faculty of bioscience engineering …...experiment for a period of 6 months was designed to...

FACULTY OF BIOSCIENCE ENGINEERING

Interuniversity Programme

Master of Science in Food Technology (IUPFOOD)

Accelerated shelf life study to assess the stability of

carotenoids in relation to lipid oxidation and their

bioaccessibility in shelf stable fruit and vegetable based

systems

Promoter: Prof. Dr. Ir. Marc Hendrickx Dissertation presented in fulfillment of the requirements for the degree of Department of Microbial and Molecular Systems Master of Science in Food Technology Center for Food and Microbial Technology Jeritah Tongonya

September 2015

This dissertation is part of the examination and has not been corrected for eventual errors after

presentation. Use as a reference is only permitted after consulting the promoter, stated on the front page.

ACKNOWLEDGEMENTS

i

ACKNOWLEDGEMENTS

I thank God for His sustenance throughout my life, for taking me this far. I cannot do without Him.

Firstly, I would like to express my sincere gratitude to my promotor, Prof. Dr. Ir. M. Hendrickx for the

continuous support of my thesis work. There are times when my thesis work got tough, but your patience,

motivation, and immense knowledge, gave me hope and kept me going. Your mentorship, not only during

my thesis work, but also as my lecturer in various courses, gave me a strong foundation for my future

aspirations as an academic.

Besides my promotor, I would like to thank Prof. A. Van Loey and Prof T. Grauwet for their insightful

comments and encouragements. The brief conversations and smiles went a long way in making the master

thesis a worthwhile challenge.

My heartfelt gratitude goes to my daily supervisor, Leonard Mutsokoti. Thank you for being my

supervisor. With your guidance, I am a better person, both academically and personally. In particular,

thank you for awakening the spirit of confidence in me which was dormant. To Dr A. Panozzo, I truly

appreciate your input in my thesis work and I learnt a lot from the few encounters I had with you.

To Heidi and Margot, your patience and ever present help in the lab was very welcome and I truly

appreciate it. It made my day to day tasks manageable, given I was not familiar with most of the

laboratory equipment in the beginning. Katrien and Lut, your warmth and kindness made administrative

concerns easy. Your door was always open, thank you very much.

The Laboratory of Food Technology has a great team of researchers. My gratitude goes to the post-

doctorates, PhD students, and my colleagues. We had lighter moments, both in and outside the lab

environment during the various extracurricular activities which include the laboratory weekend. This gives

me good memories which I will always cherish.

Last but not the least, I would like to thank my mother and father, my brothers and sister for supporting

me emotionally throughout the thesis work and my life in general.

ABSTRACT

ii

ABSTRACT

To date, carotenoid stability studies during storage have been done in both model and real food systems.

In the case of shelf stable fruit and vegetable based food systems, most studies focused on products that

have been processed in the absence of oil. Additionally, information on carotenoid bioaccessibility of

shelf stable food products during storage is scarce. Therefore, in the present study, an accelerated shelf life

experiment for a period of 6 months was designed to investigate the stability of lycopene and β-carotene in

tomato, as well as α- and β-carotene in carrot purees containing 5 % (w/w) olive oil. Furthermore, the

relation between carotenoid degradation and lipid oxidation as well as changes in carotenoid

bioaccessibility during storage were also considered. Hereto, tomato and carrot purees were in-pack

thermally processed (Tprocess=117 °C, F0= 5 minutes), and stored in the dark at isothermal temperatures of

20, 30, and 40 °C. Carotenoids were quantified by RP-HPLC-DAD. Lipid oxidation during storage was

monitored by measuring the peroxide value using the ferric thiocyanate spectrophotometric method and

hexanal by the HS-SPME-GC-MS standard addition method. Purees stored at 20°C were subjected to a

standardized static in vitro digestion method for the determination of carotenoid bioaccessibility.

Under the conditions of the study, most of the carotenoids were transferred to the oil phase and therefore,

carotenoid changes during storage were well represented by changes in the puree as a whole. The results

revealed that during storage, carotenoids and lipids in both carrot and tomato matrices were stable to

degradation. Although hexanal concentrations detected were small, temperature had an effect on hexanal

production, but no effect on both peroxide formation and carotenoid degradation. Furthermore, carotenoid

bioaccessibility was not influenced by storage time. On account of the insignificant lipid oxidation and the

observed carotenoid stability, the results suggested a direct relationship between these processes. By

acknowledging that carotenoid degradation and lipid oxidation depend largely on the food system and

storage conditions, it was concluded that the chosen food systems, processing and storage conditions in

the present study were sufficient to suppress lipid oxidation and carotenoid degradation.

TABLE OF CONTENTS

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................................... i

ABSTRACT .................................................................................................................................................. ii

TABLE OF CONTENTS ............................................................................................................................. iii

LIST OF FIGURES ...................................................................................................................................... vi

LIST OF TABLES ...................................................................................................................................... vii

LIST OF NOTATIONS ............................................................................................................................. viii

GENERAL INTRODUCTION ...................................................................................................................... 1

PART 1: LITERATURE REVIEW ............................................................................................................... 2

1. Carotenoids and lipids: Changes during processing and storage ........................................................... 3

1.1 Carotenoids....................................................................................................................................... 3

1.1.1 Chemistry, structure, solubility, size and shape ........................................................................ 3

1.1.2 Carotenoid localization.............................................................................................................. 4

1.1.3 Mechanism of and the factors influencing carotenoid degradation ........................................... 5

1.1.4 Carotenoid bioaccessibility ....................................................................................................... 7

1.1.5 Carotenoid degradation during storage ..................................................................................... 8

1.2 Lipids ................................................................................................................................................ 9

1.2.1 Structure and chemistry of acylglycerols .................................................................................. 9

1.2.2 Mechanism of lipid oxidation .................................................................................................. 10

1.2.3 Factors influencing lipid oxidation .......................................................................................... 12

1.2.3.1 Fatty acid composition ......................................................................................... 12

1.2.3.2 Temperature and oxygen concentration ............................................................... 12

1.2.3.3 Metals ................................................................................................................... 13

1.2.3.4 Light ..................................................................................................................... 13

TABLE OF CONTENTS

iv

1.2.3.5 Antioxidants ......................................................................................................... 13

2. PRINCIPLES OF ACCELERATED SHELF LIFE TESTING ........................................................... 15

2.1 Factors influencing shelf life .......................................................................................................... 15

2.1.1 Intrinsic factors ........................................................................................................................ 16

2.1.2 Extrinsic factors ....................................................................................................................... 16

2.2 Modes of food quality deterioration during storage ....................................................................... 17

2.2.1 Microbiological reactions ........................................................................................................ 17

2.2.2 Chemical reactions .................................................................................................................. 18

2.2.3 Physical reactions .................................................................................................................... 18

2.2.4 Enzymatic reactions ................................................................................................................ 18

2.3 Designing shelf life testing experiments ........................................................................................ 19

2.4 Basic principles of accelerated shelf life testing of foods .............................................................. 20

2.5 Approaches to accelerated shelf life testing ................................................................................... 20

2.5.1 Initial rate approach ................................................................................................................. 20

2.5.2 Kinetic model approach........................................................................................................... 21

3. CONCLUSION .................................................................................................................................... 23

PART II: EXPERIMENTAL WORK .......................................................................................................... 24

3. RESEARCH PLAN.............................................................................................................................. 25

4. MATERIALS AND METHODS ......................................................................................................... 27

4.1 Materials ......................................................................................................................................... 27

4.1.1 Raw material preparation ........................................................................................................ 27

4.1.2 Puree preparation ..................................................................................................................... 27

4.2 Thermal treatment .......................................................................................................................... 28

4.3 Sample storage and sampling plan for shelf life experiment ......................................................... 28

4.4 Experimental analysis .................................................................................................................... 29

TABLE OF CONTENTS

v

4.4.2 Carotenoid analysis ................................................................................................................. 29

4.4.3 Lipid oxidation analyses .......................................................................................................... 30

4.4.3.1 Determination of peroxide value. ......................................................................... 30

4.4.3.2 Determination of hexanal ..................................................................................... 31

4.4.4 Standardized static in vitro digestion for carotenoid bioaccessibility ..................................... 32

4.5 Data analysis .................................................................................................................................. 34

5. RESULTS AND DISCUSSION .......................................................................................................... 35

5.1 Effect of storage temperature and time on carotenoid stability ...................................................... 35

5.1.1 Percentage carotenoid transfer to the oil fraction .................................................................... 35

5.1.2 Carotenoid concentration changes in tomato puree during storage ......................................... 36

5.1.3 Carotenoid concentration changes in carrot puree during storage .......................................... 40

5.2 Effect of storage temperature and time on lipid oxidation ............................................................. 43

5.2.1 Changes in peroxide value during storage............................................................................... 43

5.2.2 Changes in hexanal during storage .......................................................................................... 44

5.2.3 Relation between carotenoid stability and lipid oxidation during storage .............................. 46

5.3 Changes in carotenoid bioaccessibility as influenced by storage time at 20 °C ............................. 46

6. GENERAL CONCLUSIONS .............................................................................................................. 48

REFERENCES ......................................................................................................................................... 50

LIST OF FIGURES

vi

LIST OF FIGURES

Figure 1. Structural formulas for β-carotene, α-carotene and lycopene ........................................................ 4

Figure 2. Possible scheme for carotenoid degradation (Rodriguez-Amaya et al. 2001) ............................... 6

Figure 3. Triacylglycerol molecule. * represents the chiral center. .............................................................. 9

Figure 4. Generalized scheme for autoxidation of lipids (Fennema 1996). ................................................ 11

Figure 5. Schematic representation of the experimental set-up ................................................................... 26

Figure 6. Percentage transfer of all-E-lycopene ( ) and all-trans-β-carotene ( ) from tomato puree and

all-trans-β-carotene ( ), all-trans-α-carotene ( )) from carrot puree to oil. Error bars represent standard

deviations. .................................................................................................................................................... 36

Figure 7. Changes in the concentration of (A) all-trans-lycopene and (B) all-trans-β-carotene as well as

cis isomers of lycopene: (C) 5-cis, (D) 9-cis and (E) 13-cis, expressed as absolute concentration, µg/g

puree, in tomato puree during storage at 20 °C ( ), 30 °C ( ) and 40 °C ( ). Error bars represent standard

deviations. .................................................................................................................................................... 37

Figure 8. Changes in the concentration of (A) all-trans-β-carotene and (B) all-trans-α-carotene and the cis

isomers of β-carotene: (C) 9-cis, (D) 13-cis and (E) 15-cis expressed as absolute concentration, µg/g

puree, in carrot puree during storage at 20 °C ( ), 30 °C ( ) and 40 °C ( ) ................................................. 41

Figure 9. Changes in hexanal concentration in, (A) tomato puree and (B) carrot puree during storage at 20

°C ( ), 30 °C ( ) and 40 °C ( ). .................................................................................................................... 44

Figure 10. Effect of thermal processing on hexanal concentration in the carrot and tomato purees:

untreated ( ) and treated ( ). ........................................................................................................................ 45

Figure 11. Percentage bioaccessibility as a function of storage time at 20 °C. In A: Tomato puree % BAC;

tomato all-trans-β-carotene ( ), all-trans-lycopene ( ); B: Carrot puree % BAC; carrot all-trans-β-carotene

( ), all-trans-α-carotene ( ). Statistical differences in % BAC shown by a/a` on the graph, otherwise not

statistically different. Error bars represent standard deviations ................................................................... 47

LIST OF TABLES

vii

LIST OF TABLES

Table 1. Fatty acid composition of some edible oils (n.d. means not detected) .......................................... 10

Table 2. Structures of major fatty acids found in vegetable oils ................................................................. 10

Table 3. Sampling times at each storage temperature for both carrot and tomato purees. Shaded area

represents a sampling moment ..................................................................................................................... 28

Table 4. Sample type and weight and the corresponding amounts of NaCl, extraction buffer, and MilliQ

water for carotenoid extraction..................................................................................................................... 29

Table 5. Composition (% w/w) of the Simulated Gastric Fluids (SGF) and Simulated Intestinal Fluid

(SIF) stock solutions..................................................................................................................................... 33

Table 6. Least squares linear regression of the changes in absolute carotenoid concentration in tomato

purees during storage. Slope values which are significantly different from 0 (P < 0.05) are indicated by *

...................................................................................................................................................................... 38

Table 7. Least squares linear regression of changes in absolute carotenoid concentration in carrot puree

during storage. Slope values which are significantly different from 0 (P < 0.05) indicated by *. ............... 42

LIST OF NOTATIONS

viii

LIST OF NOTATIONS

ASLT – accelerated shelf life testing

BAC – carotenoid bioaccessibility

BHT – butylated hydroxytoluene

EDTA – ethylenediaminetetraacetic acid

EI – electron ionization

EVOO – extra virgin olive oil

HPH – high pressure homogenization

HS-SPME-GC-MS – headspace-solid phase microextraction-gas chromatography-mass spectrometry

MPa – megapascal

PUFAs – polyunsaturated fatty acids

PV – peroxide value

RP-HPLC-DAD – reverse phase-high performance liquid chromatography-diode array detector

rpm – revolutions per minute

SD – standard deviation

SGF – simulated gastric fluid

SIF – simulated intestinal fluid

SIM – selected ion monitoring

GENERAL INTRODUCTION

1

GENERAL INTRODUCTION

The increased awareness of health benefits associated with certain carotenoids such as lycopene and β-

carotene has brought a surge of interest in identifying specific food formulations and processing of fruit

and vegetable based products so that carotenoid bioaccessibility can be enhanced (Liu 2003; Van Duyn &

Pivonka 2000; Astorg 1997; Hornero-Méndez & Mínguez-Mosquera 2007). In this regard, the inclusion of

a lipid substrate to fruit and vegetable based food formulations during processing (high pressure

homogenization and/or thermal processing) can result in food products with a lipid phase rich in

carotenoids prior to ingestion (Knockaert et al. 2014). This, in turn, can result in enhanced carotenoid

bioaccessibility (Colle et al. 2013). However, the type of lipid can influence not only carotenoid

bioaccessibility (Colle et al. 2012) but also stability, in particular during storage. This is because lipids can

undergo lipid oxidation, which is a source of radicals that accelerate carotenoid degradation (Bonnie &

Choo 1999). The oxidative stability of the lipid substrate is therefore important and depends on factors

such as the degree of unsaturation of fatty acids and presence of other compounds that may inhibit lipid

peroxidation during storage (Colle et al. 2011; Parker et al. 2003; Zambiazi 1997). In this context, it is

crucial to understand the mechanisms of carotenoid degradation and lipid oxidation during storage. Such

knowledge is important for the effective control of carotenoid changes during storage of shelf stable fruit

and vegetable based formulations containing a lipid substrate, to ensure a nutritional and safe product with

acceptable organoleptic properties. Therefore, the aim of this master thesis was to investigate carotenoid

stability in relation to lipid oxidation during storage of shelf-stable tomato and carrot purees containing 5

% extra virgin olive oil (w/w). Furthermore, changes in the bioaccessibility of all-trans-lycopene and all-

trans-β-carotene in tomato as well as all-trans-α- and β-carotene in carrot purees during storage at 20 °C

were also considered.

This master thesis contains two main parts namely: a literature review (Part I) and experimental work

(Part II).

PART I. LITERATURE REVIEW

2

PART 1: LITERATURE REVIEW

PART I. CAROTENOIDS AND LIPIDS: CHANGES DURING PROCESSING AND STORAGE

3

1. Carotenoids and lipids: Changes during processing and storage

Carotenoids are phytochemicals which are red, yellow and orange pigments, present in algae,

microorganisms and photosynthetic tissues of plants (Stahl & Sies 1996; Boon et al. 2010). The two major

classes of carotenoids are carotenoid hydrocarbons called carotenes and oxygenated hydrocarbons called

xanthophylls (Paiva & Russell 1999). Examples of carotenoids include zeaxanthin, lycopene, lutein, β-

carotene and astaxanthin (Edge et al. 1997). Tomatoes, watermelons, and grapefruit are some of the

common sources of lycopene (Jeffery et al. 2012), while orange fruits and vegetables like carrots,

pumpkins, papaya and mango are some of the common sources of β-carotene (Liu 2004). In this chapter,

the physico-chemical properties of and their influence on both lipids and carotenoids stability during

processing and storage will be described. Emphasis will be on β-carotene, lycopene and edible vegetable

oils. Thereafter, the mechanisms of carotenoid degradation and lipid oxidation during storage and

processing will be discussed.

1.1 Carotenoids

1.1.1 Chemistry, structure, solubility, size and shape

Structurally, carotenoids are tetraterpenes characterized by a system of conjugated double bonds with

delocalized ᴨ -electrons (Stahl & Sies 1996; Boon et al. 2010). Lycopene, β-carotene and α-carotene are

both carotenes, that have the same molecular formulas, but differ in their structures (figure 1). On the one

hand, lycopene is an acyclic, open chain molecule with 13 double bonds, of which 11 are conjugated,

while β-carotene is a dicyclic compound with 11 double bonds and 9 fully conjugated double bonds (Arab

et al. 2001; Stahl & Sies 1996). β-carotene and α-carotene both have 9 fully conjugated double bonds and

2 β-ionone rings. However, β-carotene has 2 β-ring double bonds with reduced overlap, whereas

α-carotene has only one (Anguelova & Warthesen 2000). Consequently, this difference in molecular

structure results in different biochemical properties. For example, while β-carotene and α-carotene have

pro-vitamin A activity, lycopene lacks in this property because of the absence of the β-ionone ring

structures (Shi & Le Maguer 2000). Additionally, the longer conjugated polyene chain in lycopene gives it

higher reactivity compared to β- and α-carotene (Woodall et al. 1997). The polyene chain present in

carotenoids is a highly reactive, electron rich system that is susceptible to attack by electrophilic reagents.


4

Figure 1. Structural formulas for β-carotene, α-carotene and lycopene

In nature, carotenoids exist in the all-trans configuration, and they can isomerize to cis configuration

under the influence of heat, light or certain chemical reactions e.g. quenching of the singlet oxygen species

(Schieber & Carle 2005; Stahl & Sies 1996). The isomeric forms of lycopene common in foods are 5-cis,

9-cis, 13-cis and 15-cis with stability sequence being 5-cis, > all-trans >9-cis > 13-cis >15-cis in organic

solvents (Singh & Goyal 2008; Lambelet et al. 2009). For β-carotene, its common isomeric forms are 9-

cis, 13-cis, and 15-cis. In general, the cis- configuration can be incorporated better into oil and

hydrocarbon solvents than the all-trans configuration (Shi & Le Maguer 2000; Schieber & Carle 2005).

Moreover, cis-isomers have been reported to be better absorbed by the human body compared to all-trans

lycopene( Lin & Chen 2005; Colle et al. 2010).

The molecular structure of carotenoids determines their physical and chemical properties which in turn

determines carotenoid function (Britton 1995). Carotenoid interaction with cellular and subcellular

structures is governed by their size, shape and presence of functional groups. For example, the conjugated

double bond system determines the photochemical properties like color and chemical reactivity which can

be linked to antioxidant properties (Stahl & Sies 1996; Britton 1995).

1.1.2 Carotenoid localization

In biological systems, carotenoids are localized in subcellular organelles (plastids), that is the chloroplasts

and chromoplasts. In the chloroplasts, they exist mainly in associated form with the hydrophobic areas of

protein structures whereas in the chromoplasts, they are deposited in crystalline form (El-Agamey et al.

2004; Schieber & Carle 2005; Britton 1995). The immediate environment of the carotenoid has a profound

All-trans-lycopene

All-trans-β-carotene

All-trans-α-carotene


5

effect on its properties, which in turn governs how they interact with other molecules in the system (Shi &

Le Maguer 2000), indicating that carotenoids may exhibit different properties depending on the

environment. Carotenoids in their naturally occurring location in intact cells are more stable than those

that would have been isolated or in organic solutions or oil (Minguez-Mosquera & Gandul-Rojas 1994).

Nguyen & Schwartz (1998) observed that lycopene in organic solvents isomerized readily as a function of

time even in the absence of light and presence of antioxidants, as compared to lycopene in tomato matrix.

Thermal isomerization and degradation of β-carotene in carrot puree containing oil was enhanced

compared to carrot puree without oil (Knockaert et al. 2014). In the presence of oil, the β-carotene crystals

will be dissolved in the oil droplets, thus making them more susceptible to degradation at high temperature

(Knockaert et al. 2014).

1.1.3 Mechanism of, and the factors influencing carotenoid degradation

As a result of the highly saturated nature of carotenoids, they are highly susceptible to isomerization and

oxidation (Rodriguez-Amaya et al. 2001). Nonetheless, the most generalized alteration occurring in

carotenoids is oxidative degradation (Minguez-Mosquera & Gandul-Rojas 1994). The possible scheme for

carotenoid degradation is shown in figure 2. As illustrated in figure 2, carotenoids in the all-trans

configuration can either start with isomerization to the cis-configuration before undergoing oxidation or

they are directly oxidized in their all-trans configuration. The resultant products are epoxy-carotenoids and

apocarotenoids, which further breakdown to low molecular compounds like epoxides, carbonyl

compounds and β-ionone (Boon et al. 2010). As a consequence, color and bioactivity are lost, in addition

to production of rancid flavors (Rodriguez-Amaya et al. 2001; Anguelova & Warthesen 2000).

Carotenoid oxidation degradation pathways include autoxidation, photo-degradation, and free radical

mechanism (Boon et al. 2010). Autoxidation of carotenoids occurs in the presence of oxygen, and occurs

at a faster rate if carotenoids are present in organic solvents (Boon et al. 2010). It is an autocatalytic

reaction as reported by Mordi et al. (1993), proceeding via the free radical chain reaction mechanism

evidenced by inhibition of reaction by butylated hydroxytoluene (BHT). With respect to photo-

degradation, it occurs in the presence of light, which excites sensitizers like chlorophyll leading to

formation of reactive oxygen species like singlet oxygen (Krinsky 1989).


6

Figure 2. Possible scheme for carotenoid degradation (Rodriguez-Amaya et al. 2001)

The protection mechanism against singlet oxygen is believed to be both a physical component as well as a

chemical reaction between the carotenoid and the singlet oxygen (Krinsky 1989). The physical quenching

is the most favored, resulting in the formation of a neutral carotenoid whereas the chemical reaction

results in carotenoid degradation forming degradation products like epoxides.

The free radical mechanism occurs in the presence of radical species. A possible source of radical species

can be lipid oxidation reactions in fruit and vegetable food systems containing a lipid substrate. Lipid

oxidation can result in formation of hydroxyl, lipid alkyl and the peroxyl radicals (Choe & Min 2006).

However, it is proposed that carotenoids react with radicals by addition, electron transfer and hydrogen

abstraction (Britton 1995; Haila et al. 1997; Krinsky & Yeum 2003). The addition of the radical to the

carotenoid results in a carotenoid-adduct radical whereas, the electron transfer results in the formation of

carotenoid radical cations and anions (Britton 1995; Haila et al. 1997; Krinsky & Yeum 2003).

Factors influencing carotenoid stability include light, heat and oxygen (Sharma & Le Maguer 1996;

Ribeiro et al. 2003; Xianquan et al. 2005). Light results in the photo-degradation of carotenoids (Boon et

al. 2010). In model dispersions, β-carotene degradation in light has been shown to follow first order

kinetics (Villota & Hawkes 2006). Shi et al. (2003) observed that the loss in total lycopene increased

significantly with increase in intensity and duration of light irradiation and light irradiation was more

detrimental than heat treatment. Henry et al. (1998) studied the stability of carotenoids to oxidation and

thermal degradation. Their study revealed that lycopene was more susceptible to degradation than β-

carotene. Anguelova & Warthesen (2000) observed a similar trend during oxidation in methyl linoleate at


7

37 and 60 °C. However, this result was explained by the differences in the antioxidant capacity of

lycopene and β-carotene, where (section 1.1.1) lycopene has twice as much singlet oxygen scavenging

capacity compared to β-carotene due to its molecular structure (Anguelova & Warthesen 2000; Miller et

al. 1996; Di Mascio et al. 1989). Heat stability studies on α- and β-carotene indicated that β-carotene is

about 1.9 times more susceptible to heat damage than α-carotene during normal blanching and cooking

operations (Villota & Hawkes 2006). This can also be attributed to structural differences (section 1.1.1),

where both the double bonds in the β-ionone rings are part of the chromophore of β-carotene, whereas

only one is part of the chromophore of α-carotene (Miller et al. 1996). Shi et al. (2003) observed that an

increase in temperature from 90 °C to 150 °C resulted in a 35 % decrease in total lycopene content in

tomato puree, which was attributed to thermal degradation in the presence of oxygen at temperatures

above 100 °C. The cis isomers were formed during the first 1 to 2 hours and decreased with further

incubation time, indicating that thermal treatment promoted cis-isomerization. However, lycopene showed

greater stability when thermally treated at temperatures below 100 °C, but duration of heat treatment had a

profound effect on lycopene degradation.

1.1.4 Carotenoid bioaccessibility

Nutrient content may be obtained from food composition analysis, but the availability of the

micronutrients (e.g. carotenoids) for absorption in the gut depends on several factors (Parada & Aguilera

2007). These factors include species of carotenoid, amount of carotenoid ingested, and matrix in which the

carotenoid is incorporated (van het Hof et al. 2000). Carotenoid bioavailability is the fraction of ingested

carotenoids that is utilized for normal physiological functions (West & Castenmiller 1998). Thus, it

includes nutrient absorption, metabolism, tissue distribution and bioactivity (Moelants et al. 2012; West &

Castenmiller 1998; Yeum & Russell 2002). With respect to fruit and vegetable based food products,

nutritional quality does not only depend on the nutrient content but also on the nutrient bioaccessibility

which is defined, in the context of carotenoids, as that fraction of ingested carotenoids that are released

from the food matrix in the gastrointestinal tract during digestion and available for intestinal absorption

(Lemmens et al. 2014; Parada & Aguilera 2007). Nevertheless, it is known that despite the prevalence of

carotenoids in fruits and vegetables, their absorption during digestion is low and can be highly variable

(Camara et al. 1995). This is because in order to confer their health effects, carotenoids must first be

released from the food matrix, incorporated into the lipid phase of chyme followed by transfer into mixed

micelles in the small intestine before being taken up by the body and finally reach their site of action


8

(Castenmiller et al., 1999). However, incorporation of carotenoids into the lipid phase has been reported to

be limited due to an interplay of food matrix related factors that hamper carotenoid release and the

conditions of low gastric acidity that limits carotenoid incorporation into the oil phase during digestion.

Processing (e.g. high pressure homogenization, thermal) of fruits and vegetables and in the presence of a

lipid substrate are known to enhance carotenoid bioaccessibility (van het Hof et al. 2000; Knockaert,

Pulissery, et al. 2012; Colle et al. 2013). In this regard, processing of fruit and vegetables in the presence

of oil could facilitate carotenoid incorporation into oil, thus presenting the possibility of introducing the

lipid phase rich in carotenoids, prior to ingestion, which in turn can become available for incorporation

into mixed micelles.

1.1.5 Carotenoid degradation during storage

The environmental storage conditions (e.g. storage temperature and light illumination) and properties of

the food system (e.g. water activity and oxygen concentration) influences carotenoid degradation reactions

and their stability. Lin & Chen (2005) in their study on stored tomato juice at 4 °C, 25 °C and 35 °C for a

period of 12 weeks observed that light exposure promoted formation of 9-cis and 13-cis β-carotene. For

lycopene, 15-cis isomer was the major isomer formed at 4 °C in the dark while 9-cis and 13-cis isomers

were favored at 25 °C, and 5-cis and 13-cis isomer dominated at 35 °C. Furthermore, storage at 35 °C was

destructive to all-trans β-carotene and its cis-isomers, and the degradation of cis-isomers proceeded faster

than the formation. Furthermore, the loss of all-trans-lycopene was higher than all-trans β-carotene at the

end of storage. In another study Sharma & Le Maguer (1996) reported that lycopene loss was maximum

under air and light at 25 °C, thus confirming the detrimental effect of light and oxygen on lycopene loss in

stored tomato-based products.

The effect of water activity of a food system on carotenoid degradation has been reported in previous

studies. For example, Ferreira & Rodriguez-Amaya (2008) observed an almost complete loss of lycopene

in low moisture model systems after 10 days of storage under light exposure regardless of solid support

used, with β-carotene exhibiting greater stability in the model systems compared to lycopene which is

consistent with observations from other studies. The protective effect of food matrix on the extent of

carotenoid degradation has been reported. For example, Ribeiro et al. (2003) observed that lycopene

degradation depended on the food system, stability being greatest in orange juice, followed by skimmed

milk and water. The composition of food system, in particular the presence of phenolic compounds and

vitamin C, was found to influence the extent of degradation due to their antioxidant properties.


9

1.2 Lipids

Lipids consist of a broad group of compounds that are generally soluble in organic solvents but only

sparingly soluble in water (Nawar 1996). The general classification of lipids based on their structural

components comprised simple lipids, compound lipids, and derived lipids (O’Keef 2008). Simple lipids

include acylglycerols and waxes, and they can be hydrolyzed into two different components, namely an

acid and an alcohol. Compound lipids include phosphoacylglycerols, sphingomyelins, cerebrosides, and

gangliosides. These yield 3 or more compounds upon hydrolysis (O’Keef 2008). Derived lipids are not

simple or compound lipids, which includes carotenoids, steroids and other fat soluble vitamins

(Nawar 1996).

1.2.1 Structure and chemistry of acylglycerols

Acylglycerols are mono-, di-, and triesters of glycerol and fatty acids (Nawar 1996), which are designated

as neutral lipids. Edible fats and oils such as olive oil, palm oil, coconut oil and sunflower oil consist

nearly completely of triacylglycerols (Belitz et al. 2009). Fatty acids can be esterified on the primary and

secondary hydroxyl groups of glycerol (O’Keef 2008). Figure 3 represents the structure of a typical

triacylglycerol molecule where R1 represents the carbon backbone of the fatty acid esterified on the

glycerol molecule. The properties of triacylglycerols are determined by the fatty acid composition.

Figure 3. Triacylglycerol molecule. * represents the chiral center.

Table 1 shows the fatty acid composition of some edible fats and oils (Colle et al. 2012) while Table 2

shows the structures of some common fatty acids found in vegetable oils (Belitz et al. 2009). Fatty acids

are aliphatic monocarboxylic acids which can be liberated by hydrolysis of fats and oils and differ in

chemical structure (O’Keef 2008; Belitz et al. 2009).They can have a saturated or unsaturated carbon

backbone (Nawar 1996). On the one hand, saturated fatty acids do not contain a double bond, and are

commonly unbranched, straight chain molecules with an even number of carbon atoms (Belitz et al.

2009). On the other hand, unsaturated fatty acids contain at least one allyl group in their acyl residue


10

Table 1. Fatty acid composition of some edible oils (n.d. means not detected)

Fatty acid (%) Palm oil Cocoa butter Olive oil Sunflower oil

C12 0.2 nd 0.0 0.0

C14 1.0 0.1 0.0 0.1

C16 43.0 24.9 11.4 6.0

C18 4.5 37.8 2.9 4.0

C18:1 40.4 33.3 74.4 28.6

C18:2 9.0 2.7 8.9 60.0

C18:3 0.1 1.2 0.6 0.3

C20 0.4 nd 0.5 0.1

C20:1 0.1 nd 0.3 0.1

C22 0.1 nd 0.2 0.8

Others 1.2 nd 0.8 nd

Table 2. Structures of major fatty acids found in vegetable oils

Abbreviated

designation Structure Common name

16:0

Palmitic acid

18:0

Stearic acid

18:1(9)

Oleic acid

18:2(9,12)

Linoleic acid

18:3(9,12,15)

Linolenic acid

1.2.2 Mechanism of lipid oxidation

Deterioration of foods by lipid oxidation generally shows an induction period, where the length of the

induction period is shortened by factors such as the presence of pro-oxidants and temperature increase

(Gordon 2004). Subsequently, the reaction proceeds rapidly after the induction period. In general, lipid

oxidation is a free radical chain reaction consisting of three stages namely, initiation, propagation and

termination (figure 4). However, the precise mechanism depends on the nature of reactive species and

their environment (Decker & McClements 2000; Schaich 2010). As shown in figure 4, the autoxidation

reaction of lipids starts with the initiation reaction. At this stage, the initial free radicals like alkyl radicals


11

are formed by the abstraction of a hydrogen atom from an unsaturated molecule like fatty acids of an

allylic methylene group (Simic 1981; Angelo & Vercellotti 1996).

Figure 4. Generalized scheme for autoxidation of lipids (Fennema 1996).

This initial reaction can be catalyzed by heat, light, or radiation (Angelo & Vercellotti 1996). In the

propagation stage, the resulting alkyl radical reacts with molecular oxygen to form a peroxyl radical,

which in turn abstracts another hydrogen from the backbone of an unsaturated compound of a lipid

substrate to form a peroxide ad another alkyl radical (Angelo & Vercellotti 1996). In the termination

reactions, radicals can react to form stable products. As depicted in figure 4, as long as oxygen is present

in the system as well as a source of alkyl radicals, the reaction will propagate. Ultimately, the

hydroperoxides formed degrade into secondary oxidation products like aldehydes, alcohols and epoxides.

Hydroperoxides breakdown is accelerated by the presence of transition metals, and heat (Decker &


12

McClements 2000). It is important to note that autoxidation of saturated fatty acids is extremely slow,

remaining practically unchanged at room temperature (Nawar 1996)

1.2.3 Factors influencing lipid oxidation

Factors which influence the rate of lipid oxidation include fatty acid composition, temperature and oxygen

concentration, metals, light and antioxidants (Nawar 1996; Gordon 2004).

1.2.3.1 Fatty acid composition

The rate of lipid oxidation is influenced by the number and position of double bonds. Consequently,

polyunsaturated fatty acids (PUFAs) readily get oxidized compared to monounsaturated and saturated

fatty acids. Saturated and monounsaturated fatty acids are more resistant to free radical attack (Halliwell &

Chirico 1993). Both the rate of formation and the amount of primary oxidation products accumulated

increases with an increase in degree of unsaturation (Choe & Min 2006). Additionally, though it is mostly

applicable to oil in water emulsions, the closer the double bond is to the methyl end, the more stable it is

to lipid oxidation (Decker & McClements 2000). As a consequence, rate of oxidation is faster when there

are PUFAs present in the food system (Decker & McClements 2000). In a study by Velasco et al. (2004)

where the oxidative stability at 60 °C of rapeseed and sunflower oils was investigated, rate of oxidation

was higher for sunflower compared to rapeseed oil. This was attributed to the fatty acid composition of

sunflower oil, which contains a higher proportion of linoleic fatty acid (63 %), compared to rapeseed oil

(21 %). Likewise, virgin olive oil is reported to have a high resistance to oxidative deterioration mainly

due to its fatty acid composition, which is rich in oleic fatty acid (74%) and naturally present antioxidants

such as phenols (Velasco & Dobarganes 2002).

1.2.3.2 Temperature and oxygen concentration

Due to the solubility of oxygen in oil, oxygen is always available in sufficient amounts to react with lipids,

unless measures are taken to exclude it (Decker & McClements 2000). However, at sufficiently high

oxygen concentrations, lipid oxidation rate is independent of oxygen concentration (Choe & Min 2006).

Nonetheless, oxygen concentration becomes rate limiting when oxygen content is low during lipid

oxidation, thus it will be independent of lipid concentration (Schaich 2010). The type of oxygen (singlet or

molecular oxygen), also affects the rate of oxidation reaction (García-Torres et al. 2009). Unless altered by

processing or storage conditions, molecular oxygen is most commonly found in stored processed fruits

and vegetables and the reaction rate with singlet oxygen is faster with singlet oxygen (García-Torres et al.


13

2009). Triplet oxygen reacts with lipid radicals whereas singlet oxygen reacts directly with lipids (Choe &

Min 2006).

With respect to temperature, a temperature increase causes a very large decrease in induction period

(Gordon 2004). This is because low to moderate heat breaks oxygen-oxygen bonds thus generating

radicals which will remove hydrogens from neighboring lipids to form alkyl radicals. Subsequently, these

radicals will initiate reactions in the early stages of lipid oxidation (Schaich 2010).

1.2.3.3 Metals

Redox-active metals like copper, iron, nickel, cobalt, are the most important initiators of lipid oxidation

(Schaich 1992). They exert influence on both the initiation and propagation stages, and are effective

catalysts even in trace amounts. If present even at concentrations as low as 0.1ppm, they decrease the

length of the induction period and increase the rate of lipid oxidation (Nawar 1996). They form alkyl

radicals both directly by oxidizing double bonds in unsaturated fatty acids and indirectly by oxidizing

other molecules to produce radicals that remove hydrogen from unsaturated lipids (Schaich 2010; Gordon

2004). They also catalyze the lipid oxidation reaction by breaking down lipid hydroperoxides to form

peroxyl and alkoxyl radicals (Decker & McClements 2000). Thanonkaew et al. (2006) reported catalytic

effect of Fe (II) to be more effective compared to Cu (II) and Fe (III) in muscle protein of cuttlefish.

1.2.3.4 Light

Light influences rate of lipid oxidation at low wavelengths (< ~254 nm), ultra violet light can abstract

hydrogen atoms to form alkyl radicals from an unsaturated lipid (Schaich 2010). Light also breaks down

peroxides to form radicals. Visible light (wavelength > 400 nm) initiates lipid oxidation indirectly through

photosensitizers such as chlorophyll. In this case, the photosensitizer absorbs light and transfer the

excitation energy to molecular bonds in lipids to form free radicals directly or to oxygen to form singlet

oxygen which then adds to double bonds and forms hydroperoxides in unsaturated fatty acids without

intermediate radicals (Schaich 2010). Radicals will be generated subsequently by decomposition.

1.2.3.5 Antioxidants

Antioxidants are substances that can delay the onset of autoxidation by extending the induction period or

slowing down the rate (Nawar 1996; Choe & Min 2006). Antioxidant act by controlling pro-oxidants and

oxidation of substrates (lipids and oxygen), as well as inactivation of free radicals (Decker & McClements


14

2000). For example, Anguelova & Warthesen (2000) observed that butylated hydroxyltoluene (BHT) and

α-tocopherol effectively decreased hydroperoxides formation during oxidation of methyl linoleate at

60 °C, which in turn resulted in a significant decrease in lycopene, α- and β-carotene degradation

Antioxidants can be classified as primary or secondary antioxidants. On the one hand, primary

antioxidants are chain breakers, which are capable of accepting free radicals (peroxyl and alkyl radicals) to

delay the initiation step or interrupt propagation step of autoxidation. They convert the radicals into more

stable, radical or non-radical products (Decker & McClements 2000). Chain breaking antioxidants differ

in their effectiveness not only because of their chemical properties, but also because of their physical

location within a system, whether hydrophilic (polar) or lipophilic (non-polar). However, lipophilic

primary antioxidants are more effective in oil-in-water emulsion systems. On the other hand, secondary

antioxidants retard lipid oxidation by chelating transition metals, replenishing hydrogen to primary

antioxidants, oxygen scavenging, and deactivation of reactive species e.g EDTA. Nonetheless, none of the

mechanism for secondary antioxidants involves conversion of free radicals to more stable products

(Decker & McClements 2000).

.

PART I. PRINCIPLES OF ACCELERATED SHELF LIFE TESTING

15

2. PRINCIPLES OF ACCELERATED SHELF LIFE TESTING

Food quality is a collection of properties influencing the degree of excellence of a food product or its

suitability for a particular use. (Abbott 1999; Taoukis et al. 1997). Thus, the quality of fruit and vegetable

based products encompasses sensory properties, nutritive value, chemical constituents, mechanical

properties, functional properties and defects (Abbott 1999). Nevertheless, fruit and vegetables based

products are mainly consumed for their nutritive value and sensory properties (Aamir et al. 2013). For

instance, the consumption of tomato and tomato based products has been associated with a lower risk of

cancer, due to the presence of bioactive compound, lycopene. However, food systems are complex, being

physicochemically and biologically active. As highlighted in chapter 1, processing initiates chemical and

physical changes to the food and these changes continue during storage. This is as a response to the

different environmental conditions the processed food system is exposed throughout the food supply chain

and the interaction between food components (Taoukis et al. 1997). The food supply chain refers to all the

processes that food material go through beginning at the farm up to the consumers table. The processes

include farm production, processing, distribution, marketing, retailing and consumption (Verkerk et al.

2009). As a result, the quality attributes of food change as a function of time and storage conditions

mostly reducing in levels. This means that food has a specific time period within which it is rendered

acceptable, based on some defined quality attributes, under specified storage conditions as it goes through

the food supply chain: this is termed its shelf life. Therefore, shelf life determination is vital.

In this chapter, the factors influencing quality attributes of fruit and vegetable based products will be

discussed. This will be followed by a description of the modes of food quality deterioration. Aspects to

consider when designing a shelf life experiment will be explained followed by a discussion of the different

approaches to accelerated shelf life testing. Lastly, the basic principles of kinetic modelling as applied to

food quality attributes will be highlighted.

2.1 Factors influencing shelf life

The overall quality of fruit and vegetable based products is a combination of several factors changing

during its shelf life as a result of diverse reactions, mostly deteriorative, which affect quality. Factors

which influence the extent of the reduction in quality level can be categorized into intrinsic and extrinsic

factors (Kilcast & Subremaniam, 2000), which are discussed separately in the subsequent sections.


16

2.1.1 Intrinsic factors

Intrinsic factors are final product properties which include product pH, food composition, water activity

and redox potential (Oey et al. 2008; Kilcast & Subremaniam, 2000). Variables such as product

formulation, raw material type and quality can influence these factors. However, food product formulation

is a process which includes identification of the key functional attributes the final product is expected to

have, then identification of the structural properties (based on food composition) likely to produce the

desired functionality (Avramenko & Kraslawski 2008). On the one hand, structural properties include

composition of the food product, which in essence are the raw materials or ingredients. On the other hand,

the desired food functionality may be nutritional, for example, a product designed to deliver maximum

carotenoid bioaccessibility or micronutrients.

Dissolved oxygen is one factor which affects nutritional content and organoleptic properties of fruit and

vegetable products. In a critical review, García-Torres et al. (2009) reported that dissolve oxygen affect

vitamin C, color due to non-enzymatic browning and aroma in fruit juices. With regards to the water

activity, it has been observed that carotenoids exhibit maximum stability to degradation over the water

activity range 0.341 – 0.54 (Lavelli et al. (2007).

2.1.2 Extrinsic factors

Extrinsic factors are external conditions the food product is exposed to as it moves through the food

supply chain (Kilcast & Subremaniam, 2000). These include processing conditions (Oey et al. 2008)

storage temperature, packaging and light exposure. Palmers et al. (2014) observed that intensity of

processing conditions for fruit and vegetable purees affected their safety measured by furan concentration

in the end product. High pressure high temperature treatments resulted in lower furan concentrations

compared to thermally treated purees attributed to differences in process intensities between the two

processes. Some methods of juice packing aim to reduce the exposure of product to oxygen, through the

use of high barrier materials such as glass or foil laminates in brick packs. Brick packs also have light

barrier properties (García-Alonso et al. 2009). A study by Lin & Chen (2005) concluded that for tomato

juice processed without lipids, the amounts of all-trans and cis isomers of lycopene, β-carotene and lutein

decreased with increasing storage time, and light enhances carotenoid degradation. Moreover, the authors

observed faster carotenoid degradation with increasing storage temperature. In the same study, it was

observed that different carotenoids exhibit different sensitivities to storage conditions and this was


17

attributed to different structural properties of lycopene, β-carotene and lutein. Thus, it is important to

control storage conditions and processing conditions to fully realize the nutritional properties associated

with fruit and vegetable based food products.

2.2 Modes of food quality deterioration during storage

Prior to the development of a specific procedure for shelf-life evaluation, it is important to have a good

understanding of the different reactions that cause food deterioration. The interaction of intrinsic and

extrinsic factors discussed in the previous section may either stimulate or inhibit a number of reactions

which in turn, can limit the shelf life of a food product (Kilcast & Subramaniam 2000). The overall

deteriorative reactions in fruits and vegetables and their products leading to quality changes during storage

can be classified as chemical, microbiological, physical and enzymatic reactions (Singh 1994, Kilcast &

Subremaniam, 2000, Dauthy 1995).

2.2.1 Microbiological reactions

The microbial groups important in foods are bacteria, yeasts, molds and viruses (Ray & Bhunia 2013).

They can cause foodborne disease and, food spoilage if measures for their control are not taken.

Nevertheless in some cases, their beneficial aspects in food production (e.g. in food fermentation

processes like wine production) is known. Variables which can affect microbial profiles and hence shelf

stability include initial microbiological quality of raw materials, product pH, product water activity, the

temperature time combination during process and post process handling such as storage conditions

(Efiuvwevwere & Atirike 1998; Ray & Bhunia 2013). However, foods on the one hand, can be classified

as non-perishable, semi-perishable and perishable depending on stability to microbiological spoilage. For

perishable products, microbiological reactions may be controlled by use of low temperature to limit

growth. Sterilization (110 to121 °C) or ultra-high temperature (140 to 160 °C) are thermal treatments

which may be used to produce commercially sterile food products which are shelf stable (Aamir et al.

2013). Hermetically sealed and heat processed foods are generally regarded as non-perishable provided

post process contamination is avoided (Dauthy 1995). On the other hand, foods can also be classified as

low acid or high acid, which in turn determines the treatment needed for final product stability and safety

during storage. The product pH affects the microorganisms which can grow, spore germination, and

sensitivity to heat treatment. Clostridium botulinum cannot grow and produce toxin at pH less than 4.6,

and its spores cannot germinate (Pawsey 2002). For product safety and production of a commercially


18

sterile product, at least a 12D thermal process is required for low acid foods to inactivate the most heat

resistant spore forming pathogen Clostridium botulinum (Campanella & Peleg 2001).

2.2.2 Chemical reactions

Chemical deteriorative changes involve the internal food components and the external environment (Singh

1994). These components include unsaturated fatty acids, polyene chains present in carotenoids, hydroxyl

groups present in vitamin C. Consequently, chemical reactions such as lipid oxidation (section 1.2),

carotenoid oxidation (section 1.1.3), and polymerization occur leading to color loss and vitamin loss.

(Kilcast & Subremaniam, 2000; Dauthy 1995).

2.2.3 Physical reactions

The major cause of physical deteriorative changes is moisture migration in fresh produce and dried food

products during storage (Kilcast & Subremaniam, 2000; Dauthy 1995). The driving force for moisture

migration is differences in water activity between the storage environment and the food product. In shelf

stable fruit and vegetable based products, e.g. in-pack-sterilized purees, the use of packaging with good

barrier properties limits this mode of deterioration during long term storage. This may be achieved by use

of packaging material like glass. Besides being odourless and chemically inert with most food products,

glass is impermeable to gases and vapors and therefore maintains product freshness for a long period of

time(Marsh & Bugusu 2007).

2.2.4 Enzymatic reactions

Enzymes are essential catalysts for metabolic reactions in fruits and vegetables (Whitaker 1991).

Nevertheless, resultant effects can be desirable (e.g. during ripening of fruits) or undesirable (e.g.

detrimental changes in texture and colour (Terefe et al. 2014; Dauthy 1995). For example, enzymes such

as polyphenol oxidase, peroxidase, lipoxygenase, and phenolase can lead to the initiation of deterioration

reactions affecting product colour, flavor or nutritional changes (Gonçalves et al. 2010). Pectin

methylesterase (a pectic enzyme) affects viscosity of tomato based products such as tomato puree and

cloudiness in fruit juices such as orange juice (Cano 2003; Terefe et al. 2014). This is because the

viscosity of tomato based purees is highly dependent upon the degree of polymerization of the pectic

substances (Lopez et al. 1998). Thus pectic enzymes inactivation is necessary to have a product with a

stable consistency during storage. One possible way to achieve this is by thermal treatment. For example,


19

tomatoes can be rapidly thermally treated at temperatures in the range 82–104 °C immediately following

chopping or crushing to inactivate pectic enzymes (Lopez et al. 1998).

2.3 Designing shelf life testing experiments

An experiment to estimate product shelf life can be designed provided the knowledge of the deterioration

reactions and the factors influencing them is known. Consequently, based on a particular deteriorative

reaction (s) limiting a food product shelf life, a shelf life testing experiment is designed. Real time stability

tests or accelerated stability tests can be used for shelf life estimation (Anderson 1991). Real time stability

tests involve the storage of a product at recommended storage conditions, allowing for sufficient time to

cause significant product degradation, whereas accelerated stability tests involve storage of product at

elevated storage conditions to speed up deteriorative reactions (Magari 2003; Anderson 1991). The stress

conditions may be temperature, relative humidity and pH. Therefore, accelerated stability test allows for

understanding of storage characteristics, in particular of long shelf life products within a relatively short

space of time (Magari et al. 2002; Kilcast & Subremaniam, 2000).

For an efficient design of a shelf life testing experiment, there are some aspects to be considered (Fu &

Labuza 1997; Pedro 2006; Guillet & Rodrigue 2010; Martins et al 2008; Silva & Gibbs 2004; Kirkwood

1984). The goal of the study needs to be clearly stated based on an identified quality parameter to be

monitored. Subsequently, the responses to be measured, and assay methods for response measurement

have to be clearly identified because not all methods measure exactly the same thing. However, the

appropriate determination of storage conditions is critical since the accuracy of the prediction models

depends on the data collected. Finally, the study duration period, product sample, control needs and

sampling frequency can be specified. Tydeman & Kirkwood (1984) suggested that the study period should

be long enough to allow at least 25 %, Taoukis et al (1997) suggest 50 % preferably 75% degradation of

the quality attribute under study to get a precise prediction model. If the accelerating factor is temperature,

it is known that the precision of predicted degradation rates can be improved by using a wider temperature

range and increasing the number of samples tested (Tydeman & Kirkwood 1984). The sampling frequency

may be determined by the expectation of deterioration rate from previous studies (Kilcast &

Subremaniam, 2000; Fu & Labuza 1997). As a guideline, it is not recommended to have equally spaced

sampling times. It might also prove useful to store a larger number of samples than originally planned for


20

contingency sake (Guillet & Rodrigue 2009). Finally, the data will be analyzed using the appropriate

kinetic model(s), and this will be discussed in the subsequent sections.

2.4 Basic principles of accelerated shelf life testing of foods

The objective of accelerated shelf life testing is to store the food product under abuse conditions with

periodic examination for the period of the study for data extrapolation to actual storage conditions. The

key to this premise is the assumption that the deteriorative process mechanism limiting shelf life remains

the same under the two conditions (Kirkwood 1984). Therefore, accelerated shelf life testing (ASLT)

encompasses any method with the ability to evaluate a food product stability, based on data obtained in a

significantly shorter time than the actual time with the manipulation of certain conditions, and periodic

product analysis until the end of study (Mizrahi 2004; Hough et al. 2006; Ragnarsson & Labuza 1977). Of

the accelerating factors, temperature is found to be the most important (Ragnarsson & Labuza 1977). The

principles of accelerated shelf life testing are applicable to any deteriorative process as long as it has a

valid kinetic model, whether physical, chemical or microbiological (Mizrahi 2004). For temperature,

results can be modeled using the relationship between temperature and reaction rate (Ee et al. 2002).

When conducting a constant stress level storage test, the food is subjected to constant storage conditions at

several stress level variables to determine the deterioration mechanism and validation of quality loss

kinetics, with stress levels greater than 3 ( Martins et. al 2008).

2.5 Approaches to accelerated shelf life testing

ASLT can be approached in different ways, e.g. the initial rate and kinetic model approaches, with the

aim of obtaining reliable deterioration data in a short period, model identification and eventually

prediction of actual shelf life of the product (Mizrahi 2004).

2.5.1 Initial rate approach

The initial rate approach is a method where the extent of food deterioration is measured at actual product

storage temperature (Mizrahi 2004). As a consequence, it requires a highly sensitive analytical method to

monitor the deterioration marker, with the capability of detecting minute changes after a relatively short

storage time. Nonetheless, there is need to know or evaluate how the deterioration process behaves as a

function of time. For chemical reactions, the reaction order is sufficient for this. As an example, in fruits


21

and vegetable based food products, vitamin concentration or carotenoid concentration (C) as a quality

parameter may be monitored as a function of time. The kinetic equation may be represented as follows:

[1]

Where n is the reaction order, k is the kinetic constant, t is the time. For a zero order reaction, the kinetic

model needed to employ the initial rate approach to accelerated shelf life testing and the extrapolation

process will be as follows:

[2]

Where C0 is the initial concentration. The data obtained from the initial measurements will be used for the

determination of k. The reaction order can be obtained from literature for many chemical reactions that

take place in food systems. Many deterioration reactions in fruit and vegetable based food products follow

zero order or first order kinetics. Formation of primary lipid oxidation products follows pseudo zero order

kinetics while formation of secondary lipid oxidation products, followed a pseudo first order kinetics

(Gómez-Alonso et al. 2004). With respect to lycopene and β-carotene degradation, first order kinetics are

usually used to model these reactions (Randolini 2008; Koca et al. 2007; Sharma & Le Maguer 1996;

Ferreira & Rodriguez-Amaya 2008; Anguelova & Warthesen 2000). However, if the reaction order is

unknown, an accelerated test procedure may be used to evaluate the reaction order empirically, where any

convenient kinetically active factor to accelerate the deterioration process may be used. The advantage of

the initial rate approach is the capability of obtaining kinetic data on actual storage conditions in a

relatively short time, but this requires a very sensitive and accurate analytical method, with the capability

of detecting minute changes in the product. In the absence of a sensitive and accurate analytical method,

the deterioration process should be allowed to progress for long. Thus in most instances, the deterioration

process needs to be accelerated to overcome the shortcoming of the analytical methods (Mizrahi 2004).

2.5.2 Kinetic model approach

A prerequisite for this model is a valid kinetic model for the deterioration process (Mizrahi 2004). The

initial step in this approach is the selection of the desired kinetically active factors for acceleration of the

deterioration process, for example temperature (Martins et al. 2005; Corradini & Peleg 2007).

Subsequently, a kinetic study of the deterioration process at such levels that the deterioration rate is faster


22

will be conducted. Nevertheless, the reaction mechanism should not change at the chosen levels otherwise

extrapolation to normal storage will yield erroneous results.

A general and comprehensive kinetic model includes all the factors that may affect their rate, which are

compositional such as concentration of reactive compounds, inorganic catalyst, reaction inhibitors, pH,

water activity, and environmental conditions like temperature, relative humidity, total and partial pressure

of different gases present, and light (Taoukis, et al 1997). However, for shelf life prediction, the kinetic

model should include only those factors changing during storage, which can include temperature,

composition, moisture content, and light intensity (Mizrahi 2004). A model for ASLT, should contain

those factors changing during storage and those used to accelerate the rate of reaction (Mizrahi 2004). The

most frequently used single accelerating factor is temperature with the use of the Arrhenius model (Nelson

& Labuza 1994; Labuza 1984). The Arrhenius model relates the rate of a chemical reaction to temperature

in systems where the reaction is not limited by diffusion. The Arrhenius model is shown in equation 3:

[3]

Where k is the reaction rate constant at a given temperature, k0 is the pre-exponential factor, Ea is the

activation energy, T is the temperature in Kelvins, R is the universal gas constant. The activation energy

(Ea) gives a measure of the temperature sensitivity of the reaction (Achour, et al. 2001). It is very specific

for each food system and can be determined empirically although it remains constant for a given system as

long as the mechanism does not change (Agnarsson & Labuza 1976). However, it has been proposed that

activation energy parameters may be derived using linear or non-linear regression. On the one hand, linear

regression entails a graphical representation of log(K) versus (1/T) to determine the activation energy

(Achour, et al. 2001). For this, at least 3 different temperatures are needed and a 2 step procedure is

applied where initially the rate constants are derived and then regressing them versus temperature (Van

Boekel 2008). However, linear regression approach may result in wide confidence intervals for the

parameters being determined and it is undesirable since it results in imprecise predictions. On the other

hand, the non-linear regression approach may result in better parameter estimates, where the rate constant

k, is substituted in the appropriate rate equation (Taoukis, P. S., Labuza, T. P., & Saguy et al. 1997).


23

3. CONCLUSION

Shelf stable fruit and vegetable based food products are expected to have a long shelf life, of up to a year

or even beyond, during which they should maintain their safety, nutrition, and sensorial quality attributes.

Specific food formulations and certain food processing methods are necessary to realize fully the

nutritional benefits from consumption of fruit and vegetable products. Processing of fruits and vegetables

in the presence of oil can facilitate the transfer of phytonutrients like carotenoids to oil, which can then

become available for incorporation into mixed micelles during digestion, thus enhancing their

bioaccessibility. Nevertheless, the presence of the lipid substrate during processing can be a source of

oxidizing agents that can influence carotenoid degradation during storage. As discussed in literature,

earlier studies on carotenoid degradation have been carried out for different storage and processing

conditions and in different foods or model systems. To the best of our knowledge, no studies have been

done for real food systems containing oil during storage. Nevertheless, in most cases, it has been

highlighted that the extent of carotenoid degradation is dependent upon the composition of food matrix.

Thus, by applying the principles of accelerated shelf life testing, insight into the stability of carotenoids in

specific shelf stable food systems containing oil during storage can be gained. Additionally, the effect of

storage time on carotenoid bioaccessibility can be assessed.

PART II. EXPERIMENTAL WORK

24

PART II: EXPERIMENTAL WORK

PART II. RESEARCH PLAN

25

3. RESEARCH PLAN

Based on the literature review, an accelerated shelf life storage experiment for a period of 6 months in the

dark at 20, 30, and 40 °C was designed to investigate the stability of carotenoids (lycopene, β-carotene,

and α-carotene) in tomato and carrot purees containing 5 % (w/w) extra virgin olive oil (EVOO). In this

way, different carotenoids from two different matrices could be investigated. Furthermore, the relation

between carotenoid degradation and lipid oxidation as well as changes in carotenoid bioaccessibility

during storage at 20 °C were also considered. The specific objectives were to determine during storage:

the effect of temperature and time on carotenoid stability

the effect of temperature and time on lipid oxidation

changes in carotenoid bioaccessibility as influenced by storage at 20 °C.

The research plan that was followed to achieve the objective is summarized in figure 5. Briefly, starting

from the same batch of pre-treated vegetable matrices, singular purees containing 5% EVOO (w/w) were

prepared by high pressure homogenization (HPH) at 100 MPa. Glass jars (100 ml volume, 95 mm height,

and 45 mm diameter) were filled with 90 ± 0.5 g (headspace of about 15 mm) of the purees and closed

with metal lids. The jars were then thermally processed (Tprocess = 117 °C, F0 =5 min) followed by storage

at 20, 30 and 40 °C. Samples were then collected according to a sampling frequency plan for each

temperature and product type over a period of 6 months, and stored at -80 °C until further analysis. The

analyses started with thawing of samples at 4 °C overnight followed by recovery, by ultra-centrifugation,

of the oil fraction on which both carotenoid content and primary lipid oxidation products, the peroxide

value, was measured. On the puree as a whole, carotenoid content and secondary lipid oxidation products,

hexanal, and carotenoid bioaccessibility were also measured. Carotenoids were quantified by reverse

phase-high performance liquid chromatography coupled with diode array detector (RP-HPLC-DAD)

(Palmero et al. 2014), while peroxide value was determined spectrophotometrically by the ferric

thiocyanate method (Hornero-Méndez et al. 2001). Hexanal was determined by the headspace solid phase

micro-extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) standard addition method

(Kebede et al. 2015). A standardized in vitro digestion method was used to determine carotenoid

bioaccessibility according to Minekus et al. (2014). The percentage carotenoid transfer to oil fraction was

also calculated.

PART II. RESEARCH PLAN

26

Figure 5. Schematic representation of the experimental set-up.

High pressure homogenization (100 MPa)

Thermal treatment (F0 = 5 min T

process = 117 °C)

Sampling and storage @ -80 °C

Storage in the dark 20 °C, 30 °C, 40 °C for 6 months

Ultracetrifugation (65 000g 1 hr 8 min)

Data Analysis

Carotenoids (RP-HPLC-DAD)

Peroxide value (Ferric thiocyanate method)

Carrot pre-treatment (95 °C, 20 min); Tomato blanching (95° C, 5min),

freezing liquid N2, store @ -40 °C

Carrot Puree Tomato Puree

High pressure homogenization (100 MPa)

5% EVOO

OIL

Washing, cutting, vacuum packing

Thawing overnight (4 ºC);

Blending carrot with deionized water (1:1); tomato blending

Puree

Carotenoid

bioaccessibility Hexanal

(HS-SPME-GC-MS)

Carrot or

Tomato

PART II. MATERIALS AND METHODS

27

4. MATERIALS AND METHODS

The materials and methods section will give a detailed description of the approach used for the realization

of the research objectives. It is divided into 5 sections namely: sample preparation, thermal treatment,

storage and sampling, experimental analyses and data analysis.

4.1 Materials

All chemicals and reagents used were of analytical or HPLC-grade. Olive oil (extra virgin) was kindly

donated by Vandemoortele (Ghent, Belgium). Red ripe tomatoes (Lycopersicon esculentum cv Prunus)

and orange carrots (Daucus carota cv Nerac) were obtained fresh from a local shop in Belgium and stored

at 4 °C for 1 day prior to use. Extra virgin olive oil was chosen because it is widely used in food

formulations.

4.1.1 Raw material preparation

The fruits and vegetables were sorted and washed under running water. Carrots were peeled, cut into

cylinders, while tomatoes were cut into thirds. The pieces were vacuum-packed in low density polythene

bags (300 g ± 5 g). Tomatoes were blanched at 95 °C for 8 minutes, whereas carrots were pre-treated at 95

°C for 20 minutes in a water bath. The bags were immediately placed in ice bath for cooling.

Subsequently, samples were frozen in liquid nitrogen and stored at -40 °C until puree preparation.

4.1.2 Puree preparation

Prior to puree preparation, the bags were thawed overnight at 4 °C in the cold room. In the case of carrots,

deionized water 1:1 (w/w) was added and blended for 1 minute in a kitchen blender. Subsequently, EVOO

(5 % w/w) was added and blended further for 10 seconds prior to the homogenization step. The carrot

puree/oil mixture was high pressure homogenized at 100 MPa over one cycle (Panda 2K, Gea Niro Soavi,

Mechelen, Belgium) for matrix disruption to aid carotenoid release and stabilize the puree/oil mixture.

In the case tomato, the blanched pieces were blended in a kitchen blender for 1 minute. The resultant

puree was sieved (pore diameter 1 mm) to remove seeds and excess skin. The tomato puree was high

pressure homogenized at 100 MPa during one cycle (Panda 2K, Gea Niro Soavi, Mechelen, Belgium).

Subsequently, EVOO (5 % w/w) was added to the high pressure homogenized puree, mixed in a kitchen


28

blender for 10 sec, and the resultant mixture further high pressure homogenized under the same conditions

as above. The homogenized puree/oil mixtures were subsequently filled into glass jars (100 ml volume, 95

mm height, and 45 mm diameter), 90 ± 0.5 g per jar maintaining a headspace of about 15 mm and closed

with metal lids prior to thermal treatment.

4.2 Thermal treatment

A water cascading retort (Barriquand Steriflow Retort, France) was used for the thermal treatment, in the

pilot plant for the Laboratory of Food Technology, Katholieke University Leuven. The thermal treatment

was done simultaneously for both tomato and carrot purees. The glass jars were loaded into the retort and

sterilized at a process temperature of 117 °C targeting an F0 value of 5 minutes, with a holding time of

29.9 minutes. The temperature time profile in the coldest spot within the product was recorded using

thermocouples at different positions inside the retort. After the thermal treatment, samples were

subsequently cooled in ice water to 4 °C before storage.

4.3 Sample storage and sampling plan for shelf life experiment

Thermally treated glass jars were placed in incubators at 3 different isothermal temperatures, namely 20,

30, and 40 °C for a total storage period of 24 weeks in the dark. The sampling plan is shown in table 3.

The samples were randomly selected from storage at specific moments according to the sampling plan.

Subsequently, the puree from each glass jar was equally distributed between two falcon tubes (50 mL) at 4

°C, headspace flushed with nitrogen gas, frozen in liquid nitrogen, and stored at -80 °C until further

analysis. The samples were then analyzed at once.

Table 3. Sampling times at each storage temperature for both carrot and tomato purees. Shaded area represents a sampling

moment

Storage time

(weeks) 0 1 2

4

6

8

12

16

20

24

20 °C

30 °C

40 °C


29

4.4 Experimental analysis

The experimental analysis started with thawing of samples at 4 °C overnight. The thawed purees was

ultra-centrifuged at 65 000 g for 1 hour and 8 minutes to recover the oil fraction. Carotenoid analysis and

secondary oxidation products analysis were done on the puree, whereas primary lipid oxidation analysis

was done on the recovered oil fraction. Percentage carotenoid transfer to oil was calculated according to

equation 6.

4.4.2 Carotenoid analysis

Carotenoid quantification in digest and micelles from in vitro digestion, puree and oil was according to the

method described by Palmero et al. (2014). Depending on the sample, a specific amount of sample (table

4) and a corresponding amount of NaCl and extraction buffer consisting of 50 % hexane, 25 % acetone, 25

% ethanol (v/v/v), and 0.1 % BHT (w/v) were added. The mixture was then mixed for 20 min at 4 °C.

Subsequently, a corresponding amount of MilliQ water (18.2 MΩ) (see table 4), was added to the mixture

and stirred for 10 min at 4 °C. The mixture was transferred to a glass tube or separating funnel and

allowed for phase separation. The organic phase containing the carotenoids was separated from the

aqueous phase and filtered (Chromafil PET filters, 0.20 µm pore size, 25 mm diameter, Macherey –

Nagel, Duren Germany) into HPLC vials.

Table 4. Sample type and weight and the corresponding amounts of NaCl, extraction buffer, and MilliQ water for carotenoid

extraction.

Sample type Sample

weight (g) NaCl (g)

Extraction

buffer (mL)

MilliQ water

(mL)

Puree 1 0.25 25 7.5

Oil 0.25 0.1 10 3

Digest 5 mL 0.25 25 7.5

Micelles 0.25 25 7.5

The identification and quantification of carotenoids were performed using a HPLC system equipped with

a C30-column (3μm×150mm×4.6mm, YMC Europe, Dinslaken, Belgium) and a diode array detector

% carotenoid transfer

[6]


30

(Agilent Technologies 1200 Series, Dinslaken, Belgium). During the analyses the temperatures of the

auto-sampler and the column were kept at 4 °C and 25 °C, respectively. A linear gradient, using methanol

(A), methyl-t-butyl-ether (B) and reagent grade water (18.2MΩ) (C), was applied. The starting conditions

were 81% A, 15% B and 4% C and the ending conditions corresponded to 16% A, 80% B and 4% C. The

flow rate was set at 1 mL/minute. Identification was performed at 472 nm for all-trans-lycopene and the

cis isomers and at 450 nm for all-trans-β-carotene and the cis isomers, as well as all-trans α-carotene on

the basis of retention times and spectral characteristics of pure standards (Sigma-Aldrich, Bornem,

Belgium). All-trans lycopene, all-trans-β-carotene, all-trans α-carotene as well as cis isomers were

quantified with the use of the corresponding calibration curves. Quantification of cis isomers for which the

pure standards were not available was made using the calibration curve for the all-trans standard.

Carotenoid extraction was performed with two replicates and each extract analyzed once. The carotenoids

separated and identified were all-trans-lycopene, 5-cis, 9-cis-, 13-cis-lycopene, and all-trans-β-carotene

for tomato puree. As for carrot puree, all-trans-β-carotene, 9-cis, 13-cis, 15-cis-β-carotene, and all-trans-

α-carotene were separated and identified.

4.4.3 Lipid oxidation analyses

4.4.3.1 Determination of peroxide value.

The method was based on Hornero-Méndez et al. (2001) with some modifications. The analysis was done

in subdued light. An oil sample (0.01 g – 0.05g) was weighed into a 10 ml screw caped tube and 1 mL of

chloroform/acetic acid (2:3) added for sample dissolution. Subsequently, 100 µl Fe (II) was added to the

mixture, vortexed for 15 seconds (Labdancer S40, IP 40, made in Germany), and left in the dark for 10

min. MilliQ water, (2 mL) followed by 4 mL of hexane (containing 7 ppm BHT w/v) were added. The

organic phase was discarded. Two successive extractions using hexane, with removal of organic phase

containing pigments, were done to ensure complete pigment removal from sample. The resultant aqueous

phase was bubbled for 10 seconds with N2 to remove excess hexane. To determine Fe (III), 1 mL of the

aqueous phase was transferred to a disposable plastic microfuge, and mixed with 100 µL of saturated

ammonium thiocyanate solution. The mixture was allowed to react for 10 minutes, after which 1 mL was

transferred to a quartz cuvette, and absorbance measured at 470 nm using a spectrophotometer (Amersham

Biosciences Ultrospec 2100 Pro UV Vis Spectrophotometer) against a water blank. A reaction blank

containing all the reagents, except sample was performed, and the resultant absorbance value was


31

subtracted from that of sample. All absorbance values were corrected with absorbance values measured at

670 nm.

Fe (II) stock solution was prepared fresh every time by gently mixing a solution of 0.4 g BaCl2.2H2O in 50

mL of 0.4 N HCl with a solution of 0.5 g FeSO4.7H2O in 50 mL deionized water. The resultant solution

was then filtered and stored under cover.

Quantification of peroxides was done by making use of a calibration curve of Fe (III). Hereto, different

volumes of the prepared Fe (III) solution were transferred to screw capped test tubes and diluted with a

mixture of chloroform and acetic acid (2:3). Subsequently, 1 mL from each of the serial dilutions was

pipetted into a microfuge tube and 100 µL of saturated NH4SCN solution added to each, and the mixture

left for 10 minutes. To quartz cuvette tubes, 1 mL of the mixture was transferred and absorbance at 470

nm against the mixture of chloroform/acetic acid was measured and also corrected against absorbance

value at 670 nm. The peroxide value was then determined according to equation 7:

7

where As and Ab are the corrected absorbances of the sample and blank respectively, 55.85 is the atomic

weight of Fe(s), s the slope of the Fe(III) calibration curve, ms is sample weight, and 2 is a factor to convert

milliequivalents (mequiv) of Fe(s) to mequiv of peroxide. The peroxide value procedure was performed

with two replicates (each replicate analyzed once).

4.4.3.2 Determination of hexanal

The vegetable purees were thawed overnight at 4 °C, divided into four portions of 2.5 g each, and added

into 10 ml amber glass vials (VWR International, Radnor, PA, USA). To each vial, 2.5 mL saturated salt

solution was added. The vials were tightly closed using screw-caps with silicon septum seal (Grace,

Columbia, MD).

Under the fume hood, different volumes (0, 100, 200, and 300 µL) of hexanal working solution (1 µg/mL)

were added using a chilled gas-tight syringe through the septum into each of the 4 prepared vials. To

obtain a standard volume, different of amounts of deionized water were added into the vials through the

septum. The vials were then vortexed and taken for analysis.

PV (meq peroxide /kg sample)


32

For separation a method by Kebede et al. (2015) was chosen with some modifications. The prepared

samples were transferred to a cooling tray of the autosampler which was maintained at 10 °C. Headspace

analysis was conducted on a gas chromatography (GC) system (6890N, Keysight Technologies, Diegem,

Belgium), coupled to a mass selective detector (MSD) (5973N, Keysight Technologies, Diegem,

Belgium), and equipped with a CompiPAL autosampler (CTC Analytics, Zwingen, Switzerland).

Targeting hexanal in the tomato and carrot puree samples, an HS-SPME-GC-MS method was optimized

beforehand. In the selected method, the samples were incubated at 40 °C for 20 min under agitation at 500

rpm. Subsequently, extraction of the volatiles was performed using HS-SPME fiber coated with

divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) (StableFlex, Supelco, Bellefonte,

PA). The SPME fiber was inserted into the heated (230 ⁰C) GC injector port for 2 min to desorb the

volatile compounds. Prior to extraction, the fibers were conditioned and regenerated according to the

manufacturer’s guidelines in the conditioning station of the autosampler. Injection of the samples to the

GC column was performed in split (1/5) mode.

Chromatographic separation was carried out on an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 l

μm film thickness, Agilent Technologies, Santa Clara, CA) with helium as carrier gas at a constant flow of

1.3 mL/min. The stationary phase was (5%-phenyl)-methylpolysiloxane and -60 to 325/350 ⁰C

temperature limits. The GC oven temperature was programmed from a starting temperature of 40 °C,

which was maintained for 2 min, to 172 °C at 4 °C/min, then ramped to 300 °C at 30 °C/min and kept

constant at 300 °C for 2 min before cooling back to 40 °C. Mass spectra were obtained in electron

ionisation (EI) mode at 70 eV, with a scanning range of m/z 35–400 and a scanning speed of 3.8 scans per

second. The selected ions were m/z 44, 56, 72, and 82. MS ion source and quadrupole temperatures were

230 °C and 150 °C, respectively. The dwell time was 10 sec. Scan and selected ion monitoring (SIM) were

used as data acquisition modes.

4.4.4 Standardized static in vitro digestion for carotenoid bioaccessibility

A method according to Minekus et al.(2014) with some modifications, was chosen for the determination

of carotenoid bioaccessibility in tomato and carrot purees as a function of storage time. The simulated

digestion fluids namely, Simulated Gastric Fluids (SGF) and Simulated Intestinal Fluid (SIF), were made

up of the corresponding electrolyte concentrations (table 5), enzymes, CaCl2 and water. The digestion

process started with simulation of the gastric phase. To 5 mL of tomato or carrot puree, 5 ml of a 5 %

olive oil in water emulsion (1 % phosphatidyl choline, high pressure homogenized at 100 MPa), and 7.5


33

mL of SGF electrolyte stock were added in a brown falcon tube (50 mL). 1.6 mL of porcine pepsin stock

solution (25 000 U/ml, made-up in SGF electrolyte stock solution) was then added followed by 5 µL

CaCl2(H2O) (0.3 M). In order to bring the pH of the mixture to pH 3 ± 0.05, 10 – 15 µL of HCl (2M) was

added. This was followed by the addition of appropriate amount of demineralized water to bring the final

volume of the gastric chyme to 20 mL. Subsequently, the headspace of the tubes was flushed with

nitrogen for 10 seconds and the mixture incubated in the dark during 2 hours at 37 °C while rotating end

over end (40 rpm).

Table 5. Composition (% w/w) of the Simulated Gastric Fluids (SGF) and Simulated Intestinal Fluid (SIF) stock solutions.

Constituent SGF SIF

PH 3 PH 7

KCl 0.064 0.063

KH2PO4 0.015 0.014

NaHCO3 0.263 0.893

NaCl 0.345 0.281

MgCl2(H2O)6 0.003 0.008

(NH4)2CO3 0.006 -------

Following simulation of the gastric phase was the simulation of the intestinal phase. This started with

mixing the gastric chyme obtained in the previous phase (20 mL) with 11 mL of SIF electrolyte stock

solution. CaCl2(H2O) with a concentration of 0.3 M (40 µL) and 1.46 mL of demineralized water were

added, followed by 2.5 mL of bile solution (160 mM made in SIF electrolyte stock solution). Enzyme

solution (5 mL) consisting of pancreatin stock solution (800 U/mL based on trypsin activity, 1 600 U/mL

based on lipase activity, 1.4 % α-tocopherol, and 0.6 % pyrogallol, made in SIF electrolyte stock solution)

were then added. One or two drops of NaOH (1 M) were occasionally required to adjust the pH of mixture

to pH 7 ± 0.05. Finally, the headspace was flushed with nitrogen for 10 seconds and then incubated at 37

°C, end-over-end rotation at 40 rpm in the dark.

At the end of the simulation of the intestinal phase, part of the digest was ultra-centrifuged (65 000 g for 1

hour 8 minutes) in order to obtain the micellar phase. The ultra-centrifuged digest and micellar phase were

analyzed for carotenoids (section 4.4.2). The bioaccessibility procedure was performed with three


34

replicates (each replicate analyzed once) and the experiment repeated twice. Carotenoid bioaccessibility

(% BAC) was calculated according to equation 8.

(8)

4.5 Data analysis

Statistical analyses of the experimental data obtained was performed using the statistical software package

SAS (version 6.1, SAS Institute Inc, Cary, N.C., USA). Least squares linear regression was applied to

check for slopes significantly different from zero for carotenoid concentration data as a function of storage

time and temperature. The Tukey’s Studentized Pairwise Test was used to check for statistically different

percentage bioaccessibility values for purees stored at 20 °C. The level of significance used was 95 %

(P < 0.05)

Carotenoid bioaccessibility

PART II. RESULTS AND DISCUSSION

35

5. RESULTS AND DISCUSSION

The principles of accelerated shelf life testing were applied to design a storage experiment for shelf stable

fruit and vegetable based systems containing 5 % (w/w) EVOO over a period of 6 months. The goal was

to investigate the stability of carotenoids in relation to lipid oxidation, and carotenoid bioaccessibility.

Carrot and tomato purees were chosen as the relevant case studies, as sources of α-carotene, β-carotene

and lycopene.

Separately, carrot and tomato purees were prepared for storage as described in sections 4.1 and 4.2.

Briefly, vegetables were pre-treated, blended to make puree with 5 % (w/w) EVOO, HPH, thermally

treated in glass jars, and stored at 20, 30, and 40 °C for a period of 6 months in the dark. Sampling was

done according to a sampling plan in section 4.3. Samples were transferred into falcon tubes and stored at

-80 °C until further analyses. Carotenoid content and lipid oxidation, were determined as described in

sections 4.4.1 and 4.4.2 respectively. Furthermore, a standardized static in-vitro digestion method

(section 4.4.4) was used to determine carotenoid bioaccessibility as a function of storage time for samples

stored at 20 °C.

The results from the analyses will be presented and discussed under 3 sections namely:

i. the effect of storage temperature and time on carotenoid stability (section 5.1)

ii. the effect of storage temperature and time on lipid oxidation (section 5.2)

iii. carotenoid bioaccessibility as influenced by storage time at 20 °C (section 5.3)

5.1 Effect of storage temperature and time on carotenoid stability

5.1.1 Percentage carotenoid transfer to the oil fraction

In order to determine the amount of carotenoids in the oil phase, the percentage carotenoid transfer from

puree to oil as a result of the subsequent HPH and in-pack thermal treatment as described in section 4.1.2

and 4.2 respectively, was calculated. The major carotenoids namely all-trans-lycopene and all-trans-β-

carotene in tomato puree, and all-trans-β-carotene and all-trans-α-carotene in carrot puree were

considered. Carotenoid extraction was done in duplicate per sample and analyzed once. The percentage

carotenoid transfer was calculated using equation 6 and is illustrated in figure 6. It can be observed that

the percentage carotenoid transfer was highest for all-trans-α-carotene and all-trans-β-carotene and least


36

for all-trans-lycopene. Thus, carotenoid transfer was least efficient for lycopene compared to the other

carotenoids. In addition, percentage β-carotene transfer was not influenced by the food matrix. There were

two possible explanations for this observation. Firstly, this could be attributed to the differences in

molecular structure, where because of the β-ionone rings present in all-trans-β and α-carotene but absent

in all-trans-lycopene, all-trans-β and α-carotene could be incorporated into the oil phase without

aggregating (Nguyen et al. 2001).

Figure 6. Percentage transfer of all-E-lycopene ( ) and all-trans-β-carotene ( ) from tomato puree and all-trans-β-

carotene ( ), all-trans-α-carotene ( )) from carrot puree to oil. Error bars represent standard deviations.

Secondly, it can be hypothesized that this observation was due to differences in carotenoid localization in

the original raw material (Nguyen et al. 2001). Even after HPH followed by thermal treatment (which

disrupts the food matrix), lycopene crystals can still remain enveloped and associated with the thylakoid

membrane, hence restricting mobility during transfer, whereas β-carotene molecules are dissolved in lipid

material of plastoglobuli making transfer relatively efficient (Nguyen et al. 2001). Based on this result,

carotenoid content during storage was determined on the puree as a whole without phase separation, since

most of the carotenoids were in the oil fraction, as indicated by the high percentage carotenoid transfer

(figure 6).

5.1.2 Carotenoid concentration changes in tomato puree during storage

The changes in concentration of all-trans-lycopene and all-trans-β-carotene as well as 5-cis, 9-cis and 13-

cis-lycopene isomers in tomato puree during storage are illustrated in figure 7.

0

50

100

150

Tomato puree Carrot puree

Car

ote

no

id t

ransf

er (

%)


37

Figure 7. Changes in the concentration of (A) all-trans-lycopene and (B) all-trans-β-carotene as well as cis isomers

of lycopene: (C) 5-cis, (D) 9-cis and (E) 13-cis, expressed as absolute concentration, µg/g puree, in tomato puree

during storage at 20 °C ( ), 30 °C ( ) and 40 °C ( ). Error bars represent standard deviations.

Carotenoid extraction was done in duplicate per sample and analyzed once per extract. Individual points

represent a mean of the two replicates. It can be observed from figure 7 that carotenoid concentration did

not change with storage time and temperature. Although no noticeable changes in carotenoid

0

5

10

15

20

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)

Storage time (weeks)

A

0

5

10

15

20

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


B

0

5

10

15

20

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


C

0

5

10

15

20

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


D

0

5

10

15

20

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


E


38

concentration could be detected visually (figure 7), some statistically significant changes in carotenoid

concentration were found for some carotenoids from linear regression analysis on the data (P < 0.05),

summarized for tomato purees in table 6. For example, 5-cis-lycopene at all temperatures, and at 40 °C for

9-cis, 13-cis-lycopene and all-trans-β-carotene.

By comparing the concentration at day 0 with that at the end of the storage period (24 weeks), under the

conditions in the present study, results showed that there was no significant decrease in carotenoid content

for all-trans-lycopene, while at 40 °C was there a statistically significant decrease in carotenoid

concentration for all-trans-β-carotene (5.82 to 5.56 µg/g). Significant increases in 5-cis-lycopene

concentration, 1.89 to 3.43 µg/g, 1.89 to 2.61 µg/g and 1.89 to 3.37µg/g, at 20, 30 and 40 °C respectively

were also found. However, the effect of storage temperature on the increase in 5-cis-lycopene was not

evident. In addition, a significant decrease in 9-cis-lycopene (5.03 to 3.98 µg/g) and a significant increase

in 13-cis-lycopene (2.13 to 2.64 µg/g) at 40 °C were also found.

Table 6. Least squares linear regression of the changes in absolute carotenoid concentration in tomato purees during storage.

Slope values which are significantly different from 0 (P < 0.05) are indicated by *.

Slope

Isomer 20 °C 30 °C 40 °C

All-trans-lycopene -0.028 -0.031 -0.050

5-cis-lycopene 0.068* 0.076* 0.100*

9-cis-lycopene -0.006 -0.01 -0.032*

13-cis-lycopene 0.005 0.011 0.017*

All-trans-β-carotene -0.048 -0.031 -0.049*

The stability exhibited by all-trans-lycopene in the present study, on one hand contrasts and on the other

hand supports observations from some earlier studies. On one hand, in a study by Lin & Chen (2005),

approximately 90% losses in all-trans-lycopene and all-trans-β-carotene at 35 °C were reported.

Moreover, final carotenoid content decreased with increasing temperature. On the other hand, Lavelli &

Giovanelli (2003) observed that carotenoids were stable during 3 months storage at 30, 40, and 50 °C, of

thermally processed shelf stable tomato products without oil. This discrepancy in results can be explained

by possible differences in sample preparation, which in turn influences both amount of oxygen present and

water activity of the food system. During processing and storage, it is important to minimize the

concentration of oxygen in the food system, for example by vacuum deaeration or gas sparging, as it is


39

known to affect carotenoid stability (Boon et al. 2010; Illingworth & Bissell 1994; García-Torres et al.

2009). In the current study, in-pack thermal processing was employed and headspace was minimized in

the packaged purees, and in contrast, Lin & Chen (2005) used a plate heat exchanger for thermal

treatment, which possibly increases chances of further exposure of final product to oxygen. In this regard,

there could be differences in the amount of oxygen dissolved in and in the headspace of the treated purees

between the present study and the study by Lin & Chen (2005). An advantage of employing in-pack

thermal processing is that there is no further exposure of product to oxygen after packaging and

processing. There is a high probability that oxygen in the product will get used up during processing.

Thus, it can be hypothesized that the amount of oxygen dissolved and in the headspace of treated purees in

the present study was very minimal during storage. With regards to water activity, the study by Lin &

Chen (2005) was on tomato juice which invariably, contains more water compared to tomato puree in the

present study. Ribeiro et al. (2003) observed that lycopene in emulsions diluted with water degraded faster

than in an undiluted emulsion because a greater amount of water content corresponds to more dissolved

oxygen. Thus, because the samples (tomato juice) in the study by Lin & Chen (2005) were more dilute

compared to samples in the present study (tomato puree), it can be a possible reason for the observed

lycopene degradation in the former study.

As indicated earlier, in the current study, all-trans-β-carotene underwent a small but significant decrease

in concentration only at 40 °C. This is a possible indication of effect of temperature on β-carotene

stability. In the present study, the susceptibility to degradation of β-carotene and lycopene was different to

what has been reported previously in literature. Some authors have concluded that lycopene is more

susceptible to degradation compared to β-carotene because of structural differences (Ferreira &

Rodriguez-Amaya 2008; Woodall et al. 1997; Anguelova & Warthesen 2000). Based on their results, it

was expected that lycopene should have slightly degraded instead of β-carotene in this current work. As a

result, no clear explanation could be established for this observation. Although there was a significant

decrease in concentration, it is important to note that the extent of degradation was relatively small, with

only 0.26 µg/g being lost.

Though it is reported in literature that 5-cis-lycopene formation is not favored during processing due to the

large rotational barrier for the conversion of all-trans to 5-cis-lycopene (Guo et al. 2008), the significant

increase in 5-cis-lycopene in this present study at all storage temperatures could be explained by its low

energy level compared to the other isomers present (Guo et al. 2008). Guo et al. (2008) also reported that


40

the formation of 13-cis-isomer can be favored during storage at room temperature due to the low rotational

barrier during its formation from all-trans-lycopene compared to 9-cis-lycopene. Thus, the observed

increase in 13-cis-isomer could be ascribed to its formation from all-trans-lycopene isomerization. With

respect to 9-cis lycopene, it can be hypothesized that the decrease in concentration observed could be due

to degradation during storage.

Moreover, the observations in the current study indicated the protective effect of the food matrix to

carotenoid degradation. In this regard, the food matrix can have naturally occurring antioxidants like

phenolic compounds and α-tocopherol in the case of tomato (Frusciante et al. 2007), which can protect

carotenoids from degradation (Ferreira & Rodriguez-Amaya 2008; Ribeiro et al. 2003).

5.1.3 Carotenoid concentration changes in carrot puree during storage

Results of the changes in the concentrations of all-trans-β-carotene and all-trans-α-carotene as well as 9-

cis, 13-cis, and 15-cis-β-carotene isomers in carrot puree during storage are depicted in figure 8. Similarly

to what was observed in the tomato purees (figure 7), in figure 8, there was no observable changes in

carotenoid concentration as a function of storage time and temperature. Although no noticeable changes in

carotenoid concentration could be detected visually (figure 8), statistically significant changes (P < 0.05)

in carotenoid concentration were found for some carotenoids from linear regression analysis on the data

(table 7). For example, statistically significant differences were found for 9-cis-β-carotene at all

temperatures, and at 40 °C for all-trans-α-carotene. The stability exhibited by all-trans-β-carotene in the

present study on one hand, was in agreement and on the other hand contrasted some observations from

earlier studies. On one hand, Provesi et al. (2011) and Vásquez-Caicedo et al. (2006) reported stability of

all-trans-β-carotene in pumpkin puree during 180 day storage at 23 °C, and in mango puree during 168

day storage at 25 °C respectively. On the other hand, Chen et al. (1996) observed all-trans-β-carotene

degradation in carrot juice during 3 months storage at 4 °C, 20 °C, and 35 °C in the dark with extent of

degradation increasing with storage temperature. Provesi et al. (2011) linked the all-trans-β-carotene

stability to the exclusion of oxygen and protection from light, as well as thermal inactivation of

microorganisms and enzymes. Similarly, Vásquez-Caicedo et al. (2006) also concluded that oxygen

exclusion and headspace minimization is crucial for carotenoid stability during storage.


41

Figure 8. Changes in the concentration of (A) all-trans-β-carotene and (B) all-trans-α-carotene and the cis isomers of

β-carotene: (C) 9-cis, (D) 13-cis and (E) 15-cis expressed as absolute concentration, µg/g puree, in carrot puree

during storage at 20 °C ( ), 30 °C ( ) and 40 °C ( )

0

10

20

30

40

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


A

0

10

20

30

40

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


B

0

10

20

30

40

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


C

0

10

20

30

40

0 5 10 15 20 25C

on

cen

trat

ion

(µ

g/g

)


D

0

10

20

30

40

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

)


E


42

Table 7. Least squares linear regression of changes in absolute carotenoid concentration in carrot puree during storage. Slope

values which are significantly different from 0 (P < 0.05) indicated by *.

Slope

Isomer 20 °C 30 °C 40 °C

All-trans-β-carotene -0.018 -0.149 0.082

9-cis-β-carotene 0.073* 0.032

* 0.120

*

13-cis-β-carotene -0.034 -0.068 0.006

15 cis-β-carotene 0.014 0.126 0.029

All-trans α-carotene 0.040 0.005 0.120*

In comparison to Chen et al. (1996), the differences in observed carotenoid stability can possibly be due to

differences in sample preparation. Thus, it can be hypothesized that the observed all-trans-β-carotene

stability during storage could be attributed to the low oxygen concentrations in the samples for the present

study. Similarly to tomato puree, the differences in water content between carrot juice and carrot purees

was another contributing factor, which follows that a high water content results in a greater amount of

dissolved oxygen in the system. Furthermore, the superior stability exhibited by the carotenoids in carrot

puree compared to carotenoids studied in model systems as reported in literature suggested the protective

effect of the food matrix due to the presence of other phytonutrients with antioxidant capacity like

phenolic compounds (Klaiber et al. 2005; Hager & Howard 2006).

As indicated earlier, in the current study, all-trans-α-carotene underwent a small but significant increase in

concentration only at 40 °C. By comparing the concentration at day 0 with that at the end of the storage

period (24 weeks), an increase of 2.84 µg/g was observed for all-trans-α-carotene. A possible explanation

for this observation was the co-elution of all-trans-α-carotene with other unidentified isomers during

analysis.

Significant increases in concentration for 9-cis were 3.55 to 5.32 µg/g, 3.55 to 7.63 µg/g, and 3.55 to 8.09

µg/g at 20, 30, and 40 °C respectively. It could be observed that temperature had an effect on extent of

isomerization, which increased with an increase in temperature. These increases in 9-cis-β-carotene could

be attributed to isomerization from all-trans-β-carotene. Guo et al. (2008) reported that 9-cis formation

from all-trans-β-carotene is thermodynamically favored compared to 13-cis and 15-cis, thus the increase

in 9-cis concentration during storage compared to 13-cis and 15-cis-β-carotene.


43

5.2 Effect of storage temperature and time on lipid oxidation

5.2.1 Changes in peroxide value during storage

The peroxide values of the recovered oils from carrot and tomato purees at the beginning, middle and end

of the storage period were below the detection limit (0.044 mequiv peroxide/kg of sample) (Hornero-

Méndez et al. 2001) for all the samples. Hydroperoxide extraction from sample was done in duplicate and

absorbance of extract measured once. Consequently, temperature had no effect on primary lipid oxidation

during storage. The peroxide value for fresh EVOO was 3.03 ± 0.32 mequiv peroxide/kg sample (± SD)

which is in agreement with a peroxide value for fresh olive oil as reported by García et al. (1996).

The results from PV measurement indicate that, under the conditions of the study, tomato and carrot

purees were stable to lipid oxidation since no primary lipid oxidation products were detected. Normally,

when a food system undergoes lipid oxidation, it is expected that its peroxide value increases for a certain

period of time, as a result of formation of hydroperoxides, and then starts to decrease as a result of

degradation to secondary lipid oxidation products (García et al. 1996). The observations made in the

current study can be attributed to two factors: the nature of the lipid substrate, and possibly the low

amounts of oxygen both dissolved in the sample and in the product headspace. Firstly, the lipid substrate

present in the purees in the present study (EVOO), has a high resistance to oxidative deterioration due to

the high proportion of monounsaturated fatty acids compared to polyunsaturated fatty acids in its fatty

acid composition (Zarrouk et al. 2009), as well as the presence of minor antioxidants like phenolics

(Velasco & Dobarganes 2002; Frankel 2010). The fatty acid composition of EVOO suppresses the

occurrence of lipid oxidation (Schaich 2010; Choe & Min 2006). Secondly, since oxygen is needed to

react with radicals formed in order to form hydroperoxides, it can also be hypothesized that the limited

amount of oxygen in the thermally treated samples during storage suppressed hydroperoxide formation in

detectable amounts in accordance with Hahm & Min (1995).

Though the measured peroxide value for fresh EVOO was 3.03 ± 0.32 mequiv peroxide/kg sample, it is

possible that the hydroperoxides originally present in the samples were degraded during the thermal

treatment. This is because at low or moderate temperatures, hydroperoxide formation is higher than

decomposition, but decomposition occurs at a faster rate at elevated temperatures (Velasco & Dobarganes

2002; Madhavi et al. 1995). Thus, during the thermal treatment (117 °C) under the present study, two

processes could occur, namely: alkyl radical formation and hydroperoxide breakdown. In this context, it


44

can be hypothesized that the residual oxygen in the headspace and dissolved in the samples was consumed

by radicals formed during the thermal treatment followed by the subsequent degradation of the

hydroperoxides. This explains why at day 0 no peroxides were detected in the sample and there is a high

probability that limited oxygen was left for autoxidation reactions during storage. This observation

supports the results in section 5.2, where it has been concluded that carotenoids were stable during

storage. This is because in the absence of light, but with molecular oxygen present in a system,

hydroperoxides can be formed and serve as a source of peroxyl radicals which react with carotenoids,

leading to carotenoid degradation (Boon et al. 2010). However, in the absence of hydroperoxides, as is the

case in the present study, carotenoids degradation reactions were effectively suppressed.

5.2.2 Changes in hexanal during storage

The results of the changes in hexanal content of the purees during storage as determined by the standard

addition method (section 4.4.3.2) are depicted in figure 9. It can be observed in both graphs that there was

a slight increase in hexanal concentration followed by a decrease at 30 and 40 °C during week 20 to 24.

Figure 9. Changes in hexanal concentration in, (A) tomato puree and (B) carrot puree during storage at 20 °C ( ), 30

°C ( ) and 40 °C ( ).

The increase was higher at 40 °C compared to 30 °C. However, for tomato puree in figure 9A, hexanal

production at 40 °C was much higher than in carrot puree in figure 9B. This trend is an indication that

there was production of hexanal during storage, in particular at 30 and 40 °C in both carrot and tomato

purees. As described before (section 5.2.1), decomposition of hydroperoxides to volatile carbonyl

compounds is favored at elevated temperatures.By comparing hexanal concentration at day 0 and week 24,

the increase was relatively small, 0.26 to 0.57 µg/g puree in tomato puree and 0.18 to0.32 µg/g puree in

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

pu

ree)


A

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25

Con

cen

trat

ion

(µ

g/g

pu

ree)


B


45

carrot puree. These maximum hexanal concentrations observed in the purees during storage are

insignificant to describe the overall progression of lipid oxidation in the samples when compared to

literature. In a study by Sanches-Silva et al. (2004), a hexanal concentration of 0.5-0.6 µg/g sample was

reported for fresh, non-oxidized potato crisps. Moreover, in oxidized low fat dehydrated foods, rancid

flavors started to be detected at levels of 5 ppm (Fritsch & Gale 1977). Additionally, Oey et al. (2008)

reported that at hexanal concentrations below 1.2 µg/g, the fresh flavor of tomatoes is imparted. This

indicates that in the current study, even if there was an increase in hexanal concentration during storage of

the purees, it was negligible. However, it is important to note that the hexanal could have been formed

from the breakdown of small quantities of peroxides in the sample which were not detectable by the

method described in section 4.4.3.1. The results confirm previous observations in section 5.2.1, otherwise

a significant increase in hexanal would have been observed had the hydroperoxides formation been

significant. The decrease in hexanal concentration in the last week of storage could probably be due to

hexanal breakdown to other products.

Hexanal production as a result of HPH and thermal treatment of tomato and carrot purees is illustrated in

figure 10. Hexanal was already detected in untreated homogenized puree samples (figure 10), its

concentration being higher in tomato compared to carrot samples. It follows that even during storage

tomato puree had a higher hexanal content compared to carrot puree.

Figure 10. Effect of thermal processing on hexanal concentration in the carrot and tomato purees: untreated ( ) and

treated ( ).

Furthermore, the increase in hexanal in the treated samples (64.7% and 37.0% for carrot and tomato

purees respectively) indicates that the thermal treatment resulted in the formation of additional hexanal

(Kebede et al. 2014). The presence of hexanal in untreated purees was attributed to the presence of fatty

0

0.1

0.2

0.3

0.4

0.5

Carrot Tomato

Con

cen

trat

ion

. (µ

g/g

pu

ree)


46

acid substrates originally present in plant tissue. Upon matrix disruption during cutting of vegetables

before the blanching process, compartmentalized enzymes and substrates get into contact resulting in the

production of volatiles (El Hadi et al. 2013).

The differences in enzymes present in the two matrices could explain the higher hexanal content that was

observed in tomato puree. In fact, tomato contains lipoxygenase, an enzyme which catalyzes the oxidation

of polyunsaturated fatty acids in plant tissues (Shook et al. 2001; Oey et al. 2008), whereas this enzyme is

absent in carrot (Güneş & Bayindirli 1993). Additionally, there was an increase in hexanal concentration

as a result of thermal treatment. The increase in hexanal concentration that was observed in figure 10

could be ascribed to the breakdown of hydroperoxides that resulted from the thermally-induced oxidation

of unsaturated fatty acids present in the tissue and the EVOO (Christensen et al. 2007).

5.2.3 Relation between carotenoid stability and lipid oxidation during storage

The observations in the current study, suggest a relation between carotenoid stability and lipid oxidation.

With insignificant lipid oxidation occurring in both matrices, carotenoid degradation did not occur,

indicating a direct relationship between the two processes. This is in agreement with earlier studies. For

example, though in a model system, Anguelova & Warthesen (2000) also observed that in the presence of

peroxyl radicals, up to 84%, 68%, 50% lycopene, β-carotene and α-carotene losses, respectively, were

observed. Boon et al. (2010) and Woodall et al. (1997) pointed out that carotenoids possess antioxidant

properties which makes them react with radical species and in the process get degraded. In this regard, in

the absence of hydroperoxides (a source of peroxyl radicals) in the carrot and tomato purees during

storage, carotenoid degradation could not be observed in the present study.

5.3 Changes in carotenoid bioaccessibility as influenced by storage time at 20 °C

The results of the changes in carotenoid bioaccessibility (% BAC), for all-trans-β-carotene and all-trans-

lycopene in tomato and all-trans-α- and β carotene in carrot purees that were stored at 20 °C at the

beginning, middle and end of the storage period are illustrated in figure 11.


47

Figure 11. Percentage bioaccessibility as a function of storage time at 20 °C. In A: Tomato puree % BAC; tomato

all-trans-β-carotene ( ), all-trans-lycopene ( ); B: Carrot puree % BAC; carrot all-trans-β-carotene ( ), all-trans-α-

carotene ( ). Statistical differences in % BAC shown by a/a` on the graph, otherwise not statistically different. Error

bars represent standard deviations.

In tomato purees, shown in figure 11A, the % BAC was higher for all-trans-β-carotene (about 33%)

compared to lycopene (about 20%) at all the storage time moments considered. In carrot puree (figure 11

B), % BAC was higher for carrot all-trans-β-carotene (30-36%) compared to all-trans-α-carotene (30-

32%). Comparing figures 11 A and B, it can be observed that in both food matrices, all-trans-β-carotene

was more bioaccessible compared to the other carotenoids. Moreover, similarly to carotenoid transfer

(section 5.1.1), all-trans-β-carotene % BAC was not influenced by the matrix. It can be observed (figure

11) that in both matrices, considering both carotenoids in each matrix, there was no significant difference

in % BAC during storage. Benlloch-Tinoco et al. (2015) observed a similar trend for all-trans-β-carotene

in kiwi puree stored at 10 °C for 63 daysIt is important to note that data which expresses carotenoid

bioaccessibility in the same way as in the present study was not available in order to make direct

comparisons of the obtained results with literature. Nevertheless, some general comparisons can be made.

For example, Colle et al. (2013b)a reported all-trans-lycopene bioaccessibility values of around 16 % in

high pressure homogenized and thermally treated tomato pulp containing 5% olive oil. For all-trans-β-

carotene, Knockaert et al. (2012) observed all-trans-β-carotene bioaccessibility of up to 66% of the total

β-carotene content, expressed as bioaccessible concentration µg/g dry matter. It can be concluded that

carotenoid bioaccessibility of tomato and carrot purees under the conditions of current study does not

change with time, rather it is important to ensure that carotenoid bioaccessibility is high after food

processing.

a a a

a

A AB

A A

0

10

20

30

40

50

60

0 4 12 24

BA

C (

%)


A

a' a' a' a'

A' A'

A' A'

0

10

20

30

40

50

60

0 4 12 24

BA

C (

%)


B

PART II. GENERAL CONCLUSIONS

48

6. GENERAL CONCLUSIONS

An accelerated shelf life storage experiment for a period of 6 months was designed to investigate the

stability of lycopene and β-carotene in tomato puree and β-carotene and α-carotene in carrot puree

containing 5% (w/w) EVOO. Furthermore, the relation between carotenoid degradation and lipid

oxidation as well as changes in carotenoid bioaccessibility during storage were also considered. The

purees were subjected to high pressure homogenization followed by thermal processing in the presence of

oil before storage in the dark at 20 °C, 30 °C and 40 °C.

Under the present conditions, percentage transfer to the oil was high (70 % for all-trans lycopene and

around 100 % for β- and α-carotene) indicating that carotenoid changes during storage were well

represented by changes in the puree as a whole. Furthermore, β-carotene transfer was independent of the

matrix. The extent of carotenoid transfer to oil was linked to the differences in carotenoid localization and

chemical structure. Considering carotenoid stability, storage temperature and time had no effect on the

major all-trans carotenoids in both carrot and tomato purees. The carotenoid stability observed could be

attributed to minimization of oxygen content of the food systems, absence of light during storage and the

protective effect of the food matrix. It is noteworthy that although, overall, the food systems in the present

study were stable to carotenoid degradation, some statistically significant changes in concentration of the

isomers were found. These relatively small changes during storage were observed for all-trans-β-carotene,

5-cis, 9-cis and 13-cis-lycopene in tomato, and 9-cis-β-carotene and all-trans-α-carotene in carrot.

In addition, primary lipid oxidation measured by the peroxide value were not detected in the samples.

Hexanal as a measure of secondary lipid oxidation was detected in both tomato and carrot puree before the

thermal treatment as well as during storage. The hexanal concentration was higher in tomato than in carrot

puree both before and after thermal treatment and subsequent storage. This observation was ascribed to the

differences in enzymes present in the original raw material (fresh tomatoes and carrots). The effect of

temperature on hexanal formation was observed at 30 and 40 °C, being higher at 40 °C, followed by a

decrease during week 20 to 24. However, the hexanal concentrations detected were too small for food

systems undergoing lipid oxidation as compared to literature. Considering both primary and secondary

lipid oxidation products, it was concluded that lipid oxidation did not progress to an extent significant

enough to influence carotenoid stability during storage. On account of the insignificant lipid oxidation and

PART II. GENERAL CONCLUSIONS

49

carotenoid stability observed in both tomato and carrot purees, a direct relationship between lipid

oxidation and carotenoid stability was assumed as reported in previous studies.

The results of the bioaccessibility of all-trans-lycopene and all-trans-β-carotene in tomato purees as well

as all-trans α- and β-carotene in carrot purees that were stored at 20 °C revealed that bioaccessibility did

not change with storage time.

In conclusion it can be stated that the chosen food systems and storage conditions in the present study

were sufficient to ensure stability to both lipid oxidation and carotenoid degradation. This study has also

shown that carotenoids are stable during storage and can remain in a bioaccessible form for at least 6

months in shelf stable fruit and vegetable systems containing a lipid substrate provided the latter exhibits

good stability to lipid oxidation. However, a limitation in this current study is that the oxygen

concentration before and after processing was not measured. Measuring the concentration of oxygen in the

system could help in establishing more concrete recommendations. Nevertheless, the effect of light on

carotenoid stability during storage on similar food systems as in the present study still requires to be

investigated.

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50

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