between samples under film influence. Concerning treatment

effects, a 1.5 (film A) to 2 Log10 (film B) difference was found between heat-treated and chlorine-

treated samples during storage, with heat-treated samples having lower (p<0.05) counts. From the

depicted behaviors, relevant effects are attributed to heat shock in reducing initial TAPC

contamination which enables significantly lower counts during storage regarding chlorine

decontamination. These effects are relevant concerning product microbiological shelf-life: chlorine-

treated samples packaged with film A, reached the microbial criterion (7.5 Log10 cfu.g-1

) after 7

days of storage whereas heat-treated samples packaged under the same conditions never reached

this threshold during the 14 days of storage. Samples packaged with film B (PM-MAP), the same

estimated microbiological shelf-life (7 days) is assumed for chlorine-treated samples, considering

that counts 7.2 Log10 cfu.g-1

are registered from this point on, reaching counts as high as

9 Log10 cfu.g-1

(day 14). On the other hand, heat-treated samples had reached counts of

6.4 Log10 cfu.g-1

and of 7.2 Log10 cfu.g-1

by day 14 regarding film A and B, respectively.

Significant differences were registered in LAB counts of fresh-cut shredded carrot as a result of

both applied treatments and used packaging solution (Figure 91 b). As previously demonstrated,

the established modified atmospheres inside the packages imposed by used packaging film had a

significant effect on LAB growth during storage: samples packaged with film A (conventional MAP)

registered continuous and significant outgrowth during storage, but below the respective threshold

limit of 8 Log10 cfu.g-1

(counts of 5.3 and 7.2 Log10 cfu.g-1

, p<0.05, by day 14 in heat- and chlorine-

treated samples, respectively); samples packaged with film B (PM-MAP) also registered

progressively higher counts during storage but respective counts were lower (p<0.05) than the

ones found in samples packaged with film A.


201

Figure 91 Microbial development of a) total aerobic plate counts (TAPC), b) lactic acid bacteria counts (LAB) and c) yeast and molds counts (Y&M) of fresh-cut carrot as affected by heat shock stress treatment, chlorine (industrial reference), and MAP conditions (set by the films used for



Srorage time (days) HS HIPO (industrial reference)

0 3 7 10 140

1

2

3

4

5

6

7

8

9

10

11

TA

PC

(L

og

10[c

fu.g

-1])

0 3 7 10 14

Film A Film B

Threshold limit

a)


0 3 7 10 140

1

2

3

4

5

6

7

8

9

LA

B (

Lo

g1

0[c

fu.g

-1])

0 3 7 10 14

Threshold limit

< 10 cfu.g-1 < 10 cfu.g-1

Film A Film Bb)


0 3 7 10 140

1

2

3

4

5

6

Y&

M (

Lo

g1

0[c

fu.g

-1])

0 3 7 10 14

Film A Film B

c)

< 10 cfu.g-1< 10 cfu.g-1 HS HS


202

Considering treatment effects, lower LAB counts (1 to 2 Log10 cfu.g-1

, p<0.05) were registered in

heat-treated samples regarding chlorine-treated samples but only during conventional MAP storage

(use of film A). Despite that no differences in LAB counts were found between HS and HIPO

samples during PM-MAP storage (use of film B), except on days 0 and 14, LAB development

behavior is distinct between samples from day 7 onwards: heat-treated samples show maintenance

of LAB counts till the end of storage while chlorine-treated samples show an increase pattern, but

always below the respective threshold limit. To this microbial group, film nature was of utmost

importance since higher O2 levels were achieved with film B (Part IV, section 3.2.1.1) restraining

the growth of lactic acid bacteria, aerotolerant bacteria with exclusively anaerobic (fermentative)

type of metabolism that thrive in anaerobic conditions.

As previously shown, Y&M counts of heat-treated samples were always below the quantification

limit supporting the elimination effect of the 100 C/45 s heat shock over this microbial group. In

chlorine-treated samples counts up to 4.5 Log10 cfu.g-1

(p>0.05) were found at day 14, irrespective

of packaging film (Figure 91 c). The non-significant difference found show that the modified

atmosphere established inside the packages had no influence over the development pattern of this

microbial group, confirming the previous results. Moreover, the development of Y&M in chlorine-

treated samples, where yeast are the dominant strain (Carlin et al., 1994), contributes to CO2

release to the surrounding atmosphere which aggravates the modified atmosphere composition

inside the packages, favors LAB growth and compromises the product sensorial quality.

3.2.1.11 Product shelf-life estimation

The majority of shelf-life extension studies are focused on microbiological criteria out of concerns

about product safety (presence of pathogens), particularly in vegetables, which could provide a

misleading shelf-life estimation as not all quality attributes are evaluated.

Likewise, the fresh-cut industry defines the product shelf-life (expiry date) based only on legislated

microbiological criteria and since sensory testing is expensive and time consuming, this evaluation

is assigned to the consumer. The consumer is first attracted by the appearance of the product at

the moment of purchase but later, when the package is open, is when taste and aroma are

evaluated. The visual appearance is very important since it attracts the consumer (“If it looks good,

it tastes good!”), but can be reductive as it is overdue and not always true compromising repeat

buying by consumers. These issues are further intensified considering the demands and

expectations of today's consumer regarding associated health benefits (bioactive quality), attribute

that can only be assured by labeling claims. Even though these quality responses remain unseen

by consumers, achieving higher antioxidant levels during products shelf-life is contributing to add

value to the product and could be used by processors as a marketing tool.

Considering the above, estimation of FC products shelf-life should be established taking into

account the product overall quality. Moreover, achieving shelf-life extension under this concept,

even if just by a few days, can have a significant impact on the market sustainability of FC

products. To fulfill this purpose shelf-life estimation of fresh-cut shredded carrot produced


203

according to the proposed technological approach (heat shock combined with PM-MAP) was

assessed by modeling microbiological development (Gompertz model) and phenolic changes

(proposed model by Amodio et al., 2014), including the judgment of sensorial quality attributes.

The TAPC (Log10 cfu.g-1

) experimental data of heat- and chlorine treated fresh-cut shredded carrot

samples packaged with a micro-perforated film as a function of time were fitted to the modified

Gompertz equation (Eq. 4). However, only the data fitting from heat-treated samples was

satisfactory while parameter estimation from chlorine-treated samples returned negative values for

k (initial load, Log10 cfu.g-1

) and (lag phase, days) (Gompertz parameters shown in Table App. 34,

appendix 3). The derived equation to describe the TAPC growth curve from heat-treated samples is

shown in Eq. 9 and Gompertz parameters reported in Table 33.

𝑇𝐴𝑃𝐶 = 2.3 + 5.2 ∗ 𝑒𝑥𝑝 {−𝑒𝑥𝑝 [(0.6 ∗ 𝑒

5.2) ∗ (1.1 − 𝑆𝑡) + 1]} Eq. 9

Table 33 Parameter estimates (±SE) of the modified Gompertz model and predicted shelf-life at

5 C of heat-treated fresh-cut shredded carrot samples packed with a micro-perforated film.

Gompertz parameter estimates (R2=0.978) Predicted shelf-life*

k A µmax

2.3 ± 0.4

p<0.001

5.2 ± 0.7

p<0.001

0.6 ± 0.1

p<0.001

1.1 ± 1.3

p>0.05 19.5

Gompertz equation parameters (±SE, followed by respective p-value): k, initial load (Log10 cfu.g-1); A, maximum cell increase attained at the

stationary phase (Log10 cfu.g-1); µmax, maximal growth rate (Log10 cfu.g-1 per day); , lag phase (days). * - Predicted shelf-life as the time (days) necessary to attain a 7.5 Log10 cfu.g-1 level. R2 - regression determination coefficient.

At 5 C, in the modified atmosphere provided by the micro-perforated film, the heat-treated

shredded carrot samples had an estimated lag phase of 1 day (Table 33). Despite the non-

significance of this parameter (p>0.05), the estimated lag phase was greater than the ones

reported by Corbo et al. (2006) while assessing two commercial fresh-cut shredded carrot samples

stored at 4 C, with of 2.98 x 10-13

and 1.61 x 10-13

days. The found difference indicates that the

introduction of the heat shock and the micro-perforated film were responsible for the increase of the

mesophilic population incubation phase. Regarding the k parameter (initial load), the TPAC data

fitting to the Gompertz equation returned an estimated k of 2.3 Log10 cfu.g-1

(p<0.001; Table 33)

which is also considerably lower than the ones reported by Corbo et al. (2006), ranging from 4.90

to 7.08 Log10 cfu.g-1

. The estimated k initial load also endorses the ability of the 100 C / 45 s heat

sock treatment to significantly reduce the initial contamination level (TAPC counts of

4.2 Log10 cfu.g-1

in peeled carrot and of 5.8 Log10 cfu.g-1

in raw material). Moreover, the

decontamination efficiency of a treatment, as reported by Guerzoni et al. (1996), is a key factor to

determine the shelf-life of the resulting product and that ideally a reduction of the initial level to

103 cfu.g

-1 could significantly extent the shelf-life (at 5 C) of actual commercial fresh-cut products.


204

Figure 92 shows the evolution and Gompertz projection of the TAPC microbial population during

PM-MAP storage of heat-treated samples as well as the evolution and ill-fitted Gompertz projection

of chlorine-treated samples, as a behavior indicative, for comparison purposes only.

Figure 92 Growth curve of TAPC fitted with the Gompertz equation during low temperature storage

(5 C) of heat- (HS, 100 C/45 s) and chlorine-treated (HIPO; 200 ppm/1 min) fresh-cut shredded carrot samples packaged with a micro-perforated film.

Also inscribed in Figure 92 is the considered threshold limit that defines the end of the

microbiological acceptability, i.e., the end of shelf-life. The kinetic parameters derived by the

Gompertz equation are of widespread use to calculate the shelf-life of various minimally processed

vegetables (Corbo et al., 2006; Corbo et al., 2004; Sinigaglia et al., 2003; Guerzoni et al., 1996) by

limiting the equation to a maximum acceptable contamination value. On this basis, to calculate the

products microbiological shelf-life at 5 C, a threshold limit in mesophilic (TAPC) counts of

7.5 Log10 cfu.g-1

is taken considering the HPA (2009) recommendation [counts between 106-

108 cfu.g

-1] which is also in accordance with the French regulation from the Ministere de l’Economie

des Finances et du Budget (1988) [counts of 5 x 106 cfu.g

-1] for establishing fresh-cut products

expiry date.

The necessary time at 5 C for heat-treated samples to attain the maximum acceptable

contamination value (predicted shelf-life) is referenced in Table 33 and observed in Figure 92.

According to the mean estimates from the Gompertz model fitted to the TAPC experimental data

and the set threshold limit, the microbiological expiry date of heat-treated shredded carrot samples

packed with a micro-perforated film is reached after 19 days of storage at 5 C. The predicted

19-day shelf-life for the new technological proposal, application of a pre-cut 100 C / 45 s heat

shock as a decontamination alternative and the use of micro-perforated film, demonstrates to be

HS HIPO

0 3 7 10 14 15 20 25

Storage time (days)

0

1

2

3

4

5

6

7

8

9

10T

AP

C (

Lo

g1

0[c

fu.g

-1])

Threshold limit


205

more advantageous from the microbiological standpoint than the use of the industrial standard

post-cut chlorine decontamination as it would surpass that criterion after 7 days as hinted by the ill-

fitted projection (confirming the already established 7 days shelf-life, Part IV, section 3.2.1.10).

Moreover, Corbo et al. (2006) and Guerzoni et al. (1996) reported shelf-life estimates ranging from

1.74 to 6.88 days and of 3 days, for chlorine decontaminated (100 - 150 ppm) and commercially

available shredded carrot, respectively. Therefore, the 19-day shelf-life for the current proposal

constitutes a most viable alternative for shredded carrot production since it would contribute to a

3-fold microbiological shelf-life extension regarding the industrial standard.

Then again, convenience oriented buyers make their purchasing decision based not only in the

shelf-life date but also from evaluating products external appearance, making sensorial quality a

critical factor (Beaulieu, 2010; Ragaert et al., 2004). As Piagentini et al. (2005) stated, little is

available on sensory quality modeling to predict quality degradation as a function of temperature

and time, and most often the Arrhenius or first order equations are used to model purposes.

Although mathematical models are a useful tool to predict quality loss or shelf-life (Amodio et al.,

2013), products visual and aroma attributes as well as its respective acceptance/rejection are

influenced by multiple factors, e.g. microbial growth, MAP, oxidation reactions and, ultimately, by

human perception, deeming sensory quality modelling difficult and should only be considered as a

likely behavior of the product sensorial changes during storage (van Boekel, 2008). As a result, it

was unequivocal that sensory quality was not be fitted to any possible model (e.g. zero and/or first

order, Weibullian, logistical kinetics). Thus, changes in sensorial attributes scores (Figure 89) and

in respective acceptability levels (Figure 90) of heat- and chlorine-treated samples were only

considered within the tested period.

The discussion of the sensorial scores of color, fresh-like appearance and aroma and also

respective rejection index is included in section 3.2.1.9 (Part IV) where it was found that heat-

treated shredded carrot samples packed with a micro-perforated film had a most satisfactory

sensorial quality (color, fresh-like appearance and aroma and respective acceptance) during the

entire tested storage period (14 days) than chlorine-treated samples (undoubtedly rejected by day

7) and were never rejected by the panel members. Therefore, the predicted microbiological shelf-

life of 19 days is reduced to 14 days since during this period sensorial quality is met and assured.

Having met two of the established criteria to define shelf-life, sensorial and microbiological, the

proposed technology for fresh-cut carrot production still has another criterion to comply with:

maintenance of the raw material bioactive content, namely its phenolic composition, during the

already established 14-day shelf-life. As reported, the model proposed by Amodio et al. (2014)

describes the two distinct phenomena related to phenolic changes during low temperature storage:

the de novo synthesis of phenolic compounds as a stress response and respective degradation to

oxidized compounds. The TPC data from heat- and chlorine-treated samples was fitted to Eq. 6

(Part III, section 5.5) and respective model projection is shown in Figure 93.


206

Figure 93 Changes in TPC and data fitting to the proposed equation by Amodio et al. (2014)

applied to heat- (HS, 100 C/45 s) and chlorine-treated (HIPO, 200 ppm/1 min) fresh-cut shredded

carrot samples packaged with a micro-perforated film during low temperature storage (5 C).

Despite models predictive inability (Table App. 37, appendix 3), the projection of phenolic changes

during storage (Figure 93) shows a significant difference in the phenolic accumulation pattern

between heat- and chlorine-treated samples packaged with the micro-perforated film (film B). Heat-

treated samples have a longer phenolic synthesis period enabling significant phenolic accumulation

to reach and surpass the raw material TPC level as early as day 5, situation never achieved by

chlorine-treated samples. This phenomenon can be related to the non-limiting O2 level inside the

packages as assured by the micro-perforated film and by the heat shock effects in lowering the

products respiration rate. Moreover and as previously discussed, the phenolic degradation

reactions were pushed forward as a consequence of the micro-perforated film used and of the

100 C/45 s treatment. The treatment effects as to achieve a partial inhibition of POD (Figure 86,

Part IV, section 3.2.1.6) further delayed the degradation pattern contributing to maintain the raw

material TPC levels during the 14-day shelf-life.

From the above, a reasonable shelf-life (14 days) can be obtained with appropriate combinations of

stress treatments and packaging systems while complying with three major criteria: microbiological,

sensorial and bioactive. In light of the results, the proposed technology pushes forward two food

industry axioms: i) “If it looks good, it tastes good!” and ii) the increase of the raw material quality,

emphasizing fresh-cut shredded carrot freshness, bioactive composition and the possibility of at

least doubling the shelf-life of the commercially (industrial reference) available product.

HS HIPO

0 3 5 7 10 12 14 20 25

Storage time (days)

0

20

40

60

80

100

120

140

160

180

TP

C (

mg

CA

E.1

00

g-1

)

Raw material

207

4 FINAL REMARKS

4.1 GENERAL CONCLUSIONS

… RAW MATERIAL…

Carrots (cv. Nantes) bioactive value was found to be supported by respective antioxidant

capacity mainly due to its phenolic contribution (60%) than by the carotenoid contribution

(40%). Furthermore, the found balanced mix of hydrophilic and lipophilic antioxidants

foresees quenching free radicals in both aqueous and lipid phases, which is known to be of

utmost importance to health promotion and disease prevention. Within its phenolic

composition, chlorogenic acid was the main phenolic compound found (12-17 mg.100 g-1

)

representing up to 90% of detected phenolic compounds. Carrot has 30% (dry content) of

dietary fiber and is well balanced regarding the insoluble:soluble fiber ratio (2.4) indicating

that this cultivar is a good source of dietary fiber;

The found differences in total phenolic content between cv. Nantes crop season (82.1±2.1

and 46.8±2.0 mg CAE.100 g-1

for spring and fall seasons, respectively) demonstrate the

influence (p<0.05) of climatic conditions over this bioactive marker. However, considering

registered differences between cultivars (143.8±11.4 vs. 73.5±16.8 mg CAE.100 g-1

for cv.

Navajo and cv. Nantes, respectively), crop season has only a minor effect on TPC when

compared to the genetic background influence;

The total phenolic contents in different cv Nantes carrot tissues decreased in the following

order: peel > cortical >vascular parenchymas. Spatial tissue distribution of phenolic content

across the carrot root could be interpreted in terms of increased antioxidant potential in

peripheral tissues (peels) as a part of the plants defense mechanism. In fact antioxidant

molecules, such as chlorogenic acid, inhibit the oxidation of other molecules preventing

oxidative damage either by scavenging ROS or up-regulating antioxidant defenses.

… WOUND-INDUCED PHENOLIC SYNTHESIS DYNAMIC…

It was established that wounding acts as an abiotic stress factor leading to significant

phenolic accumulation during storage (with chlorogenic acid as the main synthetized

phenolic), even at low temperature (5 C):

o Wounding triggers the de novo synthesis of PAL [from day 0 to 3] leading to significant

phenolic accumulation occurring from day 3 onwards;

o The higher ability of peels to synthetize phenolics is suggested by the highest

accumulation rates found. Nonetheless, peel removal does not compromise phenolic

accumulation levels in the fresh-cut carrot as phenolic synthesis depends on the

FINAL REMARKS

208

significant superficial area increase of the most representative carrot tissue (cortical

parenchyma);

o Regardless tissue type, PAL enzyme seems to undergo feedback inhibition as its de novo

synthesis is suppressed when phenolic levels accumulate to a critical concentration;

o O2 concentration is suggested to be a limiting factor to wound-induced phenolic synthesis

as levels below 2% suppresses significant phenolic accumulation pointing out the

importance of designing suitable MAP systems for bioactive quality recovery under the

fresh-cut product concept;

o Heat shock and UV-C abiotic stress treatments introduce changes to the phenolic

synthesis dynamic under “air” storage conditions:

Heat shock [100 C/45 s] introduces a delay in phenolic accumulation as PAL de novo

synthesis is postponed by two days and promotes reduced POD activity levels

regarding the raw material. The induced changes in both enzymatic activity levels

could either result from partial thermal inactivation or from the diversion of protein

synthesis as part of plants defense mechanism.

UV-C [pre- or post-cut 2.5 kJ.m-2

] further stimulates de novo synthesis of PAL which

enhances wound-induced phenolic synthesis by nearly two times;

Stress combination [pre-cut 100 C/45 s x post-cut 2.5 kJ.m-2

] avoids the phenolic

accumulation delay imposed by heat shock attaining a similar performance to the UV-

C single application.

… STANDARD (INDUSTRIAL) MINIMAL PROCESSING EFFECTS…

Fresh-cut carrot bioactive and sensorial quality are significantly affected just after minimal

processing:

o Peeling and decontamination procedures lead to losses of 59% and 37% in phenolic and

carotenoid contents regarding the raw material;

o Post-cut immersion operations compromise sweet taste perception (losses in soluble

solids content up to 35%) and characteristic aroma (by introducing a “disinfectant” off-

odor);

o Chlorine decontamination was insufficient to attain the considered ideal reduction (counts

of 103 cfu.g

-1).

During low temperature storage (7 days, 5 C), changes in sensorial quality were critical to

product acceptability while the bioactive quality was marginally affected:

o Losses in fresh-like aroma and fresh-like appearance occurs in the earlier stages of

storage, reaching unacceptable levels by day 5;

FINAL REMARKS

209

o Phenolic accumulation induced by shredding lead to a partial recovery (40%) of fresh

content while the carotenoid fraction was maintained stable during storage;

o Products’ microbiological quality, according to the applicable criteria, can only be assured

for about 7 days (5 C).

… HURDLE EFFECTS ON FRESH-CUT CARROT QUALITY DURING MAP STORAGE …

The first mechanism responsible for losses in FC carrot sensorial quality is the microbial

spoilage attributed to LAB development:

o The selective pressure of LAB on Y&M population is justified by the more competitive

behavior of the first microbial group and also by the fast atmosphere modification inside

the packages (achieved by the products’ characteristic high respiration rate) providing

most favorable LAB growth conditions;

Treatments’ impact in reducing initial contamination levels and consequently avoiding

fast changes of atmosphere composition inside the packages during storage is of

particular importance to prevent these quality losses;

The second appointed mechanism refers to the phenolic synthesis dynamic which does not

have any sensorial implication but has a significant impact regarding product bioactivity,

namely phenolic content recovery:

o To this mechanism the importance of oxygen levels present inside the packages is

pointed as determinant for phenolic accumulation during storage, independently of the

applied abiotic stress necessary to induce the increase of PAL activity;

FC carrot quality maintenance is limited during storage considering that both mechanisms

are inversely related to the inevitable atmosphere modification inside the packages:

o Shelf-life can be extended if two key conditions are assured: O2 levels above the

inhibitory threshold for phenolic synthesis (<2%) and CO2 levels below the threshold

promoting LAB growth (>10%).

The required steady-state equilibrium is impossible to achieve and anaerobic conditions are

rapidly established in view of the high respiratory levels of shredded carrot and the low

permeabilities of commonly used polymeric films:

o Establishment of target modified atmosphere is only possible with the use of a micro-

perforated film and/or by controlling the respiratory metabolism (e.g. active MAP);

The achieved benefits from the application of the 100 C/45 s heat shock in best retaining

fresh-like quality during storage are justified by the attained decontamination levels and by

the respective effects in delaying the modification of the gaseous composition inside the

packages (indirectly influenced by the diminished product respiratory metabolism). The

FINAL REMARKS

210

additive effects observed in the heat shock treatment combination with the micro-perforated

film are equally substantiated in light of this dynamic.

The UV-C treatment (2.5 kJ.m-2

) had an inexpressive lethal effect on microbial growth as a

single treatment. When combined with heat shock the expected effect as to anticipate

phenolic synthesis was not achieved and phenolic accumulation was probably conditioned

by the lower O2 levels inside the packages.

… MULTI-TARGET TECHNOLOGICAL ALTERNATIVE…

The use of the 100 C/45 s heat shock abiotic stress treatment allied to an alternative MAP solution

(micro-perforated film) provided a well-designed fresh-cut shredded carrot with an extended shelf-

life regarding the standard industrial production. The major benefits of this alternative technological

approach to the standard production were:

i) The significant decontamination effects sufficient to best control microbial development

during products storage which, according to the considered microbial criterion for

establishing fresh-cut products expiry date, defined a predicted shelf-life of 19 days;

ii) reduced changes regarding product sensorial color, fresh-like appearance and aroma

leading to a good FC carrot acceptability during 14 days;

iii) The significant phenolic accumulation during PM-MAP storage was sufficient to assure the

raw material phenolic content from day 5 and until the estimated 14-day shelf-life.

In comparison to the standard production of fresh-cut carrot (chlorine decontamination), this

technological approach allows extending product shelf-life by 2-fold (7 vs. 14 days) while observing

the three criteria providing fresh-cut carrot with a higher bioactive value, good sensorial quality and

a satisfactory/adequate microbiological shelf-life.

FINAL REMARKS

211

4.2 FUTURE PROSPECTS

EXPLORING ABIOTIC STRESSES

Use of controlled abiotic stresses to target secondary metabolites production results in products

increased bioactive value. Nevertheless, much is still yet to be understood on how different

stresses, as single or in combination, induce PAL expression as to synthesize the target phenolic

synthesis. Although ROS have been proposed as signaling molecules for the stress-induced

phenolic accumulation in carrot, the influence of stress induction of heat shock proteins (HSPs), the

relation with ROS and respective effects over PAL protein expression (mRNA) mechanism are still

unclear and should be further explored.

It was also highlighted that altering the atmospheric gas composition can be used to improve fresh-

cut fruits and vegetables bioactive value. Exploring what is the optimal O2 concentration as to

maximize phenolic synthesis should be pursued in order to develop MAP systems that would aid to

achieve this outcome.

EXTRACTION OF BIOACTIVE PHENOLIC COMPOUNDS FROM FRESH-CUT CARROT RESIDUES

This research area is in line with current needs to upgrade what is usually considered as a waste

and seeing it as a by-product, a raw material for other purposes. Under this concept, carrot peels

and other carrot parts that are discarded during fresh-cut carrot production could be used for the

production of value-added compounds and delivered to alternative markets, namely to chemical,

pharmaceutical and/or food industries. The high phenolic content characterized in carrot peels

justify exploring this resource as an extractable source of phenolic antioxidants, and respective

contents could even be enhanced by applying abiotic stress treatments, making this by-product a

renewed source of income. Also, carrot carotene and fiber content also make these by-products

attractive as a matrix for respective extraction. Hence, development of cost-effective industrial

biotechnology can offer attractive strategies for the valorization of the fresh-cut carrot industry by-

products, achieving even increased environmental, social and economic sustainability.

BIOAVAILABILITY AND ABSORPTION OF DIETARY ANTIOXIDANTS

An important field of research today is the control of ‘redox’ status with the antioxidants properties

of food and food components which has a substantial thrust in the food industry. Hence increasing

the existing dietary antioxidants, creating the so called nutraceuticals and functional foods, is an

area of significant interest. However, the benefits of such products have to be established on

epidemiological studies, in vitro or in vivo, proving that these compounds are capable of

neutralizing reactive oxygen species and other oxidants in the body. Scientific-based recognition of

the associated benefits from food consumption, ingredients and nutraceuticals would therefore re-

enforce fruit and vegetable consumption.

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Zwietering, M.H., De Koos, J.T., Hasenack, B.E., De Wit, J.C., van’t Riet, K. (1991). Modelling of bacterial growth as a function of temperature. Applied and Environmental Microbiology 57, 1875–1881.

REFERENCES

240

Zwietering, M.H., Jongenburger, F.M., Roumbouts, M., van’t Riet, K. (1990). Modelling of the bacterial growth curve. Applied and Environmental Microbiology 56, 1875– 1881.

APPENDIXES

243

APPENDIX 1

Supporting tables to research topic 1 – “Fresh-cut carrot: raw material bioactive quality and decay

profile”

Morphological distribution of bioactive composition along cv. Nantes carrot tissues from

two crop seasons

Table App. 1 Bioactive markers content and activity levels of enzymes involved in its synthesis and degradation of cv. Nantes carrot (whole root; raw material) as influenced by crop season (spring and fall).

Crop season TPC

(mg CAE.100 g-1)

TCC

(mg -carotene

eq.100 g-1)

PAL

(U.100 g-1)

PPO

(U.g-1)

POD

(U.g-1)

Antioxidant capacity (mg TE.100 g-1)

AOxT AOxH AOxL

Spring 82.1b±2.2 15.5b±0.3 28.9ns±2.9 40.9b±3.8 112.2b±24.2 95.5b±4.4 66.7b±5.5 28.8b±3.9

Fall 46.8a±3.5 11.2a±1.1 26.2ns±5.0 22.7a±1.9 19.8a±24.2 59.9a±0.6 35.8a±0.8 24.1a±0.3

Units expressed on carrots (whole root) fresh weight (FW) basis. AOxT- Total antioxidant capacity; AOxH- Hydrophilic antioxidant capacity; AOxL- Lipophilic antioxidant capacity. Values represent the mean of three replicates±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test); ns- non significant difference.

Table App. 2 Tissue contributions to carrots’ total bioactive markers content (TPC, TCC) and related enzymes (PAL, PPO and POD) from cv. Nantes spring and fall seasons.

Crop season Tissue Contributes (%) to:

TPC TCC PAL PPO POD

Spring

Peel 34.3c±3.0 10.1ab±0.6 24.1b±0.3 41.8b±4.6 51.7a±6.8

Cortical parenchyma 62.0b±4.6 68.6c±7.1 61.9c±1.6 54.1c±4.0 45.4a±4.0

Vascular parenchyma 7.0a±0.5 14.2b±0.8 11.9a±0.8 13.9a±1.8 1.6b±0.2

Fall

Peel 27.3c±0.7 6.2a±0.4 18.6b±0.2 38.6b±1.9 47.4a±3.6

Cortical parenchyma 62.3b±0.6 80.9d±7.5 66.8c±1.9 50.7c±6.7 50.2a±2.5

Vascular parenchyma 3.9a±0.1 9.6ab±0.7 8.0a±0.6 9.9a±0.4 2.9b±0.1

In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

244

Characterization of the raw material total phenolic content in cv. Nantes and cv. Navajo

carrot

Table App. 3 Raw material total phenolic content (TPC) data from cv. Nantes and cv. Navajo characterizations and respective mean (±SD) values.

Carrot cultivar TPC (mg CAE.100 g-1)

Characterizations means* Cultivar mean

Nantes

46.8; 76.0; 43.6; 51.8; 49.3; 47.1; 42.4; 46.8; 62.0; 43.6; 54.6; 49.3; 47.1; 93.8; 71.8; 87.7; 82.8; 84.6; 81.2; 84.5; 84.1; 83.9; 87.8; 111.6; 67.1; 69.0; 66.3; 70.7; 52.3; 70.9; 73.9; 108.3; 75.7; 80.2; 60.5; 76.9; 67.2; 76.3; 73; 80.9; 78.3; 84.2; 84.2; 85.3; 90.2; 93.2; 107.7; 95.7; 98.0; 91.3; 87.5; 90.5; 74.6; 75.2; 74.6; 72.8; 72.5; 72.8; 76.0; 76.6; 42.4; 65.4; 72.6; 61.0; 56.6; 54.9; 84.1; 83.9; 87.8 (N=69)

73.5a±16.8

Navajo

139.7; 142.1; 147.9; 123.2; 149.7; 152.7; 160; 139.9; 144.4; 137.1; 139.6; 140.1; 172.6; 146.0; 137.6; 147.1; 136.2; 163.1; 133.8; 137.6; 128.6 (N=21)

143.8b±11.4

* Spring and Fall mean values were excluded from cv. Nantes mean estimation. In the “Cultivar mean” column, different letters represent significant differences at p=0.05 (unequal N HSD test).

245

Phenolic synthesis as affected by tissue type and wounding intensity

Table App. 4 Relation between PAL activity and total phenolic content as influenced by wounding

intensities in carrot peel and inner tissues during low temperature storage (5 C, 14 days).

Sample type Storage time

(days) PAL (U.100 g-1)

TPC (mg CAE.100 g-1)

𝑃𝐴𝐿

𝑇𝑃𝐶 ratio

Peel tissues submitted to a low wounding stress (LW_P)

0 345.4g±5.0 198.5cd±6.8 -

3 499.2i±10.6 391.1f±34.2 1.3

7 495.0i±3.7 523.7g±15.3 0.9

10 435.2h±1.4 695.8i±52.9 0.6

14 213.0d±14.7 623.1hi±55.0 0.3

Peel tissues submitted to a high wounding stress (HW_P)

0 276.9e±4.9 137.3bc±6.9 -

3 290.5ef±29.4 314.6e±2.9 0.9

7 323.4fg±4.3 360.0ef±23.7 0.9

10 360.3g±22.8 629.7hi±49.2 0.6

14 319.9efg±38.6 593.2gh±39.3 0.5

Inner tissues submitted to a low wounding stress (LW_In)

0 24.2ab±2.3 26.9a±2.1 -

3 70.3c±8.1 60.4a±2.5 1.2

7 28.6abc±1.2 76.1ab±6.2 0.4

10 25.5ab±1.2 83.5ab±8.2 0.3

14 10.8a±0.8 49.5a±5.0 0.2

Inner tissues submitted to a low wounding stress (HW_In)

0 55.0bc±8.0 40.6a±3.0 -

3 184.9d±13.4 83.1ab±1.1 2.2

7 341.4g±3.6 137.6bc±2.1 2.5

10 478.0hi±1.2 232.6d±5.0 2.1

14 472.4hi±16.1 396.9f±12.4 1.2

In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

246

Phenolic synthesis as affected by peel removal and shredding

Table App. 5 Effect of peeling (P) and shredding (S) on total phenolic content (TPC), antioxidant capacity (AOx), chlorogenic acid content (CA) and PAL activity (PAL) (univariate test of significance).

Effects MS F p Effects MS F p

TPC AOx

Peeling (P) 16534 19.751 0.000014 Peeling (P) 266916 6.223 0.013534

Shredding (S) 60752 72.569 0.000000 Shredding (S) 2706488 63.098 0.000000

PxS 1545 1.846 0.175542 PxS 226 0.005 0.942227

CA PAL

Peeling (P) 209.56 7.1275 0.009278 Peeling (P) 1969.7 0.8839 0.348749

Shredding (S) 333.74 11.3512 0.001187 Shredding (S) 168374.2 75.5605 0.000000

PxS 4.51 0.1535 0.696352 PxS 1315.0 0.5901 0.443663

MS – Mean squares; F- F test; p – significance level

Table App. 6 Estimated parameters of the TPC model proposed by Amodio et al. (2014) applied

to shredded carrot samples during storage at 5 C.

Model parameter estimates S samples

[TPC0 = 71.4 ±6.1 mg CAE.100 g-1]

Speeled samples

[TPC0 = 61.6 ±3.7 mg CAE.100 g-1]

TPCpre0 987 ± 5058643 (p>0.05) 6212 ± 32015934 (p>0.05)

k1 0.0115 ± 54 (p>0.05) 0.000 ± 2 (p>0.05)

k2 0.0114 ± 54 (p<0.05) -0.071 ± 1 (p>0.05)

Model equation parameters (±SE, followed by respective p-value): TPCpre0, precursor phenolic content (mg CAE.100 g-1); k1, rate constant for phenolic de novo synthesis (day-1); k2, rate constant of phenolic oxidation (day-1).

Table App. 7 Relevant regression analysis results from fitting Eq. 6 to the TPC data from unpeeled (S samples) and peeled (Speeled samples) shredded carrot.

Regression analysis

Sample type Ē% R2 MSE Randomness of residuals

S samples 7.7 0.968 108.1 (Figure App. 1)

Speeled samples 6.0 0.985 55.83 (Figure App. 2)

Ē%, relative percent difference between experimental and predicted values (goodness of fit; Ē% =100

𝑁∗ ∑

𝑀𝑖−𝑀𝑝

𝑀𝑖

𝑖=𝑁𝑖=1 : Mi, experimental value; Mp,

predicted value; N, number of observations). Regression analysis: R2, determination coefficient; MSE, residue mean square.

Figure App. 1 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from unpeeled (S samples) shredded carrot samples.

Normal Probability Plot of Residuals

-25 -20 -15 -10 -5 0 5 10 15 20

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99

a) Randomness of residuals

60 70 80 90 100 110 120 130 140 150 160 170 180

Predicted Values

-20

-15

-10

-5

0

5

10

15

20

Re

sid

ua

l V

alu

es

b)

247

Figure App. 2 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from peeled (Speeled samples) shredded carrot samples.


-20 -15 -10 -5 0 5 10 15

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


50 60 70 80 90 100 110 120 130 140 150 160 170

Predicted Values

-15

-10

-5

0

5

10

15

Re

sid

ua

l V

alu

es

b)

248

APPENDIX 2

Supporting tables to research topic 2 - “Heat shock and UV-C stress effects on carrot quality: single

and combined application“

Preliminary study of hear shock and UV-C stress effects on fresh-cut carrot quality

Table App. 8 Bioactive and microbiological responses of fresh carrot cv. Nantes (unprocessed carrot, raw material).

Quality measurements Mean ± SD

Bioactive responses

Total phenolic contentb (mg GAE.100 g-1) 21.0±0.9

Total carotenoid contentb (mg -carotene eq.100 g) 27.5±0.4

Stress-related enzymes

Peroxidase activityb (U.g-1) 65.4±5.6

Microbiological responses

Total aerobic plate countc (Log10 cfu.g-1) 4.5±0.1

a – Data represents the mean of 27 values; b – Data represents the mean of 9 values; c – Data represents the mean of 3 values; mg GAE/100 g – mg gallic acid equivalents per 100 g.

UV-C single stress effects on the wound-induced dynamics

Table App. 9 Total phenolic and carotenoid content and PAL activity characterization of raw fresh carrot cv. Nantes (raw material, unprocessed).

Quality measurement Mean±SD

Total phenolic content (TPC, mg CAE.100 g-1) 66.3±5.9

Total carotenoid content (TCC, mg -carotene eq.100 g-1) 12.2±0.3

PAL activity (U.100 g-1) 8.1±2.6

Table App. 10 Effect parameter quadratic model estimates and respective probabilities.

Effect TPCPre-cut TPCPost-cut

Parameter p value Parameter p value

Intercept 25,1 0,000000 26,6 0,000000

UV (kJ.m-2) 12,7 0,000000 10,6 0,000002

UV2 (kJ.m-2) -2,6 0,000000 -2,2 0,000000

St (days) 5,2 0,000125 6,5 0,000001

St2 (days) 0,9 0,000000 0,7 0,000005

249

Table App. 11 Analysis of variance of the second-order polynomial model for total carotenoid content, TCCPre-cut and TCCPost-cut, of carrot subjected to UV-C treatments (dose/application site).



TCCPre-cut Regression 5.1 5 1.0 1.95† 0.109805

r2=0.21 Residual 19.0 36 0.5 r2adj=0.10 Lack of fit 6.4 3 2.1 5.6‡ 0.003210 Pure Error 12.6 33 0.4 Total 24.1 41

TCCPost-cut Regression 18.6 5 3.7 7.5† 0.000065



Table App. 12 Analysis of variance of the second-order polynomial model for hue color parameter, HuePre-cut and HuePost-cut, of carrot subjected to UV-C treatments (dose/application site).



HuePre-cut Regression 30.2 5 6.1 4.9† 0.000308


HuePost-cut Regression 7.3 5 1.5 1.7† 0.117070



250

Table App. 13 Analysis of variance of the second-order polynomial model for whiteness index, WIPre-cut and WIPost-cut, of carrot subjected to UV-C treatments (dose/application site).



WIPre-cut Regression 172.4 5 34.5 2.8† 0.000308


WIPost-cut Regression 166.3 5 33.2 4.3† 0.000886



251

Table App. 14 Mean values for total carotenoid content (TCC, mg -carotene eq.100 g-1) and color parameters of control (Ctr) and UV-C treated carrot

samples according to the RSM design independent variables (8 days, 5 C).

Sample Id

UV-C dose

(kJ.m-2)

Storage time

(days)

Application site

TCC L* a* b* C* Hue WI TCD

Ctr_0 0 0 - 10.6abcdefgh 55.3ab 24.9bcde 50.4cd 56.2cd 63.7abcd 28.0ab 4.9ns

Ctr_1 0 1 - 9.6ab 53.4ab 24.2abcde 47.9abcd 53.7abcd 63.2abcd 28.8abcd 5.7ns

Ctr_4 0 4 - 11.3cdeghij 54.9ab 24.0abcde 47.2abc 53.0abc 63.1abc 30.1abcdefg 6.5ns

Ctr_7 0 7 - 12.1hij 56.1ab 23.5abcd 47.1abc 52.6abc 63.5abcd 31.4abcdefg 5.6ns

Ctr_8 0 8 - 9.3a 55.3ab 23.0abcd 45.4ab 50.9ab 63.1abc 32.1cdefg 7.1ns

W_7 2.5 0 Pre-cut 10.3abcdef 57.3ab 24.0abcde 50.2cd 55.6bcd 64.5d 29.7abcdefg 5.3ns

W_1 0.7 1 Pre-cut 10.7abcdefgh 56.3ab 23.0abcd 47.5abc 52.7abc 64.2cd 31.4abcdefg 6.5ns

W_3 4.2 1 Pre-cut 10.6abcdefgh 57.5ab 25.3cde 49.7bcd 55.8bcd 63.1abc 29.6abcdef 6.6ns

W_5 0.1 4 Pre-cut 10.4abcdefg 56.3ab 23.3abcd 45.3ab 51.0abc 62.9abc 32.6defg 8.3ns

W_9 2.5 4 Pre-cut 11.3cdeghij 55.6ab 24.2abcde 46.8abc 52.7abc 62.7a 31.0abcdefg 5.6ns

W_10 2.5 4 Pre-cut 11.9fghij 58.5b 24.2abcde 48.3abcd 54.0abcd 63.3abcd 31.8bcdefg 5.4ns

W_11 2.5 4 Pre-cut 10.8abcdeghi 55.8ab 25.5de 49.2bcd 55.4bcd 62.6a 28.9abcde 5.3ns

W_12 2.5 4 Pre-cut 11.5deghij 57.4ab 24.7abcde 48.1abcd 54.1abcd 62.9abc 30.9abcdefg 6.7ns

W_13 2.5 4 Pre-cut 10.5abcdefg 55.5ab 23.3abcd 46.2abc 51.7abc 63.3abcd 31.4abcdefg 7.7ns

W_14 2.5 4 Pre-cut 11bcdeghi 59.1b 23.4abcd 47.1abc 52.7abc 63.6abcd 33.2fg 6.8ns

(cont.)

252

Table App. 14 (cont.) Mean values for total carotenoid content (TCC, mg -carotene eq.100 g-1) and color parameters of control (Ctr) and UV-C treated carrot

samples according to the RSM design independent variables (8 days, 5 C).

Sample Id

UV-C dose

(kJ.m-2)

Storage time

(days)

Application site

TCC L* a* b* C* Hue WI TCD

W_6 5 5 Pre-cut 10.9abcdeghi 57.6ab 22.6abc 45.3ab 50.6ab 63.4abcd 33.8g 7.3ns

W_2 0.7 7 Pre-cut 11.9ghij 54.6ab 21.9a 43.7a 48.9a 63.3abcd 32.9efg 9.2ns

W_4 4.2 7 Pre-cut 12.3ij 55.2ab 24.2abcde 48.1abcd 53.9abcd 63.2abcd 29.7abcdefg 7.9ns

W_8 2.5 8 Pre-cut 10.6abcdefgh 57.7b 24.2abcde 47.6abcd 53.4abcd 63.1abc 31.7bcdefg 6.2ns

S_7 2.5 0 Post-cut 10.1abcde 57.4ab 26.3e 52.2d 58.5d 63.3abcd 27.5a 5.2ns

S_1 0.7 1 Post-cut 9.9abcd 55.1ab 24.7bcde 49.9bcd 55.7bcd 63.7abcd 28.2abc 5.6ns

S_3 4.2 1 Post-cut 9.8abc 55.8ab 24.8bcde 49.0bcd 55.0bcd 63.2abcd 29.3abcdef 5.2ns

S_5 0.1 4 Post-cut 11.6eghij 50.9a 24.0abcde 46.6abc 52.4abc 62.7ab 27.9ab 7.7ns

S_9 2.5 4 Post-cut 11.4deghij 55.1ab 24.4abcde 48.0abcd 53.9abcd 63.1abc 29.7abcdefg 4.9ns

S_10 2.5 4 Post-cut 11.3cdeghij 55.1ab 23.6abcde 46.7abc 52.4abc 63.2abcd 30.8abcdefg 6.5ns

S_11 2.5 4 Post-cut 11.9ghij 57.7b 24.6abcde 50.0bcd 55.7bcd 63.8abcd 30.0abcdefg 4.6ns

S_12 2.5 4 Post-cut 11.4cdeghij 55.5ab 23.9abcde 47.2abc 52.9abc 63.2abcd 30.7abcdefg 5.0ns

S_13 2.5 4 Post-cut 11.1bcdeghij 53.1ab 23.9abcde 47.2abc 52.9abc 63.1abc 29.1abcde 6.3ns

S_14 2.5 4 Post-cut 12.6j 54.0ab 24.7bcde 48.3abcd 54.2abcd 63.0abc 28.6abcd 6.4ns

S_6 5 5 Post-cut 11bcdeghi 57.3ab 24.1abcde 49.0bcd 54.6bcd 63.9abcd 30.5abcdefg 5.5ns

S_2 0.7 7 Post-cut 11.9fghij 58.6b 23.5abcd 48.4abcd 53.8abcd 64.1bcd 32.0bcdefg 6.4ns

S_4 4.2 7 Post-cut 12.3ij 53.6ab 22.3ab 45.4ab 50.6ab 63.9abcd 31.2abcdefg 7.9ns

S_8 2.5 8 Post-cut 10.1abcde 52.7ab 24.1abcde 47.5abcd 53.3abcd 63.1abc 28.5abcd 5.9ns

Values represent the mean of three replicates for TCC and 15 measurements for color parameters. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test); ns – non-significant.

253

Study of heat shock [100 C/45 s] single stress effects on the wound-induced dynamics

Table App. 15 Effect of peeling and heat shock on total phenolic content (TPC) during low

temperature storage (5 C, 10 days) (univariate test of significance).

TPC

Effect df SS MS F p

Peeling 1 1020.3 1020.3 2.3713 0.130748

Heat shock 1 1858.8 1858.8 4.3200 0.043530

Peeling*Heat shock 1 15.3 15.3 0.0356 0.851295

Error 44 18931.9 430.3

Total 47 21826.2

df – degrees of freedom; SS – Sum of squares; MS – Mean squares; F- F test; p – significance at 95%.

Table App. 16 Changes in total phenolic content (TPC) of heat-treated and untreated shredded

carrot samples during low temperature storage (5 C, 10 days).

Sample Id. Peeling Heat shock (100 C/45 s) Storage time

(days)

TPC

(mg CAE.100 g-1)

NP

No peeling

No heat shock

0 38.8cdef±4.1

3 45.3fghi±3.2

7 85.3no±12.4

10 88.3o±12.5

NP_HS Heat shock

0 38.3cdef±3.0

3 41.5defg±3.8

7 60.3kl±1.4

10 63.2l±2.0

P

Peeling

No heat shock

0 28.1ab±0.5

3 34.7bcde±2.1

7 75.3mn±6.3

10 78.1mn±5.6

P_HS Heat shock

0 23.7a±0.2

3 26ab±0.7

7 59jkl±4.8

10 62.3l±5.1

Values represent the mean±SD of three replicates. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

254

Heat shock and UV-C combined stress effects on the wound-induced dynamics

Table App. 17 Raw material characterization (fresh cv. Nantes carrot, unprocessed).


Bioactive responses



Antioxidant capacity (AOxH, µg TE.100 g-1) 471.7±4.3

Stress related enzymes

Phenylalanine ammonia lyase (PAL, µmol t-cinnamic acid.h-1.100 g-1) 4.0±0.8

Peroxidase (POD, U.g-1) 34.5±1.8

Physical-chemical and sensorial responses

pH 6.2±0.0

SSC (ºBrix) 9.4±0.4

Dry matter (%, w/w) 88.1±4.4

255

APPENDIX 3

Supporting tables to research topic 3 – “Technological proposal:hurdle concept into action”

Selected heat shock and UV-C stress treatments and MAP effects on fresh-cut carrot quality

Table App. 18 Raw material characterization (fresh cv. Nantes carrot, unprocessed).


Bioactive compounds and related responses


Chlorogenic acid (CA, mg.100 g-1) 12.30±1.17


-carotene (mg.100 g-1) 16.43±0.85

Phenylalanine ammonia lyase (PAL, U.100 g-1) 28.9±2.5

Peroxidase (POD, U.g-1) 97.1±1.8

Total antioxidant capacity (AOxT, mg TE.100 g-1) 95.5±3.4

Hydrophilic antioxidant capacity (AOxH, mg TE.100 g-1) 66.7±6.2

Lipophilic antioxidant capacity (AOxL, mg TE.100 g-1) 28.8±2.8

pH/SSC

pH 6.4±0.1

Soluble solids content (SSC, ºBrix) 8.3±0.1

Microbial load

Total aerobic plate count (TAPC, Log10[cfu.g-1]) 5.8±0.1

Lactic acid bacteria (LAB, Log10[cfu.g-1]) 3.5±0.1

Yeast and mold (Y&M, Log10[cfu.g-1]) 3.2±0.2

Table App. 19 Effect of abiotic stress treatments and packaging films on O2 and CO2

concentrations during low temperature storage (5 C, 14 days) (univariate test of significance).

O2 CO2

df SS MS F p SS MS F p

T 3 599.38 199.79 5.3926 0.001404 3069.93 1023.31 11.8454 0.000000

PF 1 2646.27 2646.27 71.4249 0.000000 7359.42 7359.42 85.1895 0.000000

T*PF 3 169.61 56.54 1.5259 0.209279 565.96 188.65 2.1838 0.091463

Error 184 6817.14 37.05 15895.53 86.39

Total 191 10232.39 26890.84

T – Independent variable “Treatment”; PF – independent variable “Packaging film”; T*PF – interaction effect of “Treatment”*”Packaging film”; SS – Sum of squares; df – degrees of freedom; MS – Mean squares; F- F test; p – significance at 95%.

256

Table App. 20 Effect of abiotic stress treatments and packaging films on total phenolic content

(TPC) and on chlorogenic acid (CA) content during low temperature storage (5 C, 14 days) (univariate test of significance).


Table App. 21 Effect of abiotic stress treatments and packaging films on total carotenoid (TCC)

and -carotene content during low temperature storage (5 C, 14 days) (univariate test of significance).

TCC -carotene


T 3 3158.4 1052.8 1.445 0.231677 57.44 19.15 1.103 0.349505

PF 1 21828.7 21828.7 29.965 0.000000 38.29 38.29 2.206 0.139409

T*PF 3 694.2 231.4 0.318 0.812584 47.67 15.89 0.916 0.434809

Error 160 116554.9 728.5

2776.38 17.35

Total 167 142236.3


TPC CA


T 3 3619,7 1206,6 1,598 0,192079 186.497 62.166 7.0252 0.000181

PF 1 49251,5 49251,5 65,215 0,000000 1081.686 1081.686 122.2391 0.000000

T*PF 3 1377,0 459,0 0,608 0,610876 100.080 33.360 3.7699 0.011934

Error 160 120834,7 755,2

1415.829 8.849

Total 167 175082,9

2784.092

257

Table App. 22 Effect of abiotic stress treatments and packaging films on hydrophilic (AOxH),

lipophilic (AOxL) and total (AOxT) antioxidant capacity during low temperature storage (5 C, 14 days) (univariate test of significance).

AOxH AOxL


T 3 3158.4 1052.8 1.445 0.231677 57.44 19.15 1.103 0.349505

PF 1 21828.7 21828.7 29.965 0.000000 38.29 38.29 2.206 0.139409

T*PF 3 694.2 231.4 0.318 0.812584 47.67 15.89 0.916 0.434809

Error 160 116554.9 728.5

2776.38 17.35

Total 167 142236.3

AOxT

df SS MS F p

T 3 2924 975 1.418 0.239427

PF 1 23695 23695 34.484 0.000000

T*PF 3 404 135 0.196 0.899083

Error 160 109941 687

Total 167 136964


258

Table App. 23 Changes in total antioxidant capacity of shredded carrot samples submitted to heat shock and UV-C stress treatments, single and combined application during low temperature MAP-

and PM-MAP storage (5 C, 14 days).

Treatment Packaging film Storage time (days) AOxT (mg TE.100 g-1)

Control (C)

(untreated samples)

A

0 84.0cdefghijk±2.2

3 73.0abcdefgh±4.1

5 72.6abcdefgh±1.9

7 69.2abcd±8.9

10 70.2abcdef±10.0

12 68.4abc±2.9

14 63.0a±1.1

B

0 80.7bcdefghij±3.2

3 79.5abcdefghij±8.3

5 87.4ghijklm±1.6

7 165.3p±5.8

10 86.7efghijkl±3.5

12 86.9fghijkl±7.4

14 99.0klmno±2.4

Heat shock (H) 100 C/45 s;

single application

A

0 85.1cdefghijkl±4.6

3 76.8abcdefgh±7.4

5 78.9abcdefghij±1.3

7 152.9p±5.3

10 70.6abcdef±9.2

12 70.0abcde±1.8

14 73.7abcdefgh±0.7

B

0 85.0cdefghijkl±5.6

3 74.6abcdefgh±3.6

5 104.1mno±1.5

7 189.6q±3.7

10 88.8hijklmn±8.8

12 85.4defghijkl±2.6

14 109.9o±0.9

(cont.)

259

Table App. 23 (cont.) Changes in total antioxidant capacity of shredded carrot samples submitted to heat shock and UV-C stress treatments, single and combined application during low temperature

MAP- and PM-MAP storage (5 C, 14 days).

Treatment Packaging film Storage time (days) AOxT (mg TE.100 g-1)

UV-C (U) 2.5 kJ.m-2;

single application

A


3 72.7abcdefgh±4.1

5 68.6abc±3.2

7 80.2bcdefghij±4.2


12 70.4abcdef±3.0


B


3 76.4abcdefgh±2.9

5 81.1cdefghij±5.2

7 167.4p±1.1

10 95.1jklmno±5.3

12 99.0klmno±4.2

14 101.7lmno±0.7

Heat shock 100 C/45 s x UV-C 2.5 kJ.m-2 (HU);

combined application

A


3 77.9abcdefghi±3.4

5 64.0ab±1.9

7 104.2no±16.9

10 72.0abcdefg±6.5



B

0 81.2cdefghij±2.2

3 77.6abcdefgh±5.8

5 94.5ijklmno±1.5

7 186.2q±6.1

10 107.9o±3.5

12 88.6ghijklmn±0.1

14 88.1ghijklmn±4.1

Values represent the mean of three replicates±SD. Different letters represent significant differences at p=0.05 (Tukey HSD test).

260

Table App. 24 Effect of abiotic stress treatments and packaging films on phenylalanine ammonia

lyase (PAL) and peroxidase (POD) activities during low temperature storage (5 C, 14 days) (univariate test of significance).

PAL POD


T 3 12929 4310 1,5968 0,192274 18512,9 6171,0 136,497 0,000000

PF 1 652331 652331 241,6953 0,000000 360,5 360,5 7,974 0,005349

T*PF 3 6665 2222 0,8231 0,482905 968,8 322,9 7,143 0,000156

Error 160 431837 2699 7233,5 45,2

Total 167 1103762 27075,7


Table App. 25 Estimated parameters of the TPC model proposed by Amodio et al. (2014) applied to fresh-cut carrot samples (untreated, heat-treated, UV-treated and combined-treated) packaged

with two films (A and B) during storage at 5 C.

Model parameter estimates

Model

Ctr x film A samples

[TPC0 = 57.6 ±3.2 mg CAE.100 g-1]

Ctr x film B samples

[TPC0 = 56.3 ±2.2 mg CAE.100 g-1]

TPCpre0 0.1788 ± 126 (p>0.05) 0.0767 ± 51 (p>0.05)

k1 0.1786 ± 126 (p>0.05) 0.0766 ± 51 (p>0.05)

k2 136.4390 ± 136691 (p>0.05) 205.2 ± 174439 (p>0.05)

HS x film A samples

[TPC0 = 53.9 ±2.7 mg CAE.100 g-1]

HS x film B samples

[TPC0 = 55.4 ±3.5 mg CAE.100 g-1]

TPCpre0 0.1879 ± 158 (p>0.05) 0.0585 ± 327 (p>0.05)

k1 0.1879± 158 (p>0.05) 0.0585± 327 (p>0.05)

k2 171.5 ± 189808 (p>0.05) 263.7 ± 1779053 (p>0.05)

UV x film A samples

[TPC0 = 54.0 ±2.1 mg CAE.100 g-1]

UV x film B samples

[TPC0 = 56.2 ±2.5 mg CAE.100 g-1]

TPCpre0 0.1410 ± 61 (p>0.05) 0.0359 ± 325 (p>0.05)

k1 0.1404± 60 (p>0.05) 0.0360± 326 (p>0.05)

k2 128 ± 78433 (p>0.05) 241 ± 2688482 (p>0.05)

[HS x UV] x film A samples

[TPC0 = 52.7 ±1.6 mg CAE.100 g-1]

[HS x UV] x film B samples

[TPC0 = 50.4 ±0.6 mg CAE.100 g-1]

TPCpre0 0.1878 ± 467 (p>0.05) 0.0682 ± 142 (p>0.05)

k1 0.1878 ± 467 (p>0.05) 0.0682 ± 142 (p>0.05)

k2 174 ± 564899 (p>0.05) 290 ± 708684 (p>0.05)


261

Table App. 26 Relevant regression analysis results from fitting Eq. 6 to the TPC data from fresh-cut carrot samples (untreated, heat-treated, UV-treated and combined-treated) packaged with two films (A and B).

Regression analysis


Ctr x film A 13.4 0.853 111.46 (Figure App. 3)

Ctr x film B 13.3 0.723 259.19 (Figure App. 4)

HS x film A 15.5 0.845 179.25 (Figure App. 5)

HS x film B 20.7 0.766 541.70 (Figure App. 6)

UV x film A 18.3 0.656 164.59 (Figure App. 7)

UV x film B 14.3 0.741 361.42 (Figure App. 8)

[HS UV] x film A 25.5 0.687 552.77 (Figure App. 9)

[HS UV] x film B 19.8 0.806 571.47 (Figure App. 10)


𝑁∗ ∑

𝑀𝑖−𝑀𝑝

𝑀𝑖



Figure App. 3 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from Ctr x film A samples.

Figure App. 4 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from Ctr x film B samples.

Figure App. 5 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from HS x film A samples.


-25 -20 -15 -10 -5 0 5 10 15 20 25

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


25 30 35 40 45 50 55 60 65 70 75 80 85

Predicted Values

-25

-20

-15

-10

-5

0

5

10

15

20

25

Re

sid

ua

l V

alu

es

b)


-40 -30 -20 -10 0 10 20 30 40

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


50 55 60 65 70 75 80 85 90 95 100 105

Predicted Values

-40

-30

-20

-10

0

10

20

30

40

Re

sid

ua

l V

alu

es

b)


-35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


30 35 40 45 50 55 60 65 70 75 80 85 90 95

Predicted Values

-35

-30

-25

-20

-15

-10

-5

0

5

10

15

20

25

30

Re

sid

ua

l V

alu

es

b)

262

Figure App. 6 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from HS x film B samples.

Figure App. 7 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from UV x film A samples.

Figure App. 8 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from UV x film B samples..

Figure App. 9 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from [HSxUV] x film A samples.

Figure App. 10 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from [HSxUV] x film B samples.


-50 -40 -30 -20 -10 0 10 20 30 40

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


40 50 60 70 80 90 100 110 120 130

Predicted Values

-50

-40

-30

-20

-10

0

10

20

30

40

Re

sid

ua

l V

alu

es

b)


-30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


35 40 45 50 55 60 65 70 75

Predicted Values

-30

-25

-20

-15

-10

-5

0

5

10

15

20

25

30

Re

sid

ua

l V

alu

es

b)


-30 -20 -10 0 10 20 30 40 50 60

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


50 60 70 80 90 100 110 120

Predicted Values

-30

-20

-10

0

10

20

30

40

50

60

Re

sid

ua

l V

alu

es

b)


-40 -30 -20 -10 0 10 20 30 40 50 60 70

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


30 35 40 45 50 55 60 65 70 75 80 85 90 95

Predicted Values

-40

-30

-20

-10

0

10

20

30

40

50

60

70

Re

sid

ua

l V

alu

es

b)


-60 -50 -40 -30 -20 -10 0 10 20 30 40 50

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


40 50 60 70 80 90 100 110 120 130 140

Predicted Values

-60

-50

-40

-30

-20

-10

0

10

20

30

40

50

Re

sid

ua

l V

alu

es

b)

263

Table App. 27 Effect of abiotic stress treatments and packaging films on pH and soluble solids

content (SSC) during low temperature storage (5 C, 14 days) (univariate test of significance).

pH SSC

Df SS MS F p SS MS F p

T 3 5,288 1,763 7,13 0,000160 8,95 2,98 13,13 0,000000

PF 1 0,081 0,081 0,33 0,566807 0,23 0,23 1,01 0,317025

T*PF 3 0,372 0,124 0,50 0,682297 0,59 0,20 0,87 0,459218

Error 160 39,579 0,247 36,34 0,23

Total 167 45,320 46,11


Table App. 28 Changes in soluble solids content (SSC, ºBrix) during storage (14 days, 5 C) as affected by abiotic stress treatments and packaging film.

Treatment Packaging Film Storage time (days) SSC (ºBrix)

Control (C)

(untreated samples)

A

0 8.5bcdefgh±0.1

3 8.3abcdefgh±0.3

5 7.3a±0.4

7 7.3a±0.2

10 8.2abcdefg±0.5

12 7.7abc±0.2

14 7.3a±0.2

B

0 8.5bcdefgh±0.1

3 8.3abcdefgh±0.5

5 8.2abcdefg±0.8

7 7.5ab±0.3

10 7.3a±0.9

12 8.2abcdefg±0.2

14 8.4bcdefgh±0.0

Heat shock (H) 100 C/45 s;

single application

A

0 8.1abcdef±0.1

3 8.2abcdefg±0.0

5 8.3abcdefgh±0.5

7 8.1abcdef±0.5

10 8.2abcdefg±0.3

12 9.3h±0.3

14 9.1fgh±0.2

B

0 8.1abcdef±0.1

3 8.3abcdefgh±0.6

5 8.3abcdefgh±0.2

7 8.0abcde±0.0

10 8.2abcdefg±0.3

12 8.9efgh±0.2

14 9.0efgh±0.0

(cont.)

264

Table App. 28 (cont.) Changes in soluble solids content (SSC, ºBrix) during storage (14 days,

5 C) as affected by abiotic stress treatments and packaging film.

Treatment Packaging Film Storage time (days) SSC (ºBrix)

UV-C (U) 2.5 kJ.m-2;

single application

A

0 8.5bcdefgh±0.1

3 8.3abcdefgh±0.1

5 8.3abcdefgh±0.3

7 8.1abcdef±0.1

10 8.5bcdefgh±0.2

12 8.2abcdefg±0.2

14 8.0abcde±0.2

B

0 8.7cdefgh±0.1

3 8.3abcdefgh±0.1

5 7.7abcd±0.5

7 8.1abcdefg±0.1

10 8.6cdefgh±0.3

12 8.6cdefgh±0.2

14 7.8abcd±0.0

Heat shock 100 C/45 s x UV-C 2.5 kJ.m-2 (HU);


A

0 8.7cdefgh±0.1

3 8.2abcdefg±0.0

5 8.2abcdefg±0.2

7 8.6cdefgh±0.3

10 8.5bcdefgh±0.5

12 8.8defgh±0.2

14 8.5bcdefgh±0.2

B

0 8.1abcdef±0.1

3 8.2abcdefg±0.2

5 8.5bcdefgh±0.6

7 8.1abcdefg±0.1

10 8.8defgh±0.3

12 9.2gh±0.3

14 9.1fgh±0.2

Values represent the mean of three replicates±SD. Different letters represent significant differences at p=0.05 (Tukey HSD test).

265

Table App. 29 Effect of abiotic stress treatments and packaging films on whiteness index (WI) color

parameter during low temperature storage (5 C, 14 days) (univariate test of significance).

WI

df SS MS F p

T 3 151 50 7,3 0,000072

PF 1 1300 1300 188,5 0,000000

T*PF 3 55 18 2,7 0,046205

Error 2092 14433 7

Total 2099 15946


266

Table App. 30 Changes in color parameters during storage (14 days, 5 C) as affected by abiotic stress treatments and packaging film.

Treatment Packaging

film Storage time

(days) L* a* b* C* hue WI E

Control (C)

(untreated samples)

A

0 54.0abcde±4.4 25.8abcdefg±1.7 49.0abcdefghij±3.5 55.4abcdefgh±3.8 62.2abcdefgh±0.9 27.8bcdefg±2.0 5.1abc±2.8

3 54.6abcde±4.6 26.8bcdefgh±1.2 50.0abcdefghijk±2.9 56.8abcdefghij±3.0 61.8abcdefg±1.0 27.1abcdefg±2.0 4.9abc±3.0

5 55.7abcde±3.8 26.5bcdefgh±2.0 50.3defghijk±3.5 56.9cdefghij±3.9 62.2abcdefg±1.0 27.8bcdefg±2.2 5.2abc±2.8

7 57.3cde±3.5 26.5bcdefgh±1.8 50.7efghijk±2.2 57.3cdefghij±2.5 62.5cdefgh±1.1 28.5cdefgh±2.4 5.2abc±2.4

10 54.2abcd±4.5 26.3bcdefgh±1.9 50.2cdefghijk±3.1 56.7cdefghij±3.5 62.3bcdefgh±0.8 26.9abcd±2.1 5.2abc±2.9

12 57.6de±3.3 27.0fgh±3.3 51.2fghjk±6.4 57.9efgij±7.2 62.1abcdef±1.4 28.0cdefg±4.8 7.1bc±5.5

14 58.0e±3.1 27.8h±1.6 52.6k±2.5 59.5j±2.8 62.1abcdefg±1.1 27.1abcdef±1.8 6.5abc±3.0

B

0 54.7abcde±3.6 25.0abcde±1.6 48.8abcdefghij±2.7 54.9abcdefgh±3.0 62.9efgh±0.9 28.8cdefgh±2.1 4.1ab±2.3

3 57.4abcde±3.3 26.8bcdefgh±1.4 50.2abcdefghijk±2.0 57.0abcdefghij±2.3 61.9abcdefg±0.6 28.8cdefgh±2.1 4.9abc±2.2

5 54.3abcd±7.0 25.5abcdef±2.2 48.9abcdefgij±2.8 55.2abcdefh±3.2 62.5cdefgh±1.7 28.1cdefg±4.4 5.7abc±5.3

7 56.1abcde±4.1 25.5abcdef±1.9 48.9abcdefij±2.6 55.1abcdeh±3.1 62.5cdefgh±1.0 29.4fgh±2.5 4.8ab±2.6

10 54.3abcd±3.6 24.4a±1.6 47.6abc±2.9 53.5ab±3.2 62.9egh±0.9 29.5fgh±2.1 4.5ab±2.6

12 56.3abcde±3.9 25.7abcdefg±2.1 49.3abcdefghij±3.2 55.6abcdefgh±3.7 62.5cdefgh±1.0 29.2efgh±2.4 5.3abc±2.4

14 56.8bcde±3.3 25.6abcdefg±1.6 49.2abcdefghij±2.2 55.5abcdefgh±2.6 62.5cdefgh±0.8 29.6gh±2.4 4.3a±2.3

(cont.)

267

Table App. 30 (cont.) packaging film.

Treatment Packaging

film Storage time


Heat shock (H)

100 C/45 s;

single application

A

0 54.9abcde±3.9 26.2abcdefgh±1.7 50.6cdefghijk±2.6 57.0cdefghij±3.0 62.6cdefgh±0.8 27.2abcdefg±2.3 4.8abc±2.2

3 56.5abcde±3.2 26.9bcdefgh±1.3 51.3cdefghijk±2.4 57.9cdefghij±2.6 62.4abcdefgh±0.7 27.5abcdefgh±2.3 4.9abc±2.1

5 54.8abcde±4.2 25.8abcdefg±2.0 49.7bcdefghij±3.3 56.0abcdefgh±3.7 62.6cdefgh±1.0 27.9cdefg±2.6 5.0ab±2.9

7 55.7abcde±2.9 26.9efgh±1.6 51.6hk±2.7 58.2fgij±3.1 62.5cdefgh±0.8 26.8abcd±1.8 4.8ab±2.6

10 55.5abcde±2.9 26.5bcdefgh±2.3 49.8bcdefghij±2.7 56.4bcdefghi±3.3 62.0abcdef±1.3 28.1cdefg±2.6 4.3a±2.4

12 55.5abcde±4.2 26.5bcdefgh±1.6 50.3defghijk±2.5 56.9cdefghij±2.8 62.2abcdefg±0.9 27.6bcdefg±2.0 5.0ab±2.3

14 56.3abcde±3.7 26.3bcdefgh±1.9 50.6defghijk±3.1 57.1cdefghij±3.6 62.5cdefgh±0.8 28.0cdefg±2.3 5.1abc±2.8

B

0 53.2abc±4.8 25.9abcdefgh±2.0 49.4abcdefghij±2.8 55.8abcdefghi±3.3 62.3abcdefgh±1.2 27.0abcdef±2.8 5.3abc±2.6

3 57.8abcde±3.8 26.7abcdefgh±2.9 50.6bcdefghijk±4.1 57.2abcdefghij±4.9 62.2abcdefgh±1.3 28.7bcdefgh±3.0 6.5abc±3.4

5 54.1abcd±4.3 26.3bcdefgh±1.9 48.9abcdefgij±3.0 55.5abcdefgh±3.4 61.8abcd±0.9 27.8bcdefg±2.7 4.9ab±2.6

7 56.6abcde±3.4 26.7bcdefgh±1.9 49.9cdefghijk±3.1 56.6cdefghij±3.5 61.9abcdf±0.8 28.5cdefgh±2.2 5.0ab±2.5

10 53.3ab±4.4 25.0ab±1.7 46.8a±2.5 53.1a±2.9 61.9abcdf±1.0 29.2efgh±2.7 4.9ab±3.1

12 56.4abcde±3.9 26.1bcdefgh±1.7 49.1abcdefghij±2.7 55.6abcdefgh±3.0 62.0abcdef±1.1 29.2efgh±2.2 4.9ab±2.4

14 55.9abcde±4.7 25.1abc±2.3 47.1ab±3.3 53.4ab±3.8 62.0abcdef±1.3 30.5h±2.7 5.5abc±3.6

(cont.)

268

Table App. 30 (cont.)

Treatment Packaging

film Storage time


UV-C (U) 2.5 kJ.m-2;

single application

A

0 54.1abcde±4.2 26.0abcdefgh±1.6 49.9bcdefghijk±2.5 56.3abcdefghij±2.9 62.5cdefgh±0.8 27.2abcdefg±2.3 4.5ab±2.8

3 54.7abcde±14.8 26.8bcdefgh±6.6 52.6ghjk±5.8 59.2efgij±7.6 63.6h±5.7 24.1a±7.6 11.6d±13.1

5 55.0abcde±4.3 27.0fgh±1.7 51.1fghjk±3.0 57.8efgij±3.3 62.2abcdefg±0.8 26.6abc±2.0 5.6abc±2.4

7 56.3abcde±3.3 27.1fgh±1.5 51.6hk±2.1 58.3gij±2.4 62.4cdefgh±1.0 27.1abcdef±2.2 5.2abc±2.1

10 54.2abcd±3.6 26.8cdefgh±2.0 50.2cdefghijk±3.1 56.9cdefghij±3.6 62.0abcdef±0.8 26.8abcd±2.1 4.9ab±2.3

12 54.6abcde±7.7 27.1fgh±2.4 51.6ghk±3.9 58.3gij±4.4 62.3abcdefgh±1.1 25.7ab±3.6 7.7c±5.4

14 58.1e±2.7 27.3gh±2.2 52.6k±2.5 59.2ij±3.0 62.6defgh±1.3 27.4bcdef±2.7 6.4abc±2.7

B

0 54.5abcde±4.6 25.7abcdefgh±2.0 49.3abcdefghijk±3.1 55.6abcdefghij±3.5 62.5abcdefgh±0.9 28.0bcdefgh±3.1 5.0abc±2.8

3 57.6abcde±3.4 27.3bcdefgh±1.9 51.6defghijk±3.2 58.3cdefghij±3.6 62.1abcdefgh±0.9 27.8abcdefgh±2.6 6.0abc±2.9

5 54.1abcd±4.4 25.7abcdefg±2.0 49.0abcdefgij±3.4 55.3abcdefgh±3.8 62.3abcdefgh±1.1 27.9cdefg±1.9 4.9ab±3.2

7 55.8abcde±3.4 26.1abcdefg±2.1 49.5bcdefghij±3.1 56.0abcdefgh±3.6 62.3abcdefgh±0.8 28.5cdefgh±2.3 4.5a±2.8

10 54.3abcd±3.5 25.0ab±1.8 48.3abcde±2.9 54.4abcd±3.3 62.6defgh±0.9 28.9defgh±2.4 4.3a±2.3

12 55.9abcde±3.7 25.7abcdefg±1.9 48.7abcdefi±3.4 55.1abcdeh±3.7 62.2abcdefg±1.1 29.3fgh±2.2 4.5ab±3.3

14 56.0abcde±3.7 25.8abcdefg±2.1 48.7abcdefi±3.3 55.1abcdeh±3.7 62.1abcdef±1.2 29.3fgh±2.3 4.7ab±3.1

(cont.)

269

Table App. 30 (cont.)

Treatment Packaging

film Storage time


Heat shock

100 C/45 s x UV-C

2.5 kJ.m-2 (HU);


A

0 54.1abcde±4.8 26.9bcdefgh±1.5 51.2cdefghijk±2.8 57.8cdefghij±2.9 62.3abcdefgh±1.2 26.0abcd±2.0 5.9abc±2.0

3 57.4abcde±3.2 27.3bcdefgh±1.2 52.5ghjk±1.8 59.2efgij±1.9 62.6abcdefgh±0.8 27.0abcdefg±2.2 6.0abc±2.1

5 53.6ab±3.2 26.6bcdefgh±1.6 49.9cdefghij±2.5 56.5cdefghij±2.8 62.0abcdef±1.0 26.8abcd±2.0 4.3a±1.8

7 54.4abcd±3.9 26.8defgh±2.0 50.6defghijk±3.2 57.3defghij±3.7 62.1abcdefg±1.0 26.7abc±2.6 5.0ab±3.0

10 53.7ab±3.4 26.4bcdefgh±1.8 49.6bcdefghij±3.0 56.2bcdefghi±3.4 62.0abcdef±0.8 27.1abcdef±2.0 4.3a±2.5

12 56.3abcde±2.9 27.1fgh±2.0 50.4defghijk±3.1 57.2cdefghij±3.6 61.8abcdf±0.9 27.9cdefg±2.1 4.6ab±3.0

14 55.6abcde±4.1 26.6bcdefgh±1.6 49.5bcdefghij±2.8 56.3bcdefghi±3.0 61.8abcd±1.0 28.2cdefg±2.7 5.0ab±2.2

B

0 55.9abcde±4.8 25.5abcdefgh±2.1 50.9cdefghijk±3.3 57.0abcdefghij±3.7 63.4gh±1.4 27.8abcdefgh±2.4 6.0abc±2.7

3 56.7abcde±2.3 27.4bcdefgh±1.8 52.0fghijk±2.8 58.8efghij±3.3 62.3abcdefgh±0.6 26.9abcdefg±2.8 5.3abc±2.5

5 53.2a±4.7 25.8abcdefg±1.8 48.2abcde±3.5 54.7abcdh±3.9 61.8abcd±0.8 27.8bcdefg±1.9 5.1abc±3.7

7 55.6abcde±3.5 26.2bcdefgh±1.8 48.4abcdei±2.9 55.0abcdeh±3.3 61.6abc±1.1 29.1efgh±2.3 4.4a±2.4

10 53.4ab±4.7 25.2abcd±2.0 46.7a±3.2 53.1a±3.6 61.7abcd±1.2 29.2efgh±2.4 5.6abc±3.2

12 55.5abcde±4.5 26.0abcdefg±2.2 47.6abc±3.9 54.2abc±4.3 61.4ab±1.5 29.6gh±2.6 5.7abc±3.1

14 55.5abcde±3.8 26.2bcdefgh±2.0 48.0abcd±3.0 54.7abcdh±3.3 61.3a±1.4 29.3fgh±2.0 5.0ab±2.2

Values represent the mean of 15 meeasurments±SD. In the same column, different letters represent significant differences at p=0.05 (Tukey HSD test).

270

Table App. 31 Effect of abiotic stress treatments and packaging films on the sensorial attributes of color, fresh-like appearance and fresh-like aroma and also respective rejection index during low


Color Fresh-like appearance


T 3 20,8273 6,9424 22,715 0.000000

95,194 31,731 29,397 0.000000

PF 1 0,7508 0,7508 2,457 0,118052

1,25 1,25 1,158 0,282703

T*PF 3 0,4648 0,1549 0,507 0,677737

0,169 0,056 0,052 0,984279

Error 312 95,3562 0,3056

336,775 1,079

Total 319 117,3992

433,388

Fresh-like aroma Rejection index


T 3 183,328 61,109 39,351 0.000000

100,652 33,551 26,434 0.000000

PF 1 4,278 4,278 2,755 0,097963

2,195 2,195 1,729 0,189498

T*PF 3 0,253 0,084 0,054 0,983297

0,077 0,026 0,02 0,996063

Error 312 484,513 1,553

395,994 1,269

Total 319 672,372

498,918


Table App. 32 Effect of abiotic stress treatments and packaging films on the total aerobic plate counts (TAPC), lactic acid bacteria counts (LAB) and yeast and mold counts (Y&M) during low


TAPC LAB


T 3 127.873 42.624 11.971 0.000001

231.758 77.253 19.3191 0.000000

PF 1 0.481 0.481 0.135 0.713812

10.443 10.443 2.6116 0.108901

T*PF 3 0.179 0.060 0.017 0.997037

22.258 7.419 1.8554 0.141275

Error 112 398.791 3.561

447.861 3.999

Total 119 527.324

712.320

Y&M

df SS MS F p

T 3 99.414 33.138 23.6842 0.000000

PF 1 3.040 3.040 2.1728 0.143277

T*PF 3 3.505 1.168 0.8350 0.477391

Error 112 156.707 1.399

Total 119 262.666


271

Table App. 33 Variable and sample codes used for hierarchical cluster analysis and PCA analysis for overall quality assessment (except sensorial data).

Variables Codes

Total phenolic content TPC PAL activity PAL Total aerobic plate counts TAPC Lactic acid bacteria counts LAB Yeasts and molds counts Y&M Oxygen concentration O2 Carbon dioxide concentration CO2 pH pH General acceptance Acceptance

Samples Codes

Control (untreated) packed in film A_day 0-14 CA0; CA3; CA5; CA7; CA10; CA12; CA14 Control (untreated) packed in film B_day 0-14 CB0; CB3; CB5; CB7; CB10; CB12; CB14 Heat-treated packed in film A_day 0-14 HA0; HA3; HA5; HA7; HA10; HA12; HA14 Heat-treated packed in film B_day 0-14 HB0; HB3; HB5; HB7; HB10; HB12 ;HB14 UV-treated packed in film A_day 0-14 UA0; UA3; UA5; UA7; UA10; UA12; UA14 UV-treated packed in film B_day 0-14 UB0; UB3; UB5; UB7; UB10; UB12; UB14 HS x UV-treated packed in film A_day 0-14 HUA0; HUA3; HUA5; HUA7; HUA10; HUA12; HUA14 HS x UV-treated packed in film B_day 0-14 HUB0; HUB3; HUB5; HUB7; HUB10; HUB12; HUB14

Figure App. 11 Fresh-cut shredded carrot samples grouping as determined by cluster analysis. [CAi:

untreated samples packaged with film A; CBi: untreated samples packaged with film B; HAi: heat-treated (100 C/45 s)

samples packaged with film A; HBi: heat-treated (100 C/45 s) samples packaged with film B; UAi: UV-treated (2.5 kJ.m-

2) samples packaged with film A; UBi: UV-treated (2.5 kJ.m-2) samples packaged with film B; HUAi: combined-treated

(100 C/45 s x 2.5 kJ.m-2) samples packaged with film A; HUBi: combined-treated (100 C/45 s x 2.5 kJ.m-2) samples packaged with film B; i stands for storage day].

f

UA

14

CA

14

CA

12

UB

10

UB

12

UB

14

CB

14

CB

12

CB

10

CB

7U

A10

UA

12

CA

10

UA

7C

A7

HU

A14

HU

A12

HA

14

HA

12

HU

A10

HA

10

UA

5C

A5

UA

3C

A3

HU

B14

HB

14

HB

12

HU

B12

HU

B10

HB

10

HU

B7

HB

7U

B7

UB

5C

B5

UB

3C

B3

HU

A7

HA

7H

UB

5H

B5

HU

A5

HA

5H

UA

3H

A3

HU

B3

HB

3H

UB

0H

UA

0H

B0

HA

0U

B0

UA

0C

B0

CA

0

0

2

4

6

8

10

Lin

ka

ge

Dis

tan

ce

Group A

A1 A2 B1 B2

Group B

272

Proposed technological alternative effects on fresh-cut carrot quality and shelf-life

Table App. 34 Parameter estimates (±SE) of the modified Gompertz model and predicted shelf-life

at 5 C of chlorine-treated fresh-cut shredded carrot samples packed with a micro-perforated film.

Gompertz parameter estimates (R2=0.978) Predicted shelf-life*

k A µmax

-11.1 ± 77.4

p<0.001

21.5 ± 78.8

p<0.001

1.0 ±2.8

p<0.001

-15.7 ± 46.4

p>0.05 7.5

Gompertz equation parameters (±SE, followed by respective p-value): k, initial load (Log10 cfu.g-1); A, maximum cell increase attained at the

stationary phase (Log10 cfu.g-1); µmax, maximal growth rate (Log10 cfu.g-1 per day); , lag phase (days). * - Predicted shelf-life as the time (days) necessary to attain a 7.5 Log10 cfu.g-1 level. R2 - regression determination coefficient.

Table App. 35 Relevant regression analysis results from fitting Eq. 4 to the TAPC data from heat-treated and chlorine-treated fresh-cut shredded carrot samples packed with a micro-perforated film

during low temperature storage (5 C; 14 days).

Sample type Regression analysis

Ē% R2 MSE Randomness of residuals

HS 7.7 0.978 0.2211 (Figure App. 12)

HIPO 2.7 0.994 0.0657 (Figure App. 13)


𝑁∗ ∑

𝑀𝑖−𝑀𝑝

𝑀𝑖



Figure App. 12 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from HSxFilm B samples.

Figure App. 13 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from HIPOxFilm B samples.


-0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0

Predicted Values

-0,8

-0,6

-0,4

-0,2

0,0

0,2

0,4

0,6

0,8

Re

sid

ua

l V

alu

es

b)


-0,7 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 0,5

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


3 4 5 6 7 8 9 10

Predicted Values

-0,7

-0,6

-0,5

-0,4

-0,3

-0,2

-0,1

0,0

0,1

0,2

0,3

0,4

0,5

Re

sid

ua

l V

alu

es

273

Table App. 36 Estimated parameters of the TPC model proposed by Amodio et al. (2014) applied to heat-treated and chlorine-treated fresh-cut carrot samples packaged with a micro-perforated film

(film B) during storage at 5 C.

Model parameter estimates HS x film B samples

[TPC0 = 55.4 ±3.5 mg CAE.100 g-1]

HIPO x film B samples

[TPC0 = 36.8 ±0.6 mg CAE.100 g-1]

TPCpre0 0.0585 ± 327 (p>0.05) 0.08688 ± 205 (p>0.05)

k1 0.0585± 327 (p>0.05) 0.08688 ± 204 (p>0.05)

k2 263.7 ± 1779053 (p>0.05) 88.6 ± 297400 (p>0.05)


Table App. 37 Relevant regression analysis results from fitting Eq. 6 to the TPC data from from heat-treated and chlorine-treated fresh-cut shredded carrot samples packed with a micro-perforated

film during low temperature storage (5 C; 14 days).

Regression analysis


HS samples 20.7 0.766 541.70 (Figure App. 6)

HIPO samples 31.7

0.380 338.018

(Error! Reference source not found.)


𝑁∗ ∑

𝑀𝑖−𝑀𝑝

𝑀𝑖



Figure App. 14 a) Normal probability plot and b) randomness of residuals from the TPC model fitting from HIPOxFilm B samples.


-30 -20 -10 0 10 20 30 40

Residuals

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Ex

pe

cte

d N

orm

al

Va

lue

0.01

0.05

0.15

0.30

0.50

0.70

0.85

0.95

0.99


34 36 38 40 42 44 46 48 50 52

Predicted Values

-30

-20

-10

0

10

20

30

40

Re

sid

ua

l V

alu

es

b)

heat shock and uv-c abiotic stress treatments as alternative tools to

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