traditional meat products: improvement of quality and safety

Journal of Food Quality

Traditional Meat Products Improvement of Quality and Safety

Lead Guest Editor Marta LaranjoGuest Editors Reacutegine Talon Andrea Laukovaacute Maria J Fraqueza and Miguel Elias

Traditional Meat ProductsImprovement of Quality and Safety



Lead Guest Editor Marta LaranjoGuest Editors Reacutegine Talon Andrea Laukovaacute Maria J Fraquezaand Miguel Elias

Copyright copy 2017 Hindawi All rights reserved

This is a special issue published in ldquoJournal of Food Qualityrdquo All articles are open access articles distributed under the Creative Com-mons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Editorial Board

Encarna Aguayo SpainRiccarda Antiochia ItalyJorge Barros-Velaacutezquez SpainJoseacute A Beltraacuten SpainAacute A Carbonell-Barrachina SpainMarina Carcea ItalyMaria Rosaria Corbo ItalyEgidio De Benedetto ItalyAlessandra Del Caro ItalyAntimo Di Maro Italy

Rossella Di Monaco ItalyHuumlseyin Erten TurkeySusana Fiszman SpainAndrea Galimberti ItalyEfstathios Giaouris GreeceVicente M Goacutemez-Loacutepez SpainElena Gonzaacutelez-Fandos SpainAlejandro Hernaacutendez SpainJesuacutes Lozano SpainSara Panseri Italy

Mariacutea B Peacuterez-Gago SpainWitoon Prinyawiwatkul USAEduardo Pueacutertolas SpainJuan E Rivera MexicoFlora V Romeo ItalyJordi Rovira SpainAmy Simonne USAGiuseppe Zeppa Italy

Contents

Traditional Meat Products Improvement of Quality and SafetyMarta Laranjo Reacutegine Talon Andrea Laukovaacute Maria J Fraqueza and Miguel EliasVolume 2017 Article ID 2873793 2 pages

TheUse of Starter Cultures in Traditional Meat ProductsMarta Laranjo Miguel Elias and Maria Joatildeo FraquezaVolume 2017 Article ID 9546026 18 pages

The Effect of Drying Parameters on the Quality of Pork and Poultry-Pork Kabanosy Producedaccording to the Traditional Specialties Guaranteed RecipeMarta Chmiel Lech Adamczak Katarzyna Wroampaposnska Dorota Pietrzak and Tomasz FlorowskiVolume 2017 Article ID 1597432 7 pages

Health and Safety Considerations of Fermented SausagesAskild Holck Lars Axelsson Anette McLeod Tone Mari Rode and Even HeirVolume 2017 Article ID 9753894 25 pages

Effects of Ozone Treatments on the Physicochemical Changes of Myofibrillar Proteins from Silver Carp(Hypophthalmichthys molitrix) during Frozen StorageRongrong Zhang Shanbai Xiong Juan You Yang Hu Ru Liu and Tao YinVolume 2017 Article ID 9506596 9 pages

Effects of Micron Fish Bone with Different Particle Size on the Properties of Silver Carp(Hypophthalmichthys molitrix) Surimi GelsTao Yin Jae W Park and Shanbai XiongVolume 2017 Article ID 8078062 8 pages

Effects of Beeswax Coating on the Oxidative Stability of Long-Ripened Italian SalamiMarcello Trevisani Matilde Cecchini Daniela SiconolfiRocco Mancusi and Roberto RosminiVolume 2017 Article ID 8089135 5 pages

EditorialTraditional Meat Products Improvement of Quality and Safety

Marta Laranjo12 Reacutegine Talon3 Andrea Laukovaacute4 Maria J Fraqueza5 andMiguel Elias16

1 Instituto de Ciencias Agrarias e Ambientais Mediterranicas (ICAAM) Universidade de Evora Polo da Mitra Ap 947002-554 Evora Portugal2Instituto de Investigacao e Formacao Avancada (IIFA) Universidade de Evora Evora Portugal3Universite Clermont Auvergne INRA MEDIS Clermont-Ferrand France4Institute of Animal Physiology Slovak Academy of Sciences Kosice Slovakia5CIISA Faculty of Veterinary Medicine University of Lisbon Avenida da Universidade Tecnica Polo Universitario do Alto da Ajuda1300-477 Lisbon Portugal6Departamento de Fitotecnia Escola de Ciencias e Tecnologia Universidade de Evora Polo daMitra Ap 94 7002-554 Evora Portugal

Correspondence should be addressed to Marta Laranjo mlaranjouevorapt

Received 6 November 2017 Accepted 7 November 2017 Published 21 November 2017

Copyright copy 2017 Marta Laranjo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

What are traditional meat products Traditional meat prod-ucts are high sensory quality foods usually with high nutri-tional value produced in a small scale using ingredientsand procedures from ancient times Producers must have theability to satisfy the expectations of consumers regardingsensory nutritional aspects and safety considering that theyare increasingly more demanding and more informed turn-ing this into an important challenge Usually a meat productis defined as foods that consist of or containmeatThe flesh ofan animal typically a mammal or bird is considered as meatHowever fish meat is also present in our diet and severaltraditional processed products come from itThus traditionalmeat products sensu lato include food products derived fromboth meat and fish meat

All over the world different processes are used to pre-serve meat and the particular know-how of people livingin different regions gave rise to a great diversity of meatproducts according to their traditions and historic useThesetraditional processes their particularities and their effecton the quality and safety of meat products are importantresearch topics The scope of this edition was to disseminatehigh-quality research related with traditional meat productsand review recent developments on the quality and safetyimprovement of traditional meat products worldwide suchas the use of starter cultures or the application of newpreservation methods

Among the 16 submitted manuscripts six have beenselected to be part of this special issue

M Trevisani et al studied the effect of beeswax coatingof foods on the oxidative stability of Italian salami Beeswaxcoating constitutes a barrier to oxygen light and vapourwhich may prevent oxidation of fat and pigments andwater loss Furthermore the authors concluded that beeswaxcoating prevents case hardening and facilitated the peeling

T Yin et al have evaluated the quality of silver carp(Hypophthalmichthys molitrix) surimi (SCS) gels incorpo-rated with fish bone and found out that size reduction of thefish bone improved the quality of the SCS gel maintainingbetter gel matrices

M Chmiel et al have studied two types of kabanosysausages Traditional Specialties Guaranteed (TSG) fromPoland and concluded that their drying process could beshortened without compromising the authenticity of theproducts and with the concomitant advantages to producers

R Zhang et al investigated the effects of different ozonetreatments on the physicochemical characteristics of myofib-rillar proteins from silver carp (H molitrix) surimi duringfrozen storage

A Holck et al reviewed the possible health effects of theingredients used in fermented sausages Recent attempts toimprove sausages from the nutritional point of view includepartial replacement of saturated by unsaturated fats reducingthe use of sodium chloride or replacement by potassiumchloride and the use of selected starter cultures They alsoreview the processing and postprocessing strategies to inhibitthe growth of food pathogenic microorganisms such as

HindawiJournal of Food QualityVolume 2017 Article ID 2873793 2 pageshttpsdoiorg10115520172873793

2 Journal of Food Quality

Escherichia coli Salmonella enterica Staphylococcus aureusListeria monocytogenes Clostridium botulinum and Toxo-plasma gondii and reduce their presence in the products

The paper ldquoThe Use of Starter Cultures in TraditionalMeat Productsrdquo byM Laranjo et al reviews the role andmodeof action of bacterial and fungal starter microbiota focusingon the development of starter cultures better adapted to themeat matrix Furthermore omics approaches on starter cul-tures are revised because the use of these techniques allowsrapid screening of strains for desirable functional character-istics

Submitting authors come from five different countriesfour European (Italy Poland Norway and Portugal) and onenon-European China

We are pleased to introduce this special issue whichincludes six papers that provide new insights on the manu-facturing and processing of traditional meat products sensulato andwewish that the readers of this journal find this issueof relevance and importance to their research

Acknowledgments

We thank the authors of the manuscripts for their contri-butions as well as all the anonymous reviewers for theirvaluable participation in the evaluation process M Laranjoacknowledges a Post-Doc research grant from Fundacao paraa Ciencia e a Tecnologia (FCT) (SFRHBPD1088022015)

Marta LaranjoRegine Talon

Andrea LaukovaMaria J Fraqueza

Miguel Elias

Review ArticleThe Use of Starter Cultures in Traditional Meat Products

Marta Laranjo12 Miguel Elias13 andMaria Joatildeo Fraqueza4

1 Instituto de Ciencias Agrarias e Ambientais Mediterranicas (ICAAM) Universidade de Evora Polo da MitraAp 94 7002-554 Evora Portugal2Instituto de Investigacao e Formacao Avancada (IIFA) Universidade de Evora Evora Portugal3Departamento de Fitotecnia Escola de Ciencias e Tecnologia Universidade de Evora Polo daMitra Ap 94 7002-554 Evora Portugal4CIISA Faculty of Veterinary Medicine University of Lisbon Avenida da Universidade TecnicaPolo Universitario do Alto da Ajuda 1300-477 Lisbon Portugal


Received 3 July 2017 Revised 17 October 2017 Accepted 19 October 2017 Published 12 November 2017

Academic Editor Maria Rosaria Corbo


Starter cultures could play an essential role in the manufacture of traditional cured meat products In order to achieve objectivesrelated to meat productsrsquo quality and safety improvement the selection of particular strains constituting a starter culture shouldbe carried out in the context of its application since its functionality will depend on the type of sausage and process conditionsAlso strain selection should comply with particular requirements to warrant safety The aim of the current review is to update theknowledge on the use of starter cultures in traditional meat products with focus on dry-fermented products In this manuscriptwe will try to give answers to some relevant questionsWhich starter cultures are used and whyWhy are LAB usedWhat are theirrole and their specific mode of actionWhich other groups of microorganisms (bacteria and fungi) are used as starter cultures andhow do they act A particular revision of omics approach regarding starter cultures is made since the use of these techniques allowsrapid screening of promising wild strains with desirable functional characteristics enabling the development of starter culturesbetter adapted to the meat matrix

1 Introduction

Starter cultures or starters are individual or mixed formula-tions of selected strains with a particular enzymatic activitythat when added in a defined concentration to a substratetransform it into a food product with specific characteristics[1]This concept applied to meat products could be describedas viable microorganisms that are able to multiply themselvesinside meat products increasing their preservation control-ling their hygienic safety and potentiating their acceptabilityby consumers maintaining or improving their nutritionalquality [1]

The preliminary use of starters in meat products resultedfrom adding a portion of the final meat products to their rawmaterials meaning that part of the already fermented batchof sausage was thrown back into the new mix This alreadyfermented product contained the necessary microorganismsto start the fermentation of the new batch This is known asback-slopping or back-inoculation [2]

Fermented meat products may be manufactured withoutthe use of starter cultures although their use can help toensure safety standardising product properties (includingflavour and colour) and shorten the ripening period Nev-ertheless well-adapted and qualified presumption of safety(QPS) strains must be used and the establishment of thestarter culture must be verified in order to guarantee theexpected performance

Probiotics are live microorganisms that confer a healthbenefit to the host when administered in adequate amounts[3] Probiotics have been used in food products food sup-plements and pharmaceutical products Due to increasingconcerns over health probiotic foods (eg probiotic dairyproducts) are now accepted in theworldmarket Recently thepossibility of developing probiotic meat products has beendiscussed [4] By using probiotic starter microorganismspotential health benefits can be introduced to meat productsand it is already possible to produce probiotic meat products[5 6] Nevertheless the potentially beneficial effects on



human health from eating a probiotic sausage still needconfirmation [7 8]

The starter groups used nowadays in meat industry areby order of importance lactic acid bacteria (LAB) Gram-positive catalase-positive cocci (GCC+) (mainly staphylo-cocci) moulds and yeasts

Lactic acid bacteria (LAB) are a group of Gram-positivebacteria belonging to the Firmicutes They are catalase-negative either rod-shaped (bacilli) or spherical (cocci)characterised by an increased tolerance to acidity (low pHrange) and have a low GC (guanine-cytosine) contentAlthough many genera of bacteria produce lactic acid asa primary or secondary end-product of fermentation theterm lactic acid bacteria (LAB) is conventionally reservedfor genera in the order Lactobacillales which includes Aero-coccus Carnobacterium Enterococcus Lactococcus Lacto-bacillus LeuconostocOenococcus Pediococcus StreptococcusTetragenococcus Vagococcus and Weissella [9] As food fer-mentation agents LAB are involved inmaking yogurt cheesecultured butter sour cream sausage cucumber picklesolives and sauerkraut some speciesmay spoil beer wine andprocessed meats [10]

Gram-positive catalase-positive cocci (GCC+) are thesecond most important group of meat starters and are com-posed of nonpathogenic coagulase-negative staphylococci(CNS) The most important starters from this group arestrains belonging to the genera Staphylococcus and Kocuria[11]

At the beginning of the ripening process the surfacemycobiota is mainly composed of yeasts however as 119886

119908de-

creases moulds outcompete yeasts and predominate in thefinal product [12] Moulds colonise the surface of fermentedmeat products in some cases conferring particular charac-teristics however in other cases being considered signs ofspoilage

Yeasts are characteristic components of the mycobiotagrowing on fermented sausagesTheir origin ismainly relatedto the environment and to the meat used as raw materialsince yeasts are naturally found on freshmeatThemost com-mon genera are Candida Rhodotorula Debaryomyces andTrichosporon In fermentedmeats the lactic acid produced byLAB changes the environment favouring the development ofyeasts which use all of the nutrients and energy and grow fast[13]

Meat preservation by fermentation has been carried outfor thousands of years but the idea of starter cultures wasfirst introduced for dry sausages in the 1940s with Patent US2225783 A [14] The first commercial starter culture was astrain of Pediococcus acidilactici that was made available inthe US in 1957 [15] In Europe the first starter culture to beintroduced was strain M53 from the genus Kocuria isolatedfrom a Finnish sausage whichwas used to prevent colour andaroma defects [16]

Starter cultures play an essential role in the manufactureof fermented food products Starters composed of LABstrains produce the lactic acid that acts on meat proteinsmodifying their water biding capacity thus contributingto texture moisture content flavour and aroma of theproducts and definitively acts on its microbiological safety

Additionally microbial substances namely bacteriocinsproduced by Gram-positive species of the LAB group suchas for example nisin and other lantibiotics or pediocin-likebacteriocins have an antimicrobial role with an effect onpreservation and safety

Starter cultures have a number of advantages(i) They are of known quantity and quality(ii) They reduce the ripening time(iii) They increase safety by outcompeting undesirable

microorganisms(iv) They enable the manufacture of a product of constant

quality all year round in any climatic zone as longas proper natural conditions or fermentingdryingchambers are available

The aim of the current review is to update the knowledge onthe use of starter cultures in traditional meat products withfocus on dry-fermented products

In this manuscript we will try to give answers to somerelevant questions on this subject through the analysis ofpublished studies with some applied results Which startercultures are used and why Why are LAB used What istheir role and their specific mode of action Which othergroups of microorganisms (bacteria and fungi) are also usedas starter cultures and how do they act What is theirfunction A revision related to omics methods applied to thescreening of autochthonous strains with desirable functionalcharacteristics allowing the development of well adaptedstarter cultures to the meat matrix will be done

2 Starter Cultures inDry-Fermented Meat Products

The first generation of meat starter cultures was generallybased on microorganisms isolated from vegetable fermenta-tion such as L plantarum and members of the genus Pedio-coccus Then a second generation of starter cultures com-prising meat-borne strains such as L sakei and coagulase-negative staphylococci (CNS) was developed harbouringphenotypic traits of technological relevance [17] Morerecently efforts have been dedicated to the study of the phys-iological and technological properties of LAB and CNS iso-lated from traditional fermented sausages in order to developfunctional starter cultures that enhance safety and nutritionaladvantages while maintaining industrial performance [5 18]

The manufacturing of dry-fermented sausages involvesspontaneous fermentation commanded by bacteria (LAB)and GCC+ and less importantly by fungi namely mouldsand yeasts [19]

Most meat starter cultures commercially available arecombined cultures of LAB (mainly Lactobacillus spp andPediococcus spp) and GCC+ (primarily Staphylococcus sppand Kocuria spp) These bacteria are responsible for themicrobial reactions that occur during meat fermentationsuch as acidification catalase activity and bacteriocin pro-duction [11]

Several studies have addressed the importance of usingstarter cultures in traditional dry-fermented meat products

Journal of Food Quality 3

not only for safety or conformity reasons but also foruniformity purposes [20ndash22]

Although most studies about the use of starter culturesare on dry-fermented sausages [23ndash25] a few works on othermeat products such as hams or fresh sausages have also beenreported [26]

Inoculation of starter cultures in dry-fermented meatproducts may occur either by incorporation as an ingredientin the meat batters or by surface inoculation

Bacteria are usually incorporated in the meat batters atconcentrations between 5 and 8 log colony forming units(cfu)g [23] Yeasts may be inoculated either on the surfaceof the sausage or in the meat batter at a concentration typ-ically between 4 and 6 log cfug Moulds are always surface-inoculated due to their strictly aerobic character frequentlyby dipping in an aqueous solution of spores at concentrationsranging from 3 to 4 log sporescm2

21Their Role in Quality Improvement of Sausages The selec-tion of starter cultures for quality improvement of sausages isbased on technologically relevant traits The autochthonousmicrobiota of sausages and othermeat products as well as themicrobiota of the processing environment of the productionunits may be a good starting point for the isolation ofpotential starters because those strains are well adapted tothe meat environment [19]

Bourdichon and coworkers [27] presented a list of micro-organisms used in food fermentation in a wide range of foodmatrices (dairy products meat fish vegetables legumescereals beverages and vinegar)

211 Bacteria LAB and GCC+ When selecting starter cul-tures for dry- and semidry-fermented sausages LAB andCNS strains with useful metabolic activities and benefitsduring fermentation should be used

(1) Lactic Acid Bacteria (LAB) Lactic acid bacteria (LAB)are Gram-positive non-spore-forming cocci or bacilli witha lowGC content [28]They generally are nonrespiratory andlack catalase They produce lactic acid as one of the mainfermentation products of carbohydrates They lack genuinecatalase and do not possess cytochromes All LAB growanaerobically but unlike most anaerobes they grow in thepresence of O

2as ldquoaerotolerant anaerobesrdquo [9]

According to the current taxonomic classification theybelong to the phylum Firmicutes class Bacilli order Lacto-bacillales Six different families include all genera as shownin Table 1 (httpwwwuniprotorgtaxonomy186826)

Lactic acid bacteria are among the most importantgroups of microorganisms used in food fermentation Theycontribute to the taste and texture of fermented productsand inhibit food spoilage bacteria by producing growth-inhibiting substances and large amounts of lactic acid

Based on sugar fermentation patterns there are two broadmetabolic categories of LAB homofermentative and hetero-fermentativeThe homofermentative pathway produces basi-cally only lactic acid whereas the heterofermentative pathwayproduces CO

2and ethanol or acetate in addition to lactic

acid [9] Homofermentative LAB include some lactobacilliand most enterococci lactococci pediococci streptococcitetragenococci and vagococci that ferment hexoses throughglycolysis by the Embden-Meyerhof-Parnas pathway Onthe other hand heterofermentative LAB ferment pentosesmainly through the phosphoketolase pathway and includeleuconostocs some lactobacilli oenococci and Weissellaspecies

Relevant technological features for LAB starters includefast production of lactic acid growth at different temper-atures salt concentrations and pH values gas productionfrom carbohydrates catalase activity and hydrolysis of hydro-gen peroxide nitrate and nitrite reduction moderate prote-olytic and lipolytic enzymatic activities good performancein combined starters with other microbial components[29]

However fermentation conditions must be controlledto avoid excessive pinholes gas pockets and off-flavoursresulting from gas production from carbohydrates [30]Additionally the production of hydrogen peroxidemay resultin undesirable oxidation known as greening [30] Further-more it must be taken into account that proteolytic andlipolytic activities should be moderate to avoid undesirablesensory changes

As for the role of LAB in the quality of dry-fermentedmeat products LAB participate in the coagulation of muscleproteins by acidifying the batters which results in increasedslice stability firmness and cohesiveness of the final product[31 32] Besides they contribute to the flavour of the finalproduct through the formation of noticeable acidic andvinegary (acetic acid) tastes Moreover the existing acidicconditions may increase the activity of cathepsin D which isagain responsible for muscle proteolysis [33]

Several authors have reported the use of LAB startercultures for the production of fermented sausages [34ndash38]For example Wang and coworkers reported the inoculationwith L sakei as beneficial for microbiological quality againstthe growth of foodborne pathogens also improving sensorycharacteristics [34]

(2) Gram-Positive Catalase-Positive Cocci (GCC+) Gram-positive catalase-positive cocci GCC+ mainly nonpathogen-ic coagulase-negative staphylococci (CNS) are also impor-tant in the fermentation process of sausages since theyimprove the quality of the final product while standardis-ing the production process They enhance colour stabilitycontribute to flavour development and reduce spoilage Theones most frequently isolated from fermented sausages aresummarised in Table 2

The use of coagulase-negative staphylococci (CNS) asmeat starter cultures contributes to an adequate colourdevelopment based on their nitrate reductase activity On theother hand their catalase activity reduces oxidative damageand their metabolism contributes to flavour The flavour-generating potential of CNS is even more important whenproducing low-salt [47 48] or low-fat [49 50] sausages [51]However the full metabolic potential of CNS should befurther explored so that we may take advantage of moretechnological features of CNS [52]


Table 1 Families and genera of LAB

Family Genus Cellular morphology Sugar fermentationAerococcaceae Aerococcus Cocci-tetrads HomofermentativeCarnobacteriaceae Carnobacterium Bacilli Homofermentative

EnterococcaceaeEnterococcus Cocci Homofermentative

Tetragenococcus Cocci-tetrads HomofermentativeVagococcus Cocci Homofermentative

Lactobacillaceae Lactobacillus Bacilli Strain-dependentPediococcus Cocci-tetrads Homofermentative

LeuconostocaceaeLeuconostoc Cocci HeterofermentativeOenococcus Cocci HeterofermentativeWeissella Coccibacilli Heterofermentative

Streptococcaceae Lactococcus Cocci HomofermentativeStreptococcus Cocci Homofermentative

Table 2 Species of GCC+ isolated from fermented sausages and their role in the fermentation process

Family Genus Species Metabolic activities References

Staphylococcaceae Staphylococcus (CNS)

S xylosus(i) Nitrate reductase(ii) Proteolytic(iii) Lipolytic(iv) Catalase

[39ndash42]S carnosusS equorumS succinus

S saprophyticus

Micrococcaceae MicrococcusM luteus

M lylae

(i) Nitrate reductase(ii) Antioxidative(iii) Catalase(iv) Lipolytic(v) Proteolytic

[27 43]

KocuriaK varians

K kristinae

(i) Nitrate reductase(ii) Proteolytic(iii) Lipolytic

[42 44ndash46]

Besides contributing to flavour Staphylococcus andKocu-ria also provide nitrate-reductase and antioxidant activities[53 54]

Numerous studies addressing the use of starter cultures inmeat products have been published with both single (eitherLAB or GCC+) and mixed cultures

Several authors have reported the use of CNS starter cul-tures for the production of fermented sausages According toRavyts et al [51] the success of CNS in flavour developmentseems to be determined by acidification

Hugas and Monfort [31] highlighted the need to useselected strains of GCC+ to ensure sensory quality Besidesother authors have described the capability of S xylosus and Scarnosus strains to modulate aroma through the degradationof amino acids and free fatty acids (FFAs) [55ndash57]

Autochthonous strains of S xylosus have been recom-mended for the production of very aromatic sausages inSouthern Europe instead of the less adapted commercialstarter cultures [58]

Lusnic and colleagues have studied the effect of anadded starter culture (S xylosus and S carnosus) to a

frankfurter-type meat emulsion in degrading polychlori-nated biphenyls (PCBs) [59] Furthermore quite a few workshave been published reporting the results obtained by theutilisation of mixed starter cultures (LAB and CNS) [25 60ndash66]

Bacteriocinogenic LAB and selected strains of S xylo-sus and S carnosus are commercially available for use inimproving the safety colour and flavour of final products It isalso important to assess positive interactions such as growthand proteolytic activity among the different starter culturesstrains [67ndash70]

The effect of different starter culture combinations(Staphylococcus carnosus Pediococcus pentosaceus and Lac-tobacillus sakei) on the quality of Turkish type fermentedsausage (Sucuk) has been evaluated during ripening and itwas concluded that the use of lipolytic starter cultures (ScarnosusL sakei) would have a positive effect in acceler-ating ripening and enhancing the quality of dry-fermentedsausages [71]

Tremonte and coworkers demonstrated that S xylosusand Kocuria varians are able to stimulate the growth of L


sakei strains positively influencing the proteolytic activity ofstrains in a combined use [66]

Casquete and colleagues have emphasised the importanceof autochthonous starter cultures in improving homogeneityand safety of fermented meat products without depreciatingtheir sensory characteristics [60ndash62] Furthermore they havehighlighted the importance of choosing a starter formulationconsisting of a combination of strains that is appropriate foreach ripening procedure [60]

We may conclude that flavour and aroma of fermentedsausages result from the combined action of different bacte-ria LAB produce lactic acid and small amounts of acetic acidethanol and acetoin however the proteolytic and lipolyticactivities of both LAB and GCC+ are essential to the sensoryquality of fermented sausages

212 Fungi Yeasts and Moulds Fungi generally contributeto a characteristic flavour of some fermented meat productsYeastsmay be either inoculated in themeat batters or surface-inoculated whereas moulds are always inoculated at thesurface of sausages Surface inoculation has a further physicalprotective role

(1) Yeasts The first studies with yeasts in fermented sausageswere conducted in the first decades of the 20th century whenthe importance of the ldquofleur du saucissonrdquo was recognizedand the use of pure yeast cultures for flavouring in fermentedsausages began to be recommended Later on it was estab-lished that yeasts are part of the microbiota of fermentedsausages and their use as starter cultures was suggestedbecause the addition of selected Debaryomyces strains couldimprove the curing colour and flavour of sausages [72]

Several studies have tried to understand the role of yeastsas secondary microbiota in fermented meat products Yeaststrains belonging to the genera Debaryomyces YarrowiaPichia Rhodotorula Cryptococcus and Trichosporon havebeen isolated from meat products [73] with clear predomi-nance of the Debaryomyces genus [13]

Some yeasts have been shown to contribute to flavourand texture development throughout the curing of variousproducts [74ndash76] Moreover some studies have shown thatthe characteristic flavour of dry-cured meat products may bedeveloped through the influence of yeasts [77ndash79]

Furthermore themanufacture of dry-fermented sausageswith optimised concentrations of Debaryomyces spp in thepresence of LAB and CNS has been demonstrated to havea positive effect on the final flavour and sensory quality byinhibiting the development of rancidity and generating ethylesters that contribute to the proper sausage aroma [78]

(2) Moulds Surface moulding of fermented meat productsis considered a desirable event in most European countrieswhich include Italy Romania Bulgaria France HungarySwitzerland Southern Germany Spain Austria and Belgium[12] In fact the presence of mycelium at the surface ofsausages has several main advantages

(i) It prevents excessive drying allowing homogeneousdehydration of the product [12]

(ii) It metabolizes peroxides protecting fat from oxida-tion thus preventing rancidity [12]

(iii) It reduces O2levels on the product surface thus

avoiding oxidative processes and improving meatcolour [80]

(iv) It contributes to the flavour of the final productby breaking up fats proteins and lactic acid thusfavouring pH increase [12]

The use of moulds as a seasoning for sausage can haveboth desirable and undesirable consequences The desirableconsequences are the creation of a successful product thatappeals to consumers The undesirable consequences arehealth risks associatedwith the growth of undesirablemouldsthat produce highly toxic secondarymetabolites mycotoxinssuch as ochratoxin A (OTA) or penicillin produced byspecies of Penicillium [81]

Furthermore surface moulding of fermented meat prod-ucts was observed during storage and can be a quality prob-lem because of the undesirable effects mainly connected tothe production of off-flavours [81]

Surface mould inoculations were traditionally done withthe autochthonous mycobiota which was mainly composedof Penicillium spp Aspergillus spp or Scopulariopsis sppThe first toxicologically and technologically suitable mouldstarter culture for meat products P nalgiovense strain wasselected by Mintzlaff and Leistner in 1972 [82] Howevernowadays a wide assortment of industrialised starter culturesis commercially available as an alternative to the inoculatingmixtures composed of autochthonous strains

Some studies on the use of mould starter cultures havealready been performed [80 83] For example quality traits ofwild boarmould-ripened salamimanufactured with differentselections of meat and fat tissue and with and withoutcommercial bacterial starter cultures have been investigated[84] The use of a bacterial starter culture in the manufac-ture of mould-ripened wild boar salami resulted in signifi-cantly lower peroxide values lower TBARS concentrationsand lower amounts of biogenic amines namely histaminecadaverine and putrescine associated with better sensoryevaluation scores

Application of commercial moulds to sausage surfacesimproves primarily the safety towards regarding mycotoxinproduction Moreover the production of antibiotics namelypenicillin also needs to be controlled [82] Additionallysausage producers achieve more consistent flavour taste anddrying rate and a more uniform appearance

Table 3 shows a list of moulds found in fermented meatproducts

Among the species mentioned in Table 3 P nalgiovenseand P gladioli are currently considered safe and are commer-cially available to be used as starter cultures in meat products[12]

22 Antimicrobial Activity of Starter Cultures Bacteriocinsnatural antimicrobial peptides and the acid lactic producedfrom glucose could be used to improve the quality and safetyof meat products by avoiding the presence of pathogens suchas Listeria monocytogenes and spoilage microorganisms and


Table 3 Species of moulds usually found in dry-fermented sausages

Common species Uncommon speciesPenicillium nalgiovense P waksmanii Mucor sppP gladioli Aspergillus ochraceus Scopulariopsis sppP camemberti E herbariorum Cladosporium sppP chrysogenum E repens Eupenicillium sppP aurantiogriseum A niveus Eurotium sppP brevicompactum P citrinum Talaromyces sppP nordicum A candidus Geotrichum candidumP phoeniceum P crustosum Talaromyces wortmanniiEurotium rubrum P communeP griseofulvum A sclerotiorumP olsonii A versicolorP implicatum P aliiScopulariopsis candida P fellutanumP solitum

improving the competitiveness of their producers for survival[85]

A list of the main bacteriocins produced by LAB alongwith a list of bacteria they are effective against is summarisedin Table 4

Several L sakei and L curvatus have been reportedas bacteriocin producers and have been used as protectivecultures and their activity against L monocytogenes has beenproved in meat products [87ndash90]

Lactococcus lactis and Enterococcus spp strains isolatedfrom different food matrices have been shown to producebacteriocins [91ndash93]

Pediococcus acidilactici MCH14 pediocin-producingstrain and the pediocin PA-1 itself have been demonstratedto inhibit the growth of the foodborne pathogens L monocy-togenes and Clostridium perfringens in Spanish dry-fer-mented sausages and frankfurters [94]

Bacteriocins produced by strains of L plantarum isolatedfrom Portuguese traditional pork products have been shownto have a broad spectrum of activity [95]

LAB starter cultures have been used in the productionof Nham which is a Thai-style fermented pork sausagefor their antilisterial activity in order to reduce the severityof postacidification and increase the shelf life of Nham atambient temperature [96 97]

Additionally also S xylosus strain SX S031M12 hasbeen shown to produce a thermostable bacteriocin whichcould be used as starter culture or meat additive to preventpossible handling or meat processing contamination [98]

23 Competitiveness of Starter Cultures One of the mostimportant properties of meat starter cultures is the abilityto colonize the meat environment in competition with theautochthonous microbiota and dominating the microbialcommunity of fermented products The starter culture mustcompete with the natural microbiota of the raw materialwhich carries out the expected metabolic activities throughits growth rate and survival under the prevailing conditionsduring sausage production Low temperatures high salt

concentrations and to a lesser extent oxygen availability areamong the most important preservative conditions duringmeat fermentation [17]

The main metabolic activities and their correspondingtechnological roles for the main microbial starter groups areshown in Table 5

In general CNS are poorly competitive in the presenceof acidifying LAB strains [99] On the other hand strains ofL sakei have shown superior competitiveness which couldprobably be explained by their specialised metabolic reper-toire well adapted to the sausage environment including thearginine deiminase (ADI) pathway [100] and the utilisationof nucleosides [101]

Genus-specific and species-specific PCR and real-timeRT-PCRmethods have been used tomonitor and quantify thepopulations of the inoculated starter cultures [24] MoreoverRT-PCR-DGGE and RNA-based pyrosequencing of the 16SrRNA gene have also been used to monitor the microbiota offermented sausages [102]

24 Safety of Selected Meat Starter Cultures Meat startercultures or food cultures (FC) are safe live bacteria yeastsor moulds used in food production and they are in them-selves a characteristic food ingredient (httpwwweffcaorgcontentfood-culture) Food starter cultures (microorgan-isms) used directly in food production are regarded as foodingredients in the European Union (EU) Starters enter in acategory of food ingredients with a very long history of usein a great variety of food products If a starter is added toa food product the requirements established in the GeneralFood Law should be accomplished by the food operator Thefood cultures used as starters in the fermentation of foodsare not subject to EU premarketing regulation unless theyare regarded as being novel to the EU market and their con-sumers Many starters were selected from fermented foodsand several microorganisms are present in spontaneouslyfermented foods However regarding safety concerns anyfood cultures to be introduced in a food should be evaluatedThe approaches for assessing the safety of microorganisms


Table 4 LAB bacteriocins bacteriocin producers and susceptible pathogenic bacteria

Bacteriocin Bacteriocin producer Susceptible pathogenic bacteria

Sakacin Lactobacillus sakei

Listeria monocytogenesStaphylococcus aureusEnterococcus spp

Brochothrix thermosphactaPseudomonas sppCampylobacter sppEscherichia coliKlebsiella sppOther LAB

Plantaricin L plantarum

Listeria monocytogenesStaphylococcus aureusClostridium perfringens

Clostridium tyrobutyricumBacillus cereus

Enterococcus sppBrochothrix thermosphacta

Pseudomonas sppSalmonella sppEscherichia coliOther LAB

Curvacin L curvatus

Listeria monocytogenesStaphylococcus aureus

Brochothrix thermosphactaPseudomonas sppEscherichia coliOther LAB

Nisin Lactococcus lactis


Clostridium tyrobutyricumOther LAB

Pediocins Pediococcus sppListeria monocytogenes

Enterococcus sppOther LAB

Adapted from Fraqueza et al [86]

entering the human food chain differ considerably dependingon the applicable legislation if any

Several approaches have been delineated in order toconsider the starter cultures safe The Qualified Presumptionof Safety (QPS) list is the EFSA fast track risk assessmenttool that is used by EFSA panels when evaluating prod-ucts with microorganisms that require a premarket autho-risation (eg feed additive cultures cell factories produc-ing enzymesadditivesvitamins novel microorganisms andplant protection) This approach is restricted only to themicroorganisms related to regulated food and feed productsand is based on history of use body of knowledge andthe absence of adverse effects at the taxonomic unit level[103 104]

TheGenerally Recognized as Safe (GRAS) status is open toall types of food additives which include food cultures Thedetermination of GRAS status is made by the FDA andor

external experts and is based on the history of use body ofknowledge and the absence of adverse effects at the strainlevel

Food cultures with a long history of safe use in foodare considered as traditional food ingredients and are legallypermitted for use in foods in the EU without premarketauthorisation as described earlier As a consequence EFSApanels do not evaluate microbial strains of food culturesNevertheless the QPS list can be consulted when safetyevaluations of food culture are made

Microorganisms which are not on the QPS list are notnecessarily considered to be unsafe and their assessmentregarding antibioresistance virulence and biogenic aminecharacterization should be done

The International Dairy Federation (IDF) and the Euro-pean Food and Feed Cultures Association (EFFCA) haveproposed additional tools and methods to evaluate the safety


Table 5 Requirements for starter LAB GCC+ yeasts and moulds

Microbial group Metabolic activity Technological role

LAB

Acidification

Modulate flavour (acidtangy)Inhibit pathogensDevelop textureAccelerate drying

Proteolysis Develop flavour

Antimicrobial Inhibit pathogensExtend shelf life

Antioxidant Protect colourProbiotic Compete in the gastrointestinal tract

GCC+ Nitrate reductase Develop typical red (cured) colourDegradation of amino acids and FFAs Develop flavour

YeastsAntioxidant

Prevent rancidificationProteolyticLipolytic

Moulds Antioxidant Prevent rancidificationAdapted from [17]

of food cultures with the unique target of keeping a high levelof food safety and to protect human life and health Accordingto Laulund et al [105] whatever the strategy applied it isimperative to have an evaluation of the food culturesrsquo safetyat three levels (a) at the strain level (b) during productionand (c) in the process it is applied to and throughout the shelflife of the food

241 Assessment of Antibioresistance The One Health con-cept recognises that the health of people is connected tothe health of animals and the environment The food chainhas been recognized as one of the main routes for thetransmission of antibiotic-resistant bacteria between animaland human populations [106] Antibiotic resistant bacterialstrains may be a potential direct link between the indigenousmicrobiota of animals and the human gastrointestinal tract

Bacterial strains selected as starters with technologicalor food protective characteristics to be introduced in foodalways need to be phenotypically assessed for antibioticresistance to clinically relevant antibiotics The phenotypictesting based on determination of a minimum inhibitoryconcentration (MIC) for a selected group of antimicrobialsshould be performed The absence of phenotypic antibioticresistance is preferred but if a resistance profile is observeda proper analysis of the whole genome potentially combinedwith information that the observed resistance is not transfer-able is needed only then can the strain(s) be considered safefor use in food culture [107]

The possibility of antimicrobial resistance transfer fromviable microorganisms to other microorganisms is relatedto the genetic basis of the resistance being considered mostplausible when the resistance is mediated by addedacquiredgenes Regarding this possibility several safety assessmentshave been done by several authors on the species usuallyselected for starters such as CNS or LAB

Safety hazards associated with CNS were mostly limitedto the presence of antibiotic resistance [108] CNS strainsresistant to multiple antibiotics have been reported [109]Kastner et al [110] detected the tetracycline resistance genestetK in Staphylococcus spp starter cultures

The detection of antibiotic resistant (AR) strains amongLAB has resulted in their recognition as a reservoir of ARgenes horizontally transmissible to pathogens through thefood chain which constitutes a problem [111 112] Antibi-otic multiresistant strains of lactobacilli and other LABhave been isolated from dry-fermented meat products [113ndash120] LAB possesses a broad spectrum of natural (intrinsic)and acquired antibiotic resistance However only resistanceacquired by mutation or horizontal gene transfer poses a riskfor public health [121]

The most common resistance genes detected in LABisolated from dry-fermented sausages are the tetracyclineresistance genes tetM tetW and tetS and the genes codingfor erythromycin resistance ermB and ermC [117 120]Theseare genes linked to mobile elements and if the phenotypicexpression of antibiotic resistance is expressed their presenceis considered a hazard

242 Detection of Strains Producers of Biogenic AminesAny strains to be incorporated as starters in fermentedmeat products should be assessed for their (in)ability tomediate the production of biogenic amines Strategicallythe use of Lactobacillus spp or Pediococcus spp non-BAproducer strains could dominate and avoid the presence ofhigh contents of BA in meat products Several authors havereported the important role of starter cultures in decreasingthe content in biogenic amines [47 48 122ndash126]

243 Toxigenic Potential Among LAB enterococci play animportant role in food fermentation and may contribute to


the organoleptic uniqueness of some products but they arealso responsible for community-acquired and nosocomialinfections [118] Some of the most important virulencefactors include the production of hydrolytic enzymes namelygelatinase lipase and DNase haemolytic activity and theproduction of cytolysin the presence of adhesins and theability to form biofilms [127]

Two studies with enterococci strains isolated from severalPortuguese dry-fermented sausages revealed that althoughmeat enterococci harbour antibiotic resistance and producebiofilms a reduced number of virulence factors were detected[118 128] However a third study with Portuguese dry-fermented products from northern Portugal has detectedphenotypic and genotypic evidence of potential virulencefactors among Enterococcus spp isolates which is a reason ofconcern [129]

Some members of the CNS group primarily S epider-midis are common nosocomial pathogens and the presenceof regulatory elements involved in the control of virulence-factor synthesis has recently been identified Remarkablystrains of S xylosus were isolated from patients who had anunderlying disease while the same species has been reportedto be involved in infections of poultry [130]

Although CNS of food origin have not been found toproduce nosocomial infections some strains that produceenterotoxins have been describedVernozy-Rozand et al [131]reported enterotoxin E to be the most common enterotoxinin S equorum and S xylosus although it is reported that theoccurrence of staphylococcal enterotoxin genes in CNS fromslightly fermented sausages was rare detecting only entC inS epidermidis [132]

Absence of genes coding for staphylococcal enterotoxinsor enterotoxin-like superantigens is a requirement for strainsselected as starter cultures and the S xylosus and S carnosusstrains currently used as starter cultures or isolated fromfermented meat products generally lack toxin genes [11]

The analysis of virulence factors in strains of S epi-dermidis S simulans S xylosus S kloosii and S capraerevealed sometimes high percentage of incidence of thefollowing virulence traits production of slime120572-haemolysin120573-haemolysin DNase TNase hyaluronidase andTSST-1 andproduction of enterotoxins SEA SEB SEC and SED [133]

244 Strains with Ability of Biofilm Formation In foodindustry biofilm formation is undesirable for hygienic andsafety reasons as it can allow the attachment of food-spoilageor pathogenic microorganisms to food or food surfaces [134]Nevertheless several authors believe that colonization of foodsurfaces by starters could be desirable as it would inhibitcolonization by pathogenic or spoilage bacteria [135]

Among CNS biofilm formation has been studied in Saureus [136] S epidermidis [136] S hominis [137] S sciuri[135] and S equorum [138] S capitis S cohnii S epidermidisS lentus and S saprophyticus have all also been reported toform biofilms [139] though due to different genetic determi-nants [140] These studies concluded that in general biofilmformation is a strain-dependent characteristic Furthermorethe capacity of S xylosus to form biofilms may contribute toits survival of food processing [141] On the other hand the

inability of S carnosus to form biofilms may explain why it israrely recovered from meat processing environments [142]

LAB biofilms may be used to control the formation ofbiofilms by the foodborne pathogens Listeria monocytogenesSalmonellaTyphimurium andEscherichia coliO157H7 [143]

Genes potentially responsible for biofilm formation andcellular aggregation that may assist the organism to colonizemeat surfaces have been identified in L sakei strain 23K [144]Moreover the analysis of microenvironments through thescanning electron microscopy (SEM) evidenced the presenceof microchannels that favour microbial flow while the abilityof L sakei to form biofilm guarantees the correct colonisationof the different meat niches throughout the fermentationprocess (2017)

Biofilm formation in LAB species has been reportedto be a stress response and survival strategy in stressfulenvironments [145 146] Some reports have also describedthe genes responsible for quorum sensing adhesion andbiofilm formation [147ndash150]

Another possible biocontrol strategy to avoid the pres-ence of pathogens in meat industry could be the use ofbacteriocins and enzymes this is considered important forthe maintenance of biofilm-free systems and thus for thequality and safety of foods

25 Functional Starter Cultures Functional starter culturesare starters that have at least one functional property whichmay contribute to food safety andor offer one or moreorganoleptic technological nutritional or health advantages[151] They offer additional functionalities compared to plainstarter cultures and are a way of improving the fermentationprocess of meat products and achieving tastier safer andhealthier products

251 Bioprotective Cultures Biological preservation hasgained increasing attention as a means of naturally control-ling the shelf life and safety of foods The use of protec-tive starter cultures in the manufacture of fermented meatproducts is a well-established technology [86] Bioprotectivestarters may contribute to the safety and increase in shelf lifeof fermented meat products through the release of organicacids [152] the production of bacteriocins against importantfood pathogens mainly L monocytogenes [153] and thecontrol of biological hazards [86]

Potential protective starter cultures to use in fermentedmeat products have been identified [154] and tested [4155ndash157] The use of bioprotective starter cultures ensuressafety while increasing shelf life without compromising thenutritional value of fermented meat products or depreciatingtheir sensory quality

252 Probiotics According to the currently adopted defini-tion by the Food and Agriculture OrganizationWorld HealthOrganization (FAOWHO) [158] probiotics are defined asldquolive microorganisms which when administered in adequateamounts confer a health benefit on the hostrdquo

Probiotics are nonpathogenic health-promotingmicroor-ganisms that when ingested in defined amounts may have apositive effect on human physiology and health [29] In 1965


Lilly and Stillwell proposed probiotics to be ldquomicroorganismspromoting the growth of other microorganismsrdquo To act as safeprobiotic microorganisms strains should be of species andgenera normally present in the human gastrointestinal tract[159]

Probiotics are LAB (or bifidobacteria) mainly Gram-positive Lactobacillus species

In general health benefits of probiotic foods are based onthe presence of selected strains of LAB that having passedthrough the stomach and the small intestine survive in thelarge intestine and confer a health benefit on the host [160]

LAB with probiotic properties may have a positive influ-ence on product taste flavour and aroma as well as onfunctional and physiological properties [8]

Some LAB strains are able to produce nutraceuticalcompounds [161] Studies on Lactococcus lactis highlight thepossibility of developing LABmeat starter cultures for in situproduction of vitamins by overexpression andor disruptionof relevant metabolic genes [162ndash164]

Although dairy products are the most commonly usedfood vehicles for the delivery of probiotics several studiesdealing with the use of probiotics in fermentedmeat productsto improve their nutritional value as functional foods havebeen reported [5 154 165ndash167]

The commercial application of probiotics in meat prod-ucts is not a current procedure mostly because of technolog-ical issues Although fermented meat products are processedwithout heating probioticsmay still be inactivated due to lowpH or water activity value as well as by the presence of nativemicroorganisms or curing saltsThemost important problemis to find a compromise between technology safety qualityand health-beneficial value of food [160] For recent reviewsplease refer to Neffe-Skocinska et al [168] and Vuyst et al [8]

Some species involved in sausage fermentation such asL plantarum have been engineered to produce an excessof folate (vitamin B11) [162] This gives the possibility offortifying meat products with vitamins and other essentialcompounds thus producing healthier meat products [29]

Today the use of probiotic starters in any fermentedfood claiming health benefits should be scientifically demon-strated according to the legal requirements of EU for labelling[169 170]

3 Omics of Meat Starter Cultures

The main bacterial species used in meat fermentation areLAB and CNS Lactobacillus sakei Lactobacillus curvatusLactobacillus plantarum (mainly in Europe) and Pediococcuspentosaceus and Pediococcus acidilactici (mainly in the US)are the starters commonly used for their fermentative rolein dry-sausage production while Staphylococcus xylosus andStaphylococcus carnosus are known for their involvement inthe development and stability of colour and aroma produc-tion [171]

Using comparative genomics transcriptomics proteom-ics and metabolomics the diversity of strains naturallypresent in traditional fermented sausages is being exploredThese approaches allow rapid high-throughput screen-ing of promising wild strains with desirable functional

characteristics and a lack of negative features enablingthe development of starter cultures based on indigenoustechnological bacteria from traditional sausages which arethus better adapted to the meat matrix [22 172]

The first genome sequence of a starter to be published wasthe one of the LAB L sakei 23K [144] Despite the small sizedgenome (1883 protein-coding genes) L sakei contains sevenrRNA gene clusters [144] This redundancy may contributeto its ability to grow in complex microbial ecosystems [173]With regard to gene products the L sakei genome shares thehighest level of conservation with Lactobacillus plantarumwhich can be used as a starter in fermented meat dairy andvegetable products [144 174 175] Genome analysis revealeda specialized metabolic repertoire to adapt and grow onmeat products Important cellular functions are encodedby a redundancy of genes likely to enhance the organismrsquosrobustness and most probably help it to outgrow other com-peting bacteria As a unique ability among lactic acid bacteriaL sakei is able to use meat components such as purinenucleosides abundant in meat upon glucose depletion togrow and produce energy Genes possibly responsible forbiofilm formation and cellular aggregation which may assistin colonising meat surfaces were also identified [144]

The draft genome sequence of L sakei subsp sakei strainLS25 a commercial starter culture for fermented sausageshas been released [176] Slightly larger than the one of Lsakei 23K this genome has 1972 predicted protein-codinggenes and 7 rRNA operons [176] Compared to the L sakei23K genome [144] 1618 genes are orthologous but 250seem to be unique to LS25 including a set of genes forcarbohydrate metabolism various transporters and dehy-drogenasesoxidoreductases [176]

Complete or draft genome sequences of Pediococcuspentosaceus and Pediococcus acidilactici strains from diverseKorean fermented food products have been released butnone isolated from meat products [177ndash179]

Genomes of several strains of starter CNS have alsobeen published namely S xylosus SMQ-121 [180] S xylosusS04002 [181] and S carnosus TM300 [182]

The draft genome sequence of S xylosus SMQ-121revealed the absence of genes coding for toxins or viru-lence factors Furthermore only four antibiotic resistancegenes were found two genes encode proteins that belongto the major facilitator superfamilies involved in phenicoland fluoroquinolone resistance another gene encodes aputative aminoglycoside 31015840-phosphotransferase for resistanceto aminoglycosides and the last one encodes trimethoprimresistance Nevertheless this strain was found to be sensitiveto amikacin chloramphenicol ciprofloxacin and trimetho-prim [180]

A genome comparison of several S xylosus meat startercultures including strain S04002 with other S xylosus strainscausing cow and goat mastitis among others has shown thepresence of aroma compounds in S xylosus S04002 [181]

S carnosus TM300 genome has the highest GC contentof all sequenced staphylococcal genomes [182] It containsonly one prophage and one genomic island characterisedby a mosaic structure composed of species-specific genesAll starter cultures features such as nitratenitrite reduction


several sugar degradation pathways two catalases and nineosmoprotection systems are present It lacks most virulencefactors namely the typical S aureus toxins as well as biofilmformation genes highlighting its nonpathogenic status [182]

Following the publication of the genome sequences ofseveral strains global approaches based on transcriptomicsand proteomics have been developed in order to better under-stand the adaptation of starters to the meat environment andtheir interactions with the ecosystem and the meat substrate

Genes involved in safety and technologically relevantproperties of food associated CNS such as antibiotic resis-tance haemolysins toxins amino acid decarboxylases bind-ing proteins to extracellular matrix (ECM) lipases proteasesstress response factors and nitrate dissimilation have beendetected using DNA microarrays [183]

S xylosus C2a strain response to nitrosative [184] ornutrients and osmotic stress [185] has been investigatedthrough DNA microarrays S xylosus has been shown tocounteract nitrosative stress by developing several oxidativestress resistance mechanisms such as modulation of theexpression of genes involved in iron homeostasis detoxifyingenzymes and DNA and protein repairs [184] S xylosusadapted its metabolism to the meat nutrients and anaerobicconditions by simultaneously using glucose and lactate ascarbon sources and by using meat peptides and amino acidsS xylosus responded to the osmotic stress caused by theaddition of salt (NaCl) by overexpressing genes involvedin transport and synthesis of osmoprotectants particularlyglycine betaine and Na+ and H+ extrusion [185] To over-come the damaging effects of oxidative and nitrosative stressstaphylococci have developed protection detoxification andrepair mechanisms controlled by a network of regulators[186]

Among the overexpressed proteins in S xylosus biofilmseveral related to exopolysaccharide biosynthesis werereported [187] Furthermore with overexpression of someproteins involved in amino acids metabolism translationand secretion nitrogen metabolism appeared as quite activein sessile cells of S xylosus Additionally protein secretionsystems were also upregulated in biofilms suggesting moreactive protein trafficking in sessile S xylosus cells [187]

L sakei 23K strain global transcriptome response duringgrowth on ribose [188] andL sakeiLa22 strain transcriptomicresponse to meat protein environment [189] have beenstudied using DNA microarrays

The ribose uptake and catabolism in L sakei 23K ishighly regulated at the transcriptional level and it is closelyrelated to the catabolism of nucleosides A global regulationmechanism seems to allow fine tuning of the expression ofenzymes which control the efficient use of available carbonsources [188]

Whole-genome DNA microarrays were used to analysegene expression related to growth and survival of L sakeiLa22 when grown in a sarcoplasmic (S) or myofibrillar (M)protein-supplemented chemically defined medium (CDM)Most genes related to peptides or amino acids metabolismwere overexpressed in both mediums Still meat proteinsdo not represent a stressful environment for L sakei La22because no stress response genes were induced [189]

Next generation sequencing methods will improveknowledge related to microbiota and strain characterizationinvolved in dry-fermented meat products Future work mustbe done regarding these novel approaches and certainlynovel vision of starter behaviour on particular products willbe given

4 Conclusions

The increasing knowledge and exigence level of consumershave forced the search for high value traditional meat prod-ucts Consequently the number of production units (meattransforming) has increased sometimes in low developedregions in a bewildered way

The production of traditional meat products namelydry-fermented dry-cured sausages is still a very tradi-tional and laborious process subjected in several cases touncontrolled natural environmental conditions This poses aproblem to the producers since their meat products will notbe uniform throughout timeThus it is necessary to find solu-tions contributing to the reproducibility of products charac-teristics The use of starter cultures based on autochthonousmicrobiota selection may play here an important role Infact the use of these starters in sausages production mayimprove their sensorial characteristics and contribute to theirbiopreservation and safety extending their shelf life and toincreased meat products uniformity

Selected starter cultures provide a powerful tool fordriving the fermentation of meat products allowing desiredquality and safety targets to be reached Their use in meatfermentation results in acceleration of fermentation time animprovement of safety (by reducing undesirable microorgan-isms) and a better quality of the final product The selectionof a starter culture should be carried out in the context ofits application since functionality will depend on the type ofsausage the technology applied the ripening time and theingredients and rawmaterials used Future knowledge will begained with omics methods approach

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was funded by National Funds through FCT-Fundacao para a Ciencia e a Tecnologia under the ProjectUIDAGR001152013 M Laranjo acknowledges a postdocresearch grant from FCT (SFRHBPD1088022015)

References

[1] W P Hammes and C Hertel ldquoNew developments in meatstarter culturesrdquoMeat Science vol 49 no 1 pp S125ndashS138 1998

[2] P Kumar M K Chatli A K Verma et al ldquoQuality function-ality and shelf life of fermented meat and meat products Areviewrdquo in Critical Reviews in Food Science and Nutrition vol57 pp 2844ndash2856 2017


[3] K Arihara ldquoProbioticsrdquo in Handbook of Fermented Meat andPoultry F Toldra Ed pp 155ndash160 John Wiley amp Sons Ltd2015

[4] M I Khan M S Arshad F M Anjum A Sameen and WT Gill ldquoMeat as a functional food with special reference toprobiotic sausagesrdquo Food Research International vol 44 no 10pp 3125ndash3133 2011

[5] M Rouhi S Sohrabvandi and A M Mortazavian ldquoProbioticFermented Sausage Viability of Probiotic Microorganisms andSensory Characteristicsrdquo Critical Reviews in Food Science andNutrition vol 53 no 4 pp 331ndash348 2013

[6] A Jofre T Aymerich and M Garriga ldquoProbiotic FermentedSausages Myth or Reality Procedia Foodrdquo Procedia FoodScience vol 5 pp 133ndash136 2015

[7] Y Rivera-Espinoza and Y Gallardo-Navarro ldquoNon-dairy pro-biotic productsrdquo Food Microbiology vol 27 no 1 pp 1ndash11 2010

[8] L Vuyst G Falony and F Leroy ldquoProbiotics in fermentedsausagesrdquoMeat Science vol 80 no 1 pp 75ndash78 2008

[9] A V Wright and L Axelsson ldquoLactic Acid Bacteria An Intro-ductionrdquo in Lactic Acid BacteriaMicrobiological and FunctionalAspects S Lahtinen A C Ouwehand S Salminen and A VWright Eds pp 1ndash16 CRC Press Taylor Francis Group NewYork NY USA 2012

[10] F-K Lucke ldquoLactic acid bacteria involved in food fermenta-tions and their present and future uses in food industryrdquo in inLactic Acid Bacteria Current Advances in Metabolism Geneticsand Applications T Faruk Bozoglu and B Ray Eds pp 81ndash99Springer Berlin Heidelberg 1996

[11] P S Cocconcelli and C Fontana ldquoBacteriardquo in Handbook ofFermented Meat and Poultry F Toldra Ed pp 117ndash128 JohnWiley amp Sons Ltd 2015

[12] E Berni ldquoMoldsrdquo inHandbook of Fermented Meat and PoultryF Toldra Ed UK John Wiley Sons Ltd 2015

[13] R C S Mendonca D M Gouvea H M Hungaro A D FSodre and A Querol-Simon ldquoDynamics of the yeast flora inartisanal country style and industrial dry cured sausage (yeastin fermented sausage)rdquo Food Control vol 29 no 1 pp 143ndash1482013

[14] L B Jensen and L S Paddock ldquoSausage treatment Patent US2225783 Ardquo 1940

[15] C W Everson W E Danner and P A Hammes ldquoBacterialstarter cultures in sausage productsrdquo Journal of Agricultural andFood Chemistry vol 18 no 4 pp 570-571 1970

[16] F P Niinivaara M S Pohja and Se Komulain ldquoSome aspectsabout using bacterial pure cultures inmanufacture of fermentedsausagesrdquo Food Technology vol 18 p 147 1964

[17] G Vignolo P Castellano and S Fadda ldquoBioprotective Cul-turesrdquo in Handbook of Fermented Meat and Poultry F ToldraEd pp 129ndash138 John Wiley amp Sons Ltd 2015

[18] A Galvez R Lucas Lopez H Abriouel E Valdivia andN B Omar ldquoApplication of bacteriocins in the control offoodborne pathogenic and spoilage bacteriardquo Critical Reviewsin Biotechnology vol 28 no 2 pp 125ndash152 2008

[19] P S Cocconcelli and C Fontana ldquoCharacteristics and Appli-cations of Microbial Starters in Meat Fermentationsrdquo in MeatBiotechnology F Toldra Ed pp 129ndash148 2008

[20] T Semedo-Lemsaddek L Carvalho C Tempera et al ldquoChar-acterization and Technological Features of AutochthonousCoagulase-Negative Staphylococci as Potential Starters for Por-tuguese Dry Fermented Sausagesrdquo Journal of Food Science vol81 no 5 pp M1197ndashM1202 2016

[21] R Talon S Leroy and I Lebert ldquoMicrobial ecosystems of tradi-tional fermented meat products The importance of indigenousstartersrdquoMeat Science vol 77 no 1 pp 55ndash62 2007

[22] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[23] M Elias M E Potes L C Roseiro C Santos A Gomes andA C Agulheiro-Santos ldquoThe Effect of Starter Cultures on thePortuguese Traditional SausagePaio doAlentejo in Terms of ItsSensory and Textural Characteristics and Polycyclic AromaticHydrocarbons Profilerdquo Journal of Food Research vol 3 pp 45ndash56 2014

[24] S Fonseca L I Ivette Ouoba I Franco and J Carballo ldquoUseof molecular methods to characterize the bacterial communityand to monitor different native starter cultures throughout theripening of Galician chorizordquo Food Microbiology vol 34 no 1pp 215ndash226 2013

[25] S Fonseca A CachaldoraMGomez I Franco and J CarballoldquoEffect of different autochthonous starter cultures on the volatilecompounds profile and sensory properties of Galician chorizoa traditional Spanish dry fermented sausagerdquo Food Control vol33 no 1 pp 6ndash14 2013

[26] A GM Scannell PM Kenneally and E K Arendt ldquoContribu-tion of starter cultures to the proteolytic process of a fermentednon-dried whole muscle ham productrdquo International Journal ofFood Microbiology vol 93 pp 219ndash230 2004

[27] F Bourdichon S Casaregola C Farrokh et al ldquoFood fer-mentations Microorganisms with technological beneficial userdquoInternational Journal of Food Microbiology vol 154 no 3 pp87ndash97 2012

[28] L Morelli M L Calleagri F K Vogensen and A v WrightldquoGenetics of Lactic Acid Bacteria In Lactic Acid Bacte-riaMicrobiological and Functional Aspectsrdquo in Lactic AcidBacteriaMicrobiological and Functional Aspects S Lahtinen AC Ouwehand S Salminen and A V Wright Eds pp 17ndash37CRC Press Taylor Francis Group New York US 2012

[29] M S Ammor and B Mayo ldquoSelection criteria for lactic acidbacteria to be used as functional starter cultures in dry sausageproduction an updaterdquoMeat Science vol 76 no 1 pp 138ndash1462007

[30] S C Ricke I Z Diaz and J T Keeton FermentedMeat Poultryand Fish Products in Food Microbiology Fundamentals andFrontiers ASM Press Washington wash USA 2007

[31] M Hugas and JMMonfort ldquoBacterial starter cultures formeatfermentationrdquo Food Chemistry vol 59 no 4 pp 547ndash554 1997

[32] J A Ordonez E M Hierro J M Bruna and L de La HozldquoChanges in the components of dry-fermented sausages duringripeningrdquoCritical Reviews in Food Science andNutrition vol 39no 4 pp 329ndash367 1999

[33] K Molly D Demeyer G Johansson M Raemaekers MGhistelinck and I Geenen ldquoThe importance of meat enzymesin ripening and flavour generation in dry fermented sausagesFirst results of a European projectrdquo Food Chemistry vol 59 no4 pp 539ndash545 1997

[34] X H Wang H Y Ren D Y Liu W Y Zhu and W WangldquoEffects of inoculating Lactobacillus sakei starter cultures onthe microbiological quality and nitrite depletion of Chinesefermented sausagesrdquo Food Control vol 32 no 2 pp 591ndash5962013


[35] B T Cenci-Goga P V Rossitto P Sechi S Parmegiani V Cam-biotti and J S Cullor ldquoEffect of selected dairy starter cultures onmicrobiological chemical and sensory characteristics of swineand venison (Dama dama) nitrite-free dry-cured sausagesrdquoMeat Science vol 90 no 3 pp 599ndash606 2012

[36] S Ahmad ldquoSensory Quality of Fermented Sausages as Influ-enced by Different Combined Cultures of Lactic Acid BacteriaFermentation during Refrigerated Storagerdquo Journal of FoodProcessing Technology vol 4 2012

[37] F Ravyts and L De Vuyst ldquoPrevalence and impact of single-strain starter cultures of lactic acid bacteria on metaboliteformation in sourdoughrdquo Food Microbiology vol 28 no 6 pp1129ndash1139 2011

[38] J Garcia-Diez and L Patarata ldquoInfluence of salt level starterculture fermentable carbohydrates and temperature on thebehaviour of L monocytogenes in sliced chourico during stor-agerdquo Acta Alimentaria vol 46 pp 206ndash213 2017

[39] E H Drosinos S Paramithiotis G Kolovos I Tsikouras andI Metaxopoulos ldquoPhenotypic and technological diversity oflactic acid bacteria and staphylococci isolated from traditionallyfermented sausages in Southern Greecerdquo Food Microbiologyvol 24 no 3 pp 260ndash270 2007

[40] S C Morot-Bizot S Leroy and R Talon ldquoMonitoring ofstaphylococcal starters in two French processing plants man-ufacturing dry fermented sausagesrdquo Journal of Applied Microbi-ology vol 102 no 1 pp 238ndash244 2007

[41] C Fontana P S Cocconcelli and G Vignolo ldquoMonitoringthe bacterial population dynamics during fermentation ofartisanal Argentinean sausagesrdquo International Journal of FoodMicrobiology vol 103 no 2 pp 131ndash142 2005

[42] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[43] M C Garcia Fontan J M Lorenzo A Parada I Francoand J Carballo ldquoMicrobiological characteristics of ldquoandrollardquoa Spanish traditional pork sausagerdquo Food Microbiology vol 24no 1 pp 52ndash58 2007

[44] AMartın B Colın E ArandaM J Benito andMG CordobaldquoCharacterization ofMicrococcaceae isolated from Iberian dry-cured sausagesrdquoMeat Science vol 75 no 4 pp 696ndash708 2007

[45] C Lopez L M Medina R Priego and R Jordano ldquoBehaviourof the constitutive biota of two types of Spanish dry-sausagesripened in a pilot-scale chamberrdquo Meat Science vol 73 no 1pp 178ndash180 2006

[46] I Lebert S Leroy PGiammarinaro et al ldquoDiversity ofmicroor-ganisms in the environment and dry fermented sausages ofsmall traditional French processing unitsrdquoMeat Science vol 76no 1 pp 112ndash122 2007

[47] M Laranjo A Gomes A C Agulheiro-Santos et al ldquoImpactof salt reduction on biogenic amines fatty acids microbiotatexture and sensory profile in traditional blood dry-curedsausagesrdquo Food Chemistry vol 218 pp 129ndash136 2017

[48] M Laranjo A Gomes A C Agulheiro-Santos et al ldquoCharac-terisation of ldquoCatalaordquo and ldquoSalsichaordquo Portuguese traditionalsausages with salt reductionrdquo Meat Science vol 116 pp 34ndash422016

[49] J M Lorenzo R Montes L Purrinos and D Franco ldquoEffect ofpork fat addition on the volatile compounds of foal dry-curedsausagerdquoMeat Science vol 91 no 4 pp 506ndash512 2012

[50] A C Venturini A D Cavenaghi C J C Castillo and E MQuinones ldquoSensory and microbiological evaluation of uncured

fresh chicken sausage with reduced fat contentrdquo Ciencia eTecnologia de Alimentos vol 31 no 3 pp 629ndash634 2011

[51] F Ravyts L Steen O Goemaere H Paelinck L De Vuystand F Leroy ldquoThe application of staphylococci with flavour-generating potential is affected by acidification in fermented drysausagesrdquo Food Microbiology vol 27 no 7 pp 945ndash954 2010

[52] M S Mainar D A Stavropoulou and F Leroy ldquoExploring themetabolic heterogeneity of coagulase-negative staphylococci toimprove the quality and safety of fermented meats A reviewrdquoInternational Journal of Food Microbiology vol 247 2017

[53] R Talon D Walter S Chartier C Barriere and M C MontelldquoEffect of nitrate and incubation conditions on the productionof catalase and nitrate reductase by staphylococcirdquo InternationalJournal of Food Microbiology vol 52 no 1-2 pp 47ndash56 1999

[54] R Talon D Walter and M C Montel ldquoGrowth and effect ofstaphylococci and lactic acid bacteria on unsaturated free fattyacidsrdquoMeat Science vol 54 no 1 pp 41ndash47 2000

[55] L H Stahnke A Holck A Jensen A Nilsen and E ZanardildquoMaturity acceleration of italian dried sausage by Staphy-lococcus carnosus-Relationship between maturity and flavorcompoundsrdquo Journal of Food Science vol 67 no 5 pp 1914ndash1921 2002

[56] H C Beck A M Hansen and F R Lauritsen ldquoCatabolism ofleucine to branched-chain fatty acids in Staphylococcus xylosusrdquoJournal of Applied Microbiology vol 96 no 5 pp 1185ndash11932004

[57] P T Olesen A S Meyer and L H Stahnke ldquoGeneration offlavour compounds in fermented sausages - The influence ofcuring ingredients Staphylococcus starter culture and ripeningtimerdquoMeat Science vol 66 no 3 pp 675ndash687 2004

[58] J Samelis J Metaxopoulos M Vlassi and A Pappa ldquoStabilityand safety of traditional Greek salamimdasha microbiological ecol-ogy studyrdquo International Journal of Food Microbiology vol 44no 1-2 pp 69ndash82 1998

[59] M Lusnic T Polak L Gasperlin et al ldquoDegradation of PCBsin a frankfurter-type meat emulsion Effects of a meat starterits proteins extract and thermal treatmentsrdquo Food and ChemicalToxicology vol 50 no 8 pp 2643ndash2647 2012

[60] R Casquete M J Benito A Martın S Ruiz-Moyano AHernandez and M G Cordoba ldquoEffect of autochthonousstarter cultures in the production of ldquo salchichonrdquo a tradi-tional Iberian dry-fermented sausage with different ripeningprocessesrdquo LWT- Food Science and Technology vol 44 no 7 pp1562ndash1571 2011

[61] R Casquete M J Benito A Martin S Ruiz-Moyano J JCordoba and M G Cordoba ldquoRole of an autochthonousstarter culture and the protease EPg222 on the sensory andsafety properties of a traditional Iberian dry-fermented sausagesalchichonrdquo Food Microbiol vol 28 pp 1432-40 2011

[62] R Casquete M J Benito A Martın S Ruiz-Moyano EAranda and M G Cordoba ldquoMicrobiological quality ofsalchichon and chorizo traditional Iberian dry-fermentedsausages from two different industries inoculated withautochthonous starter culturesrdquo Food Control vol 24 no 1-2pp 191ndash198 2012

[63] I Essid and M Hassouna ldquoEffect of inoculation of selectedStaphylococcus xylosus and Lactobacillus plantarum strains onbiochemical microbiological and textural characteristics of aTunisian dry fermented sausagerdquo Food Control vol 32 no 2pp 707ndash714 2013

[64] M Bedia L Mendez and S Banon ldquoEvaluation of differentstarter cultures (Staphylococci plus Lactic Acid Bacteria) in


semi-ripened Salami stuffed in swine gutrdquoMeat Science vol 87no 4 pp 381ndash386 2011

[65] J M Aro Aro P Nyam-Osor K Tsuji K-I Shimada MFukushima and M Sekikawa ldquoThe effect of starter cultureson proteolytic changes and amino acid content in fermentedsausagesrdquo Food Chemistry vol 119 no 1 pp 279ndash285 2010

[66] P Tremonte A Reale T Di Renzo et al ldquoInteractions betweenLactobacillus sakei and CNC (Staphylococcus xylosus and Kocu-ria varians) and their influence on proteolytic activityrdquo Lettersin Applied Microbiology vol 51 no 5 pp 586ndash594 2010

[67] A Casaburi V Di Martino P Ferranti L Picariello and FVillani ldquoTechnological properties and bacteriocins productionby Lactobacillus curvatus 54M16 and its use as starter culturefor fermented sausage manufacturerdquo Food Control vol 59 pp31ndash45 2016

[68] M Z Barbosa S D Todorov I Ivanova J-M Chobert THaertle and B D G de Melo Franco ldquoImproving safety ofsalami by application of bacteriocins produced by an autochtho-nous Lactobacillus curvatus isolaterdquo Food Microbiology vol 46pp 254ndash262 2015

[69] M Simonova V Strompfova M Marcinakova et al ldquoCharac-terization of Staphylococcus xylosus and Staphylococcus carnosusisolated from Slovak meat productsrdquo Meat Science vol 73 no4 pp 559ndash564 2006

[70] A M Fiorentini M C Sawitzki T M Bertol and E SSantrsquoAnna ldquoViability of Staphylococcus xylosus isolated fromartisanal sausages for application as starter cultures in meatproductsrdquo Brazilian Journal of Microbiology vol 40 no 1 pp129ndash133 2009

[71] E B Bingol F YilmazH Yardibi et al ldquoEffect of lipolytic startercultures on ripening and quality of Turkish type fermentedsausages (sucuk)rdquo Current Opinion in Biotechnology vol 22 pS97 2011

[72] M D Selgas and M L Garcıa ldquoYeastsrdquo in Handbook ofFermented Meat and Poultry F Toldra Ed pp 139ndash146 USAWiley Blackwell 2015

[73] M Flores S Corral L Cano-Garcıa A Salvador and CBelloch ldquoYeast strains as potential aroma enhancers in dryfermented sausagesrdquo International Journal of FoodMicrobiologyvol 212 pp 16ndash24 2015

[74] B C Viljoen G A Dykes M Callis and A von Holy ldquoYeastsassociated with Vienna sausage packagingrdquo International Jour-nal of Food Microbiology vol 18 no 1 pp 53ndash62 1993

[75] B C Viljoen and T Greyling ldquoYeasts associated with CheddarandGoudamakingrdquo International Journal of FoodMicrobiologyvol 28 no 1 pp 79ndash88 1995

[76] E Miteva E Kirova D Gadjeva and M Radeva ldquoSensoryaroma and taste profiles of raw-dried sausages manufacturedwith a lipolytically active yeast culturerdquo Nahrung-Food vol 30pp 829ndash832 1986

[77] M A Dura M Flores and F Toldra ldquoEffect of Debaryomycesspp on the proteolysis of dry-fermented sausagesrdquo Meat Sci-ence vol 68 pp 319ndash328 2004

[78] M Flores M-A Dura A Marco and F Toldra ldquoEffect ofDebaryomyces spp on aroma formation and sensory quality ofdry-fermented sausagesrdquo Meat Science vol 68 no 3 pp 439ndash446 2004

[79] A Martin J J Cordoba E Aranda M G Cordoba and M AAsensio ldquoContribution of a selected fungal population to thevolatile compounds on dry-cured hamrdquo International Journalof Food Microbiology vol 110 pp 8ndash18 2006

[80] J M Bruna E M Hierro L De La Hoz D S Mottram MFernandez and J AOrdonez ldquoChanges in selected biochemicaland sensory parameters as affected by the superficial inocu-lation of Penicillium camemberti on dry fermented sausagesrdquoInternational Journal of Food Microbiology vol 85 no 1-2 pp111ndash125 2003

[81] M Papagianni I Ambrosiadis andG Filiousis ldquoMould growthon traditional greek sausages and penicillin production byPenicillium isolatesrdquo Meat Science vol 76 no 4 pp 653ndash6572007

[82] L O Sunesen and L H Stahnke ldquoMould starter cultures for drysausagesmdashselection application and effectsrdquoMeat Science vol65 no 3 pp 935ndash948 2003

[83] V Ludemann M Greco M P Rodrıguez J C Basılico and AG Pardo ldquoConidial production by Penicillium nalgiovense foruse as starter cultures in dry fermented sausages by solid statefermentationrdquo LWT- Food Science and Technology vol 43 no2 pp 315ndash318 2010

[84] P Paulsen S Vali and F Bauer ldquoQuality traits of wild boarmould-ripened salami manufactured with different selectionsof meat and fat tissue and with and without bacterial starterculturesrdquoMeat Science vol 89 no 4 pp 486ndash490 2011

[85] I F Nes and J R Tagg ldquoNovel lantibiotics and their pre-peptidesrdquo Antonie van Leeuwenhoek-Journal of Microbiologyvol 69 no 2 pp 89ndash97 1996

[86] M J Fraqueza L Patarata and A Laukova ldquoProtective StarterCultures and Bacteriocins in Fermented Meatsrdquo in FermentedMeat Products Health Aspects N Zdolec Ed pp 228ndash269CRC Press New York 2016

[87] M Hugas M Garriga M T Aymerich and J M MonfortldquoInhibition of Listeria in dry fermented sausages by the bac-teriocinogenic Lactobacillus sake CTC494rdquo Journal of AppliedBacteriology vol 79 no 3 pp 322ndash330 1995

[88] P M Foegeding A B Thomas D H Pilkington and T RKlaenhammer ldquoEnhanced control of Listeria monocytogenesby in situ-produced pediocin during dry fermented sausageproductionrdquo Applied and Environmental Microbiology vol 58no 3 pp 884ndash890 1992

[89] J C Nieto-Lozano J I Reguera-Useros M D C Pelaez-Martınez and A H De La Torre ldquoEffect of a bacteriocin pro-duced by Pediococcus acidilactici against Listeria monocytogenesandClostridiumperfringens on Spanish rawmeatrdquoMeat Sciencevol 72 no 1 pp 57ndash61 2006

[90] T Azuma D K Bagenda T Yamamoto Y Kawai and KYamazaki ldquoInhibition of Listeria monocytogenes by freeze-dried piscicocin CS526 fermentate in foodrdquo Letters in AppliedMicrobiology vol 44 no 2 pp 138ndash144 2007

[91] A Alegria S Delgado C Roces B Lopez and BMayo ldquoBacte-riocins produced bywild Lactococcus lactis strains isolated fromtraditional starter-free cheesesmade of rawmilkrdquo InternationalJournal of Food Microbiology vol 143 no 1-2 pp 61ndash66 2010

[92] C Henning D Gautam and PMuriana ldquoIdentification ofMul-tiple Bacteriocins in Enterococcus spp Using an Enterococcus-Specific Bacteriocin PCR Arrayrdquo Microorganisms vol 3 pp 1ndash16 2015

[93] I F Nes D B Diep and H Holo ldquoBacteriocin diversity inStreptococcus andEnterococcusrdquo Journal of Bacteriology vol 189no 4 pp 1189ndash1198 2007

[94] J C Nieto-Lozano J I Reguera-Useros M D C Pelaez-Martınez G Sacristan-Perez-Minayo A J Gutierrez-Fer-nandez and A H D la Torre ldquoThe effect of the pediocin PA-1


produced by Pediococcus acidilactici against Listeria monocyto-genes and Clostridium perfringens in Spanish dry-fermentedsausages and frankfurtersrdquo Food Control vol 21 no 5 pp 679ndash685 2010

[95] S D Todorov P Ho M Vaz-Velho and L M T DicksldquoCharacterization of bacteriocins produced by two strains ofLactobacillus plantarum isolated from Beloura and Chouricotraditional pork products from PortugalrdquoMeat Science vol 84no 3 pp 334ndash343 2010

[96] Y Kingcha A Tosukhowong T Zendo et al ldquoAnti-listeriaactivity of Pediococcus pentosaceus BCC 3772 and application asstarter culture for Nham a traditional fermented pork sausagerdquoFood Control vol 25 no 1 pp 190ndash196 2012

[97] P Jaichumjai R Valyasevi A Assavanig and P Kurdi ldquoIsola-tion and characterization of acid-sensitive Lactobacillus plan-tarumwith application as starter culture for Nham productionrdquoFood Microbiology vol 27 no 6 pp 741ndash748 2010

[98] A Laukova M Simonova and V Strompfova ldquoStaphylococcusxylosus S031M12 bacteriocin-producing meat starter cultureor additiverdquo Food Control vol 21 no 7 pp 970ndash973 2010

[99] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[100] T Rimaux G Vrancken V Pothakos D Maes L De Vuystand F Leroy ldquoThe kinetics of the arginine deiminase pathwayin the meat starter culture Lactobacillus sakei CTC 494 are pH-dependentrdquo FoodMicrobiology vol 28 no 3 pp 597ndash604 2011

[101] T Rimaux G Vrancken B Vuylsteke L De Vuyst and F LeroyldquoThe pentose moiety of adenosine and inosine is an importantenergy source for the fermented-meat starter cultureLactobacil-lus sakei CTC 494rdquo Applied and Environmental Microbiologyvol 77 no 18 pp 6539ndash6550 2011

[102] A Greppi I Ferrocino A La Storia K Rantsiou D Ercoliniand L Cocolin ldquoMonitoring of the microbiota of fermentedsausages by culture independent rRNA-based approachesrdquoInternational Journal of Food Microbiology vol 212 pp 67ndash752015

[103] S Barlow A Chesson J Collins et al ldquoOpinion of the ScientificCommittee on a request from EFSA related to a genericapproach to the safety assessment by EFSA of microorganismsused in foodfeed and the production of foodfeed additivesrdquoEFSA Journal vol 3 pp 1ndash12 2005

[104] A Ricci A Allende D Bolton et al ldquoScientific Opinion onthe update of the list of QPS-recommended biological agentsintentionally added to food or feed as notified to EFSArdquo EFSAJournal vol 15 p 4664 2017

[105] S Laulund A Wind P Derkx and V Zuliani ldquoRegulatory andsafety requirements for food culturesrdquo Microorganisms vol 5p 28 2017

[106] W Witte ldquoSelective pressure by antibiotic use in livestockrdquoInternational Journal of Antimicrobial Agents vol 16 no 1 ppS19ndashS24 2000

[107] G Rychen G Aquilina G Azimonti et al ldquoGuidance on thecharacterisation of microorganisms used as feed additives oras production organisms EFSA Journalrdquo Endorsed for publicconsultation on 18 2017

[108] S Even S Leroy C Charlier et al ldquoLow occurrence of safetyhazards in coagulase negative staphylococci isolated from fer-mented foodstuffsrdquo International Journal of Food Microbiologyvol 139 no 1-2 pp 87ndash95 2010

[109] G Landeta J A Curiel A V Carrascosa R Munoz and Bde las Rivas ldquoCharacterization of coagulase-negative staphy-lococci isolated from Spanish dry cured meat productsrdquo MeatScience vol 93 no 3 pp 387ndash396 2013

[110] S Kastner V PerretenH Bleuler GHugenschmidt C Lacroixand L Meile ldquoAntibiotic susceptibility patterns and resistancegenes of starter cultures and probiotic bacteria used in foodrdquoSystematic and Applied Microbiology vol 29 no 2 pp 145ndash1552006

[111] B M Marshall D J Ochieng and S B Levy ldquoCommensalsunderappreciated reservoir of antibiotic resistancerdquo Microbevol 4 no 5 pp 231ndash238 2009

[112] C Devirgiliis S Barile and G Perozzi ldquoAntibiotic resistancedeterminants in the interplay between food and gut micro-biotardquo Genes amp Nutrition vol 6 no 3 pp 275ndash284 2011

[113] T Aymerich B Martın M Garriga M C Vidal-Carou SBover-Cid and M Hugas ldquoSafety properties and molecularstrain typing of lactic acid bacteria from slightly fermentedsausagesrdquo Journal of AppliedMicrobiology vol 100 no 1 pp 40ndash49 2006

[114] S Federici F Ciarrocchi R Campana E Ciandrini G Blasiand W Baffone ldquoIdentification and functional traits of lacticacid bacteria isolated from Ciauscolo salami produced inCentral ItalyrdquoMeat Science vol 98 no 4 pp 575ndash584 2014

[115] R Comunian E Daga I Dupre et al ldquoSusceptibility totetracycline and erythromycin of Lactobacillus paracasei strainsisolated from traditional Italian fermented foodsrdquo InternationalJournal of Food Microbiology vol 138 no 1-2 pp 151ndash156 2010

[116] G Landeta J A Curiel A V Carrascosa R Munoz and BDe las Rivas ldquoTechnological and safety properties of lacticacid bacteria isolated from Spanish dry-cured sausagesrdquo MeatScience vol 95 no 2 pp 272ndash280 2013

[117] D Zonenschain A Rebecchi and L Morelli ldquoErythromycin-and tetracycline-resistant lactobacilli in Italian fermented drysausagesrdquo Journal of Applied Microbiology vol 107 no 5 pp1559ndash1568 2009

[118] T Ribeiro M Oliveira M J Fraqueza et al ldquoAntibioticresistance and virulence factors among Enterococci isolatedfrom chourico a traditional Portuguese dry fermented sausagerdquoJournal of Food Protection vol 74 no 3 pp 465ndash469 2011

[119] D Gevers GHuys F DevlieghereMUyttendaele J Debevereand J Swings ldquoIsolation and identification of tetracycline resis-tant lactic acid bacteria from pre-packed sliced meat productsrdquoSystematic and AppliedMicrobiology vol 23 no 2 pp 279ndash2842000

[120] D Gevers G Huys and J Swings ldquoIn vitro conjugal transferof tetracycline resistance from Lactobacillus isolates to othergram-positive bacteriardquo FEMS Microbiology Letters vol 225no 1 pp 125ndash130 2003

[121] M J Fraqueza ldquoAntibiotic resistance of lactic acid bacteriaisolated from dry-fermented sausagesrdquo International Journal ofFood Microbiology vol 212 pp 76ndash88 2015

[122] S Lu H Ji Q Wang et al ldquoThe effects of starter culturesand plant extracts on the biogenic amine accumulation intraditional Chinese smoked horsemeat sausagesrdquo Food Controlvol 50 pp 869ndash875 2015

[123] T Komprda D Smela P Pechova L Kalhotka J Stencl andB Klejdus ldquoEffect of starter culture spice mix and storage timeand temperature on biogenic amine content of dry fermentedsausagesrdquoMeat Science vol 67 no 4 pp 607ndash616 2004


[124] M L Latorre-Moratalla S Bover-Cid M T Veciana-Noguesand M C Vidal-Carou ldquoControl of biogenic amines in fer-mented sausages role of starter culturesrdquo Frontiers in Microbi-ology vol 3 article 169 2012

[125] M L Latorre-Moratalla S Bover-Cid R Talon et al ldquoDistri-bution of aminogenic activity among potential autochthonousstarter cultures for dry fermented sausagesrdquo Journal of FoodProtection vol 73 no 3 pp 524ndash528 2010

[126] M L Latorre-Moratalla S Bover-Cid R Talon et al ldquoStrategiesto reduce biogenic amine accumulation in traditional sausagemanufacturingrdquo LWT- Food Science and Technology vol 43 no1 pp 20ndash25 2010

[127] T SemedoM Almeida SantosM F Silva Lopes J J FigueiredoMarques M T Barreto Crespo and R Tenreiro ldquoVirulencefactors in food clinical and reference enterococci A commontrait in the genusrdquo Systematic and AppliedMicrobiology vol 26no 1 pp 13ndash22 2003

[128] S C Santos M J Fraqueza M Elias A Salvador Barreto andT Semedo-Lemsaddek ldquoTraditional dry smoked fermentedmeat sausages Characterization of autochthonous enterococcirdquoLWT- Food Science and Technology vol 79 pp 410ndash415 2017

[129] J Barbosa P AGibbs andP Teixeira ldquoVirulence factors amongenterococci isolated from traditional fermented meat productsproduced in the North of Portugalrdquo Food Control vol 21 no 5pp 651ndash656 2010

[130] F M Aarestrup Y Agersoslash P Ahrens J C Oslash Joslashrgensen MMadsen and L B Jensen ldquoAntimicrobial susceptibility andpresence of resistance genes in staphylococci from poultryrdquoVeterinary Microbiology vol 74 no 4 pp 353ndash364 2000

[131] C Vernozy-Rozand C Mazuy G Prevost et al ldquoEnterotoxinproduction by coagulase-negative staphylococci isolated fromgoatsrsquo milk and cheeserdquo International Journal of Food Microbi-ology vol 30 no 3 pp 271ndash280 1996

[132] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[133] P T Fowoyo and S T Ogunbanwo ldquoVirulence and toxigenicityof coagulase-negative staphylococci in Nigerian traditionalfermented foodsrdquoCanadian Journal of Microbiology vol 62 no7 pp 572ndash578 2016

[134] C G Kumar and S K Anand ldquoSignificance of microbial bio-films in food industry a reviewrdquo International Journal of FoodMicrobiology vol 42 no 1-2 pp 9ndash27 1998

[135] V Leriche and B Carpentier ldquoLimitation of adhesion andgrowth of Listeria monocytogenes on stainless steel surfaces byStaphylococcus sciuri biofilmsrdquo Journal of Applied Microbiologyvol 88 no 4 pp 594ndash605 2000

[136] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009

[137] P Kotilainen ldquoAssociation of coagulase-negative staphylococcalslime production and adherence with the development andoutcome of adult septicemiasrdquo Journal of Clinical Microbiologyvol 28 no 12 pp 2779ndash2785 1990

[138] S Leroy I Lebert J-P Chacornac P Chavant T Bernardi andR Talon ldquoGenetic diversity and biofilm formation of Staphy-lococcus equorum isolated from naturally fermented sausagesand their manufacturing environmentrdquo International Journal ofFood Microbiology vol 134 no 1-2 pp 46ndash51 2009

[139] A Fagerlund S Langsrud E Heir M I Mikkelsen and TMoslashretroslash ldquoBiofilm matrix composition affects the susceptibilityof food associated staphylococci to cleaning and disinfectionagentsrdquo Frontiers in Microbiology vol 7 article no 856 2016

[140] T Moslashretroslash L Hermansen A L Holck M S Sidhu K Rudiand S Langsrud ldquoBiofilm formation and the presence of theintercellular adhesion locus ica among staphylococi from foodand food processing environmentsrdquoApplied and EnvironmentalMicrobiology vol 69 no 9 pp 5648ndash5655 2003

[141] S Planchon B Gaillard-Martinie E Dordet-Frisoni et alldquoFormation of biofilm by Staphylococcus xylosusrdquo InternationalJournal of Food Microbiology vol 109 no 1-2 pp 88ndash96 2006

[142] S Planchon B Gaillard-Martinie S Leroy M N Bellon-Fontaine S Fadda andR Talon ldquoSurface properties and behav-iour on abiotic surfaces of Staphylococcus carnosus a geneticallyhomogeneous speciesrdquo FoodMicrobiology vol 24 no 1 pp 44ndash51 2007

[143] N C Gomez J M P Ramiro B X V Quecan and B DG de Melo Franco ldquoUse of potential probiotic lactic acidbacteria (LAB) biofilms for the control of Listeria monocyto-genes Salmonella Typhimurium and Escherichia coli O157 H7biofilms formationrdquo Frontiers in Microbiology vol 7 article no863 2016

[144] S Chaillou M-C Champomier-Verges M Cornet et alldquoThe complete genome sequence of the meat-borne lactic acidbacterium Lactobacillus sakei 23Krdquo Nature Biotechnology vol23 no 12 pp 1527ndash1533 2005

[145] P D Cotter and C Hill ldquoSurviving the acid test responses ofgram-positive bacteria to low pHrdquoMicrobiology and MolecularBiology Reviews vol 67 no 3 pp 429ndash453 2003

[146] M Van de Guchte P Serror C Chervaux T Smokvina SD Ehrlich and E Maguin ldquoStress responses in lactic acidbacteriardquo Antonie van Leeuwenhoek-Journal of Microbiologyvol 82 no 1-4 pp 187ndash216 2002

[147] T Fujii C Ingham J Nakayama et al ldquoTwo homologous agr-like quorum-sensing systems cooperatively control adherencecell morphology and cell viability properties in LactobacillusplantarumWCFS1rdquo Journal of Bacteriology vol 190 no 23 pp7655ndash7665 2008

[148] S Lebeer S C J De Keersmaecker T L A Verhoeven A AFadda KMarchal and J Vanderleyden ldquoFunctional analysis ofluxS in the probiotic strain Lactobacillus rhamnosusGG revealsa central metabolic role important for growth and biofilmformationrdquo Journal of Bacteriology vol 189 no 3 pp 860ndash8712007

[149] M H J Sturme J Nakayama D Molenaar et al ldquoAn agr-liketwo-component regulatory system in Lactobacillus plantarum isinvolved in production of a novel cyclic peptide and regulationof adherencerdquo Journal of Bacteriology vol 187 no 15 pp 5224ndash5235 2005

[150] G W Tannock J B Luchansky L Miller et al ldquoMolecularCharacterization of a Plasmid-Borne (pGT633) ErythromycinResistance Determinant (ermGT) from Lactobacillus reuteri100-63rdquo Plasmid vol 31 no 1 pp 60ndash71 1994

[151] F Leroy and L de Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004

[152] R Talon I Lebert S Leroy et al ldquoMicrobial ecosystem of tradi-tional dry fermented sausages in Mediterranean countries andSlovakiardquo Mediterranean Ecosystems Dynamics Managementand Conservation pp 115ndash127 2012


[153] M P Zacharof and R W Lovitt ldquoBacteriocins Produced byLactic Acid Bacteria A Review Articlerdquo in Proceedings of the3rd International Conference on Biotechnology and Food Science(edited by DAN pp 50ndash56 2012

[154] T D Klingberg L Axelsson K Naterstad D Elsser and B BBudde ldquoIdentification of potential probiotic starter cultures forScandinavian-type fermented sausagesrdquo International Journal ofFood Microbiology vol 105 no 3 pp 419ndash431 2005

[155] M Trząskowska D Kołozyn-Krajewska K M Wojciak andZ J Dolatowski ldquoMicrobiological quality of raw-fermentedsausages with Lactobacillus casei LOCK 0900 probiotic strainrdquoFood Control vol 35 no 1 pp 184ndash191 2014

[156] E Sayas-Barbera M Viuda-Martos F Fernandez-Lopez J APerez-Alvarez and E Sendra ldquoCombined use of a probioticculture and citrus fiber in a traditional sausage rsquoLonganiza dePascuarsquordquo Food Control vol 27 no 2 pp 343ndash350 2012

[157] S Ruiz-Moyano A Martın M J Benito A Hernandez RCasquete and M de Guia Cordoba ldquoApplication of Lacto-bacillus fermentum HL57 and Pediococcus acidilactici SP979 aspotential probiotics in the manufacture of traditional Iberiandry-fermented sausagesrdquo Food Microbiology vol 28 no 5 pp839ndash847 2011

[158] FAOWHO ldquoReport of a joint FAOWHO working group ofdrafting guidelines for the evaluation of probiotics in foodrdquoguidelines for the evaluation of probiotics in food London UK2002

[159] S Salminen A von Wright L Morelli et al ldquoDemonstrationof safety of probioticsmdasha reviewrdquo International Journal of FoodMicrobiology vol 44 no 1-2 pp 93ndash106 1998

[160] D Kołozyn-Krajewska and Z J Dolatowski ldquoProbiotic meatproducts and human nutritionrdquo Process Biochemistry vol 47no 12 pp 1761ndash1772 2012

[161] J Hugenholtz and E J Smid ldquoNutraceutical production withfood-grademicroorganismsrdquoCurrentOpinion in Biotechnologyvol 13 no 5 pp 497ndash507 2002

[162] W Sybesma M Starrenburg L Tijsseling M H N Hoefnageland J Hugenholtz ldquoEffects of cultivation conditions on folateproduction by lactic acid bacteriardquo Applied and EnvironmentalMicrobiology vol 69 no 8 pp 4542ndash4548 2003

[163] C Burgess M OrsquoConnell-Motherway W Sybesma J Hugen-holtz andD Van Sinderen ldquoRiboflavin production in Lactococ-cus lactis Potential for in situ production of vitamin-enrichedfoodsrdquo Applied and Environmental Microbiology vol 70 no 10pp 5769ndash5777 2004

[164] W Sybesma C Burgess M Starrenburg D Van Sinderen andJ Hugenholtz ldquoMultivitamin production in Lactococcus lactisusing metabolic engineeringrdquoMetabolic Engineering vol 6 no2 pp 109ndash115 2004

[165] C Pennacchia E E Vaughan and F Villani ldquoPotential pro-biotic Lactobacillus strains from fermented sausages Furtherinvestigations on their probiotic propertiesrdquo Meat Science vol73 no 1 pp 90ndash101 2006

[166] R Rubio T Aymerich S Bover-Cid M D Guardia J Arnauand M Garriga ldquoProbiotic strains Lactobacillus plantarum299V and Lactobacillus rhamnosus GG as starter cultures forfermented sausagesrdquoLWT- Food Science andTechnology vol 54no 1 pp 51ndash56 2013

[167] K Neffe-Skocinska D Jaworska D Kołozyn-Krajewska ZDolatowski and L Jachacz-Jowko ldquoThe effect of LAB asprobiotic starter culture and green tea extract addition on dryfermented pork loins qualityrdquo BioMed Research Internationalvol 2015 Article ID 452757 2015

[168] K Neffe-Skocinska K Wojciak and D Zielinska ldquoProbioticmicroorganisms in dry fermented meat productsrdquo in ProbioticMicroorganisms in Dry Fermented Meat Products in Probioticsand Prebiotics in Human Nutrition and Health InTech RijekaCroatia 2016

[169] Regulation (EC)No 19242006 of the European Parliament andof the Council of 20 December 2006 on nutrition and healthclaims made on foods

[170] EU ldquoCommission Regulation (EU) No 4322012 of 16 May 2012establishing a list of permitted health claims made on foodsother than those referring to the reduction of disease risk andto childrenrsquos development and healthrdquo Official Journal of theEuropean Union vol 136 pp 1ndash40 2012

[171] J Anba-Mondoloni M-C Champomier-Verges M Zagorecet al ldquoThe Genetics of Microbial Startersrdquo in Handbook ofFermented Meat and Poultry Second Edition F TOLDRA Edpp 161ndash168 Wiley Blackwell 2015

[172] F Villani A Casaburi C Pennacchia L Filosa F Russo andD Ercolini ldquoMicrobial ecology of the soppressata of Vallo diDiano a traditional dry fermented sausage from southern Italyand in vitro and in situ selection of autochthonous starterculturesrdquo Applied and Environmental Microbiology vol 73 no17 pp 5453ndash5463 2007

[173] J A Klappenbach J M Dunbar and T M Schmidt ldquorRNAoperon copy number reflects ecological strategies of bacteriardquoApplied and Environmental Microbiology vol 66 no 4 pp1328ndash1333 2000

[174] C-J Liu R Wang F-M Gong et al ldquoComplete genomesequences and comparative genome analysis of Lactobacil-lus plantarum strain 5-2 isolated from fermented soybeanrdquoGenomics vol 106 no 6 pp 404ndash411 2015

[175] W P Hammes A Bantleon and S Min ldquoLactic acid bacteria inmeat fermentationrdquo FEMS Microbiology Letters vol 87 no 1-2pp 165ndash173 1990

[176] A McLeod D A Brede I Rud and L Axelsson ldquoGenomesequence of Lactobacillus sakei subsp sakei LS25 a com-mercial starter culture strain for fermented sausagerdquo GenomeAnnouncements vol 1 no 4 Article ID e00475-13 2013

[177] S H Lee M Y Jung B Park et al ldquoComplete genomesequence of Pediococcus pentosaceus strain wikim 20 isolatedfrom Korean kimchirdquo Genome Announcements vol 4 no 6Article ID e01233-16 2016

[178] S H Dantoft E M Bielak J-G Seo M-J Chung and P RJensen ldquoComplete genome sequence of Pediococcus pentosaceusstrain SL4rdquo Genome Announcements vol 1 no 6 Article IDe01106-13 2013

[179] G-S Park S-J Hong B K Jung et al ldquoWhole genome se-quence of lactic acid bacterium Pediococcus acidilactici strainS1rdquo Brazilian Journal of Microbiology vol 48 no 3 2017

[180] S J Labrie L El Haddad D M Tremblay et al ldquoFirst completegenome sequence of Staphylococcus xylosus a meat starterculture and a host to propagate Staphylococcus aureus phagesrdquoGenome Announcements vol 2 no 4 Article ID e00671-142014

[181] E Dordet-Frisoni G Dorchies C De Araujo R Talon and SLeroy ldquoGenomic diversity in Staphylococcus xylosusrdquo Appliedand Environmental Microbiology vol 73 no 22 pp 7199ndash72092007

[182] R Rosenstein C Nerz L Biswas et al ldquoGenome analysisof the meat starter culture bacterium Staphylococcus carnosusTM300rdquo Applied and Environmental Microbiology vol 75 no3 pp 811ndash822 2009


[183] M S Resch C Nerz R Rosenstein F Gotz and C HertelldquoDNA microarray based detection of genes involved in safetyand technologically relevant properties of food associatedcoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 145 pp 449ndash458 2011

[184] A Vermassen A de la Foye V Loux R Talon and SLeroy ldquoTranscriptomic analysis of Staphylococcus xylosus in thepresence of nitrate and nitrite in meat reveals its response tonitrosative stressrdquo Frontiers in Microbiology vol 5 article no691 2014

[185] A Vermassen E Dordet-Frisoni A De La Foye et al ldquoAdap-tation of Staphylococcus xylosus to nutrients and osmotic stressin a salted meat modelrdquo Frontiers in Microbiology vol 7 articleno 87 2016

[186] R Gaupp N Ledala and G A Somerville ldquoStaphylococcalresponse to oxidative stressrdquo Front Cell Infect Microbiol vol 2p 33 2012

[187] S Planchon M Desvaux I Chafsey et al ldquoComparativesubproteome analyses of planktonic and sessile StaphylococcusxylosusC2a New insight in cell physiology of a coagulase-nega-tive staphylococcus in biofilmrdquo Journal of Proteome Researchvol 8 no 4 pp 1797ndash1809 2009

[188] A McLeod L Snipen K Naterstad and L Axelsson ldquoGlobaltranscriptome response in Lactobacillus sakei during growth onriboserdquo BMCMicrobiology vol 11 article no 145 2011

[189] H-Q Xu L Gao Y-S Jiang et al ldquoTranscriptome responseof Lactobacillus sakei to meat protein environmentrdquo Journal ofBasic Microbiology vol 55 no 4 pp 490ndash499 2015

Research ArticleThe Effect of Drying Parameters on the Quality ofPork and Poultry-Pork Kabanosy Produced according tothe Traditional Specialties Guaranteed Recipe

Marta Chmiel Lech Adamczak KatarzynaWroNskaDorota Pietrzak and Tomasz Florowski

Division of Meat Technology Department of Food Technology Faculty of Food SciencesWarsaw University of Life Sciences-SGGW 166 Nowoursynowska Street 02-787 Warsaw Poland

Correspondence should be addressed to Marta Chmiel marta_chmielsggwpl

Received 12 January 2017 Revised 6 April 2017 Accepted 19 April 2017 Published 28 May 2017

Academic Editor Maria J Fraqueza

Copyright copy 2017 Marta Chmiel et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to determine the effect of differentiated air relative humidity during the drying process on selected qualityfeatures of TSG (traditional specialties guaranteed) pork and poultry-pork kabanosy After heat treatment and 24-hour cooling at4ndash6∘C the products were placed in three chambers at 15∘C with differentiated air relative humidity 60 70 and 80 respectivelyThe drying process was carried out until all variants of kabanosy achieved the required final yield of the product (lt68) Colorcomponents water activity and shear force water protein fat and salt content and the TBARS indicator values were determinedThe drying process might be shortened (sim50) by a reduction of humidity in the drying chamber from 80 to 60 The changesin the content of chemical components in pork kabanosy compared to poultry-pork ones demonstrated the different dynamicsof the drying of the two types of kabanosy and the need for the selection of optimum drying conditions relative to raw materialcomposition

1 Introduction

Sausages are one of the oldest meat products and have beenmanufactured for nearly two thousand years They are con-sumed all over the world due to their attractive flavor profileDry and semidry sausages are considered by consumers to betwo of the most delicious and highly sought after products[1ndash3] The growing interest in this type of products resultsfrom their diversity the use of different raw materials andtheir degree of fragmentation not to mention the spices orsmoking methods used Moreover their high desirability isalso affected by their unique taste and aroma [4 5] In recentyears there has been an increase in consumer demand interms of food quality and its safety and effects on health [6]Growing consumer interest has been noted in food with asmaller level of additives [7] as well as traditional and naturalproducts especially in the countries of Eastern Europe [89] This group of products includes kabanosy which arepopular in Poland Kabanosy are a Polish traditional product

whose production history dates to the 1920s30s [10] Theword ldquokabanosrdquo probably comes from the name of the porkldquokabaninardquo which was obtained from a characteristic speciesof pig known as a ldquokabanrdquo In the nineteenth century akaban in Poland and Lithuania was a young male extensivelyfattened with potatoes to obtain a delicate meat with a highdegree of intramuscular fat (marbled meat with intramuscu-lar fat content above 3) which in turn positively affectedits juiciness and tenderness In 2011 pork kabanosy wereregistered in the European Union as a product of traditionalspecialties guaranteed (TSG) Kabanosy are long thin andevenly wrinkled pork sausages in natural casings sheepintestines The product is subjected to drying and smokingprocesses which affects the formation of their specific colorThe color of the surface of pork kabanosy should be dark redwith a hint of cherry while slightly creamy fat particles anddark redmeat pieces should be visible in the cross section [10]In the case of poultry kabanosy the color is lighter whichis the result of the use of poultry meat Kabanosy should



be characterized by the aroma of cured meat with a delicatehint of cumin and black pepper The characteristic featureof kabanosy is their unique smell which is mainly due tosmoking

The drying process also plays an important role in thedistinctive unique taste of kabanosy emphasizing the advan-tages of their aroma and taste Drying is one of the oldestmethods of food preservation [11ndash16] According to EuropeanParliament and Council Regulation (EU) number 10442011[10] pork kabanosy (TSG) should be dried for 3 to 5 daysat a temperature of 14ndash18∘C with an air relative humidity of80 to achieve the desired yield (le68)The drying processsignificantly affects the quality of the produced kabanosyand is a highly energy-consuming step that determines theeconomy of production [11 14] Therefore one aim of thisstudy was to determine the effect of differentiated air relativehumidity in the drying chamber (80 70 and 60) on thequality of pork kabanosy The consumption of poultry meathas increased rapidly all over the world in the last decadeand poultry production has become the fastest growing meatsector [17] Due to the nutritional value and low price ofpoultrymeat resulting from its high supply we also decided toproduce poultry-pork kabanosywith a chemical compositionsimilar to pork kabanosy TSG in this study

2 Materials and Methods

21 Kabanosy Production The basic raw materials for theproduction of pork kabanosy were class I pork 30 classIIA 40 class IIB 30 In the case of poultry-pork kabanosythe composition was chicken thigh meat 80 and pork jowl20 For both types of kabanosy the same spice compositionwas used (with respect to rawmaterials) that is black pepper(015) sugar (020) nutmeg (005) and cumin (007)Pork and poultry-pork kabanosy were produced in threeseries with precut (pieces measuring 5 cm) cured (2 basedon theweight of rawmaterials for 24 hours before productionunder refrigeration 4ndash6∘C) pork or poultry meat and porkjowlThe production process was conducted according to thefollowing scheme

(i) Grinding class I pork meat or part of chicken thighmeat (30) was ground on a mesh of Oslash 10mm classIIA IIB pork meat part of chicken thigh meat (50)and jowl were ground on a mesh of Oslash 8mm theraw material grinding was conducted in a MeskoWN60 laboratory grinder (Mesko-AGD Skarzysko-Kamienna Poland)

(ii) Mixing ground meat was mixed using a KenwoodMajormixer (Kenwood Havant UK) for 5minutes tothoroughlymix the ingredients the spices were addedduring the mixing

(iii) Bar stuffing andmolding sheep intestines (Oslash 22mm)were filled using a Dick manual stuffer (Friedr DickGmbH amp Co KG Deizisau Germany) and thenformed into 25 cm bars

(iv) Settling the bars were deposited on a smoking stickfor 1 hour at room temperature

(v) Heat treatment kabanosy were dried in a Jugemasmoking-cooking chamber (Jugema Sroda Wielko-polska Poland) for 15 minutes at 40∘C smoked withwarm smoke for 30 minutes at 50∘C and then bakedat 75∘Cuntil 70∘Cwas reached in the geometric centerof the bar after the treatment kabanosy were cooledfor 1 hour in an off chamber

(vi) Cooling kabanosy were cooled in a refrigerator for 24hours at 4ndash6∘C

(vii) Drying kabanosy were divided into three variantsand these were subjected to a drying process in a labo-ratory drying chamber (PHU Chłodnictwo WarsawPoland) at three different drying air relative humidi-ties (K1ndash80 plusmn 2 K2ndash70 plusmn 2 and K3ndash60 plusmn2) at the same temperature of 15∘C plusmn 2∘C Thehumidity and temperature in the chambers weremonitored using data loggers (EL-USB-2 modelsLascar Electronics Ltd Erie USA) The drying pro-cess was carried out until all variants of kabanosyachieved the TSG required [10] final yield of the prod-uct that is below 68 The final yield was controlledeach 12 h of the drying process For kabanosy dried ina chamber at 60 humidity the required final yieldbelow 68 was achieved after 24 h of the process at ahumidity of 70 after 36 h and at a humidity of 80after 48 h of drying

Before and after the drying process a range of measure-mentswere performed for each of the three production seriesfor each of the three variants of kabanosy and for both porkand poultry-pork ones The color components 119871lowast 119886lowast 119887lowast ofthe surface and cross-sectional area of the bar water activity(119886119908) and the texture that is the shear force (only after thedrying process) were measured on the unground kabanosyGround kabanosy (laboratory grinder Zelmer Diana 8868Zelmer Rzeszow Poland grid hole diameter 3mm) weresubjected to the measurements of basic chemical componentcontent that is water protein fat and salt and the TBARSindicator was also determined

22 Kabanosy Yield at Different Stages of the Production Pro-cess The yield of heat treatment after 24 hours and the finalyield after the drying process at different air humidities inthe chamber at a level of 80 70 and 60 respectively weredetermined during the process of pork and poultry-porkkabanosy production The yields were determined relative tothe initial weight before the heat treatment

23 Measurement of Color Components on the 119871lowast 119886lowast 119887lowast ScaleThe measurements of color components were performedusing a Minolta CR-200 camera (Konica Minolta WroclawPoland light source D65 2∘ observer measuring head hole8mm) calibrated according to white standard (119871lowast 9781119886lowastminus045 119887lowast 188) The measurements were taken in each ofthe 5 repetitions on the surface and cross section of kabanosytaking the average as a result of the measurement

24 Content of the Basic Chemical Components Themoisturecontent was determined according to PN-ISO 14422000 [18]


Table 1 Yield of kabanosy at different stages of the production process

Kabanosy Thermal treatment yield () Yield after cooling () Final yield ()K1 - 80 K2 - 70 K3 - 60

Pork 785 plusmn 53 749 plusmn 44 592a plusmn 68 553a plusmn 35 508a plusmn 28

Poultry-pork 842 plusmn 21 820 plusmn 35 650a plusmn 36 612a plusmn 29 607a plusmn 32aAverage values in rows marked with different letters differ significantly at 119901 le 005

by drying samples at 105∘C (SUP-65 dryer Wamed WarsawPoland)The protein content was determined by the Kjeldahlmethod according to PN-75A-040182002 (Velp ScientificaUDK 129 Distillation Unit Poland) [19] The fat content wasdetermined by Soxhlet extraction (Buchi Extraction SystemB-811 Donserv Poland) according to PN-ISO 14442000[20] The salt content was determined using potentiometricmethods according to PN-ISO 1841-22002 [21] using a 702SM Titrino (Metrohm AG Herisau Switzerland) device

25 Measurement of Water Activity (119886119908) Measurement ofwater activity was carried out using an Aqua Lab CX-2 appa-ratus (DecagonDevices Inc PullmanUSA)The samplewasprepared by cutting a flat rectangle from kabanosy and theanalysis was performed at a temperature of 250plusmn15∘C threetimes for each sample and the average was taken as a resultof the measurement

26 TBARS Indicator Determination Thiobarbituric acidreactive substances (TBARS) values were determined accord-ing to the extractionmethod of Shahidi [22] Absorbance wasmeasured at 532 nm using a spectrophotometer (Hitachi U-1100 Gemini bv Apeldoorn Netherlands) against a blankcontaining 5mL of 2-thiobarbituric acid (TBA) and 5mL of10 trichloroacetic acid (TCA) A constant coefficient of 234was employed for converting the absorbance units to TBARSvalues which were expressed as mg malondialdehyde per kgsample (mg MADkg)

27Measurement of Texture-Shear Force Measurement of theshear force was performed using a ZWICKI 1120 enduranceapparatus (Zwick Ulm Germany) Warner-Bratzlerrsquos deviceequipped with a flat knife was used for this purpose Themaximum shear force 119865max was read out at a head shift of50mmmin The samples were prepared directly before testsby cutting the kabanosy into 10 cm sections and subjectingthem to 30 minutes of conditioning at room temperatureThe measurements were taken at three points on each of theprepared sections for three different bars from each variantof kabanosy The results were then averaged

28 Sensory Evaluation The sensory quality of kabanosyafter the drying process in chambers with different levels ofhumidity was assessed using a 10-point intensity scale forsuch sensory characteristics as surface color hardness aromaand taste However overall acceptability was assessed on ahedonic scale (where 0 points corresponded to unacceptableand a 10-point evaluation as very desirable) The evalua-tion was conducted by a trained 10-person team according

to PN-ISO 41211998 [23] Kabanosy prior to the assess-ment were conditioned for half an hour at room tempera-ture

29 Statistical Analysis of the Results The results were sub-jected to statistical analysis using ANOVA analysis and adetailed Tukeyrsquos HSD test at a significance level of 120572 = 005Statistica ver 10 PL (StatSoft Inc Tulsa USA) was used Theeffect of air relative humidity in the drying chamber on yieldand on selected quality features of pork and poultry-porkkabanosy was determined

3 Results and Discussion

31 Yield of Kabanosy The yields of kabanosy at differentstages of the production process are shown in Table 1 Inthe case of both pork and poultry-pork kabanosy there wasno significant (119901 gt 005) effect derived from air relativehumidity on the final yield of these products (Table 1) Driedsausages can lose up to 30 of their weight throughout theproduction process and such sausages in the US market areconsidered to be ldquomoderatelyrdquo dry products In contrast drysausages are those that are characterized by a 40 weightloss [24] The weight loss of the product is faster in the caseof a product dried at lower relative air humidity [25] Thetendency observed in this study was therefore consistentwith the literature data The period of drying of kabanosyin the chamber with 80 humidity (yield below 68 asrequired by TGS) was considerably longer in comparisonwith the drying time of the kabanosy under a humidity of70 and 60 The use of lower relative humidity in the dryingprocess reduced the duration of the process by 50 howeverthis may result in lower product yield The observed average10-percentage point difference in the yield of pork kabanosydried at 80 and 60 relative air humidity was caused not onlyby the varying relative air humidity in the chamber but alsoby the varied duration of the process The different dryingdynamics of both types of kabanosy suggest the need forthe selection of optimum drying conditions in terms of rawmaterial composition

32 119871lowast 119886lowast 119887lowast Color Components of Kabanosy The results of119871lowast 119886lowast and 119887lowast color component measurement on the surfaceand cross sections of kabanosy are summarized in Table 2A reduction of color component 119871lowast and thus the lightnessof the surface after the drying process was noted in porkkabanosy The lowest value for this component was observedin the case of kabanosy subjected to drying in a chamberwith a relative air humidity of 60 The effect of differentrelative air humidities in terms of the color lightness on the


Table2Color

ofthes

urface

andcrosssectio

nof

pork

andpo

ultry-po

rkkabano

sybefore

andaft

erthed

ryingprocess

(a)

Kabano

sy

Surfa

cecolor

Before

drying

process

Afte

rdryingprocess

K180

K270

K360

119871lowast

119886lowast

119887lowast119871lowast

119886lowast


119886lowast


119886lowast

119887lowast

Pork

454plusmn02211plusmn0489plusmn18404

aplusmn07199

aplusmn0968aplusmn18392

aplusmn20180

abplusmn1243a

bplusmn13382

aplusmn18164

bplusmn0420bplusmn07

Poultry-po

rk505plusmn29138plusmn09129plusmn17442

aplusmn19147


aplusmn22152


aplusmn22149


(b)

Kabano

sy

Crosssectio

ncolor

Before

drying

process

Afte

rdryingprocess

K180

K270

K360

119871lowast

119886lowast


119886lowast


119886lowast


119886lowast

119887lowast

Pork


aplusmn03156


bplusmn08154

aplusmn0716bplusmn06456

bplusmn17161


Poultry-po


abplusmn19106

aplusmn0653a

bplusmn05555

aplusmn0694aplusmn1562aplusmn04524

bplusmn0698aplusmn0648bplusmn04

abAv

eragev

aluesfor

thes

amed

ifferentiatorinrowsm

arkedwith

different

lette

rsdiffersignificantly

at119901le005


surface of pork kabanosy was not significant (119901 gt 005)Similar relationships were found for poultry-pork kabanosy(Table 2) According to De Maere et al [26] reduction of the119871lowast component values during the drying process may resultfrom the reduction of water content in the product

For both the pork and poultry-pork kabanosy a decreasein the values of the 119886lowast and 119887lowast color components was observedduring the drying process in chambers with different relativeair humidity compared to the kabanosy before the dryingprocessThis applies to both the color of the bar and the crosssection of the products There was a tendency for 119886lowast and 119887lowastvalues to be lower with a decrease in relative air humidityin the drying chamber Pork kabanosy dried with an relativeair humidity of 60 were characterized by significantly (119901 lt005) lower values for components 119886lowast and 119887lowast than kabanosydried at 80 humidity (Table 2) In contrast for the poultry-pork kabanosy drying relative air humidity had no significant(119901 gt 005) effect on the values of those components (Table 2)

Measurements of color components 119871lowast 119886lowast and 119887lowast werealso taken on the cross sections of kabanosyThe color on thecross sections of kabanosy dried with a relative air humidityof 70 and 60 was characterized by significantly lowervalues for component 119871lowast compared to the values obtainedon the cross sections of kabanosy dried at 80 humidity(Table 2) In the case of poultry-pork kabanosy a significant(119901 lt 005) effect of relative air humidity was noted on thelightness of their cross section color however this effectwas ambiguous Poultry-pork kabanosy dried in a chamberwith a relative air humidity of 60 were characterized bysignificantly lower values for component 119871lowast and so they werethe darkest compared to kabanosy dried at a humidity of 70(Table 2)

Pork kabanosy dried in chambers with a relative airhumidity of 70 and 60 were characterized by significantly(119901 lt 005) lower levels of yellow color (119887lowast) in the crosssections of the bar compared to kabanosy dried at a relativeair humidity of 80 (Table 2)

In the case of poultry-pork kabanosy significant differ-entiation of this color component was observed between thevalues determined for the cross section of the product driedat 70 and 60 of relative air humidity (Table 2)

33 Content of the Basic Chemical Components in KabanosyThe average content of basic chemical components in porkkabanosy before the drying process was 521 water content248 protein content 196 fat content and 30 saltcontent During the drying process as expected there was adecrease in the water content and increase in the componentsconstituting the dry matter of the product Olivares et al[27] indicate that the concentration of protein and fat inthe product during the drying process is mainly due to thewater content decrease resulting fromweight lossThehighestaverage values for protein (295) fat (235) and salt (36)content were found in the case of drying of the pork kabanosyin a chamber with 60 relative air humidity Despite the 10-percentage point differences in the yield of products dried at80 and 60 of relative air humidity no significant differ-ences (119901 gt 005) were found in the chemical compositionof kabanosy dried in chambers with different relative air

humidity This could be due to significant differences in theprocess of drying in the various research series as evidencedby among others high standard deviations

In the case of poultry-pork kabanosy a decrease in watercontent was observed from 531 before the drying processto 458 after drying in a relative air humidity of 60Protein fat and salt content changed from 217 219 and27 respectively before the drying process to 254 256and 31 after drying in the chamber with 60 relative airhumidity Also in this case there were no significant (119901 gt005) differences of chemical composition of poultry-porkkabanosy dried in chambers of different relative air humidity(data not shown)

According to European Parliament and Council Reg-ulation (EU) number 10442011 [10] kabanosy should becharacterized by the following chemical composition proteincontent of at least 15 water up to 60 fat up to 35 andsalt maximum 35 The poultry-pork kabanosy producedin this study irrespective of the drying conditions met allthe above requirements In the case of pork kabanosy onlya slight excess of or the average salt content was found inproducts dried at 60 relative air humidity

34 Water Activity (119886119908) TBARS Indicator Value and Textureof Kabanosy The water activity of both pork and poultry-pork kabanosy decreased with a decrease in relative airhumidity in the drying chamber (Table 3) There was nosignificant (119901 gt 005) effect of drying air humidity on wateractivity in pork kabanosy Poultry-pork products dried at60humiditywere characterized by a significantly (119901 lt 005)lower water activity than those dried at 70 and 80 humidityAlso in the studies presented byCollell et al [1] a relationshipwas demonstrated between the fall in 119886119908 and water contentduring the drying process

In the present study there was no significant (119901 gt 005)effect of air relative humidity on the value of the TBARSindicator in the case of pork and poultry-pork kabanosy(Table 3)

There was no significant (119901 gt 005) effect of air relativehumidity on the shear force of either pork or poultry-porkkabanosy There was only a tendency pointing to an increasein the shear force of kabanosy with a decrease in relativehumidity in the drying chamber According to Arnau etal [14] the first method to obtain an acceptable texturewith a short drying period in dry-cured meat products isto accelerate the drying process by decreasing the relativehumidity and increasing the temperature of the drying airThis was not confirmed in our research

35 Sensory Quality of Kabanosy A reduced relative humid-ity of the air in the drying chamber did not affect such sensoryqualities of pork and poultry-pork kabanosy as hardnessaroma taste and overall acceptability (Table 4) A significant(119901 lt 005) impact on the color of pork kabanosy was notedas a result of the relative air humidity Kabanosy dried at therelative humidity of 60 received lower scores for color thankabanosy dried at a humidity of 80 An opposite trend wasobserved in the case of the color of poultry-pork kabanosybut this was not significant (Table 4) Also some trends in


Table 3 Water activity (119886119908) and TBARS indicator values in pork and poultry-pork kabanosy before and after the drying process shear forcevalue after drying process

(a)

KabanosyWater activity TBARS (mg MADkg of the product)

Before dryingprocess

After drying process Before dryingprocess

After drying processK1 80 K2 70 K3 60 K1 80 K2 70 K3 60

Pork 0943 plusmn 0007 0940a plusmn0004 0927a plusmn0011 0919a plusmn0011 054 plusmn 017 050a plusmn 011 060a plusmn 023 056a plusmn 018

Poultry-pork 0956 plusmn 0005 0940a plusmn0002 0935a plusmn0004 0926b plusmn0003 055 plusmn 008 053a plusmn 008 055a plusmn 012 055a plusmn 011

(b)

KabanosyShear force (119873)

After drying processK1 80 K2 70 K3 60

Pork 913a plusmn 149 993a plusmn 69 1034a plusmn 80

Poultry-pork 836a plusmn 179 855a plusmn 229 944a plusmn 149abAverage values for the same differentiator in rows marked with different letters differ significantly at 119901 le 005

Table 4 The effect of air relative humidity on the sensory quality characteristics of kabanosy

Characteristic (points) Kabanosy K1 80 K2 70 K3 60

Surface color Pork 94a plusmn 06 84ab plusmn 03 68b plusmn 03

Poultry-pork 74a plusmn 05 82a plusmn 03 926a plusmn 03

Hardness Pork 94a plusmn 12 84a plusmn 09 72a plusmn 06


Aroma Pork 68a plusmn 08 68a plusmn 07 66a plusmn 03


Taste Pork 74a plusmn 03 72a plusmn 08 62a plusmn 08


Overall acceptability Pork 62a plusmn 05 60a plusmn 05 72a plusmn 09


changes in the scores given in the evaluation of hardnessand taste of kabanosy were observed In the case of porkkabanosy scores given for these discriminants were lowerwith the decrease in relative humidity and in the case ofpoultry-pork kabanosy marks were higher (Table 4) Thisconfirms the need for a separate examination of the effect ofhumidity in the drying chamber depending on the type ofraw material used in kabanosy production

4 Conclusion

Pork kabanosy produced at differentiated drying relative airhumidities met the requirements for the recipe for tradi-tional specialty guaranteed (TSG) regarding the content ofbasic chemical components water protein and fat and theassumed final yield in the case of the salt content theseassumptions were only met in the case of the product driedin air at 70 and 80 relative humidity TSG assumptionsregarding the chemical composition were also fulfilled bypoultry-pork kabanosy regardless of relative air humidity inthe chamberThe final yield of the products was proportionalto the humidity prevailing in the drying chamber but this was

not significantly differentiated Kabanosy dried in air of 60humidity were thus characterized by the lowest final yieldand moreover the duration of drying in this case was theshortest No significant effect of drying relative air humiditywas found on the value of the TBARS indicator in the caseof pork or poultry-pork kabanosy however reduced wateractivity and water content were observed with a decreasein air relative humidity and analogically an increase wasobserved in protein and fat content in the products After thedrying process the products were darker both on the surfaceand in cross section (a lower value for component 119871lowast) thanbefore the process Also relative air humidity in the dryingchamber affected the color components of kabanosy Thechanges in the contents of particular chemical componentin pork kabanosy compared to poultry-pork ones point tothe different dynamics of the drying process of both types ofkabanosy and the need for the selection of optimum dryingconditions in terms of raw material composition

Differentiation in relative air humidity in the dryingchambers affected the dynamics of changes mainly in termsof the color parameters and water activity of producedkabanosy Moreover pork kabanosy dried at the relative


humidity of 60 received lower scores for color thankabanosy dried at a humidity of 80 The results obtainedin this study indicate the possibility of shortening the dryingprocess by 50 together with the reduction of humidity inthe drying chamber from 80 to 60 In turn the economiceffect of the lower humidity should be examined underproduction plant conditions


The authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] C Collell P Gou J Arnau I Munoz and J Comaposada ldquoNIRtechnology for on-line determination of superficial a119908 andmoisture content during the drying process of fermentedsausagesrdquo Food Chemistry vol 135 no 3 pp 1750ndash1755 2012

[2] W-W Ren A E-D A Bekhit F Li et al ldquoPhysicochemicalproperties of pastirma fromhorsemeat beefmutton and porkrdquoJournal of Food Quality vol 38 no 5 pp 369ndash376 2015

[3] G Tabanelli F Coloretti C Chiavari L Grazia R Lanciottiand F Gardini ldquoEffects of starter cultures and fermentationclimate on the properties of two types of typical Italian dryfermented sausages produced under industrial conditionsrdquoFood Control vol 26 no 2 pp 416ndash426 2012


[5] A Berardo B Devreese H De Maere et al ldquoActin proteolysisduring ripening of dry fermented sausages at different pHvaluesrdquo Food Chemistry vol 221 pp 1322ndash1332 2017

[6] F Toldra and M Reig ldquoInnovations for healthier processedmeatsrdquo Trends in Food Science and Technology vol 22 no 9pp 517ndash522 2011

[7] E Muguerza O Gimeno D Ansorena and I Astiasaran ldquoNewformulations for healthier dry fermented sausages a reviewrdquoTrends in Food Science and Technology vol 15 no 9 pp 452ndash457 2004

[8] Z Pieniak W Verbeke F Vanhonacker L Guerrero and MHersleth ldquoAssociation between traditional food consumptionand motives for food choice in six European countriesrdquoAppetite vol 53 no 1 pp 101ndash108 2009

[9] M Duskova J Kamenık O Sedo et al ldquoSurvival and growth oflactic acid bacteria in hot smoked dry sausages (non-fermentedsalami) with and without sensory deviationsrdquo Food Control vol50 pp 804ndash808 2015

[10] Commission Regulation 10442011 ldquoEuropean Parliament andCouncil Regulation (EU) No 10442011 of 19 October 2011recording in the register of traditional specialties guaranteed[Kabanosy (TSG)]rdquo Official Journal of the European Communi-ties vol L 27516 2011

[11] A del Olmo J Calzada and M Nunez ldquoLipolysis lipid perox-idation and texture of Serrano ham processed under differentripening temperature conditionsrdquo International Journal of FoodScience and Technology vol 51 no 8 pp 1793ndash1800 2016

[12] K Naidoo and D Lindsay ldquoSurvival of Listeria monocytogenesand enterotoxin-producing Staphylococcus aureus and Staphy-lococcus pasteuri during two types of biltong-manufacturingprocessesrdquo Food Control vol 21 no 7 pp 1042ndash1050 2010

[13] F Toldra ldquoThe role of muscle enzymes in dry-cured meatproducts with different drying conditionsrdquo Trends in FoodScience and Technology vol 17 no 4 pp 164ndash168 2006

[14] J Arnau X Serra J Comaposada P Gou and M GarrigaldquoTechnologies to shorten the drying period of dry-cured meatproductsrdquoMeat Science vol 77 no 1 pp 81ndash89 2007

[15] B A Nummer J A Harrison M A Harrison P Kendall J NSofos and E L Andress ldquoEffects of preparation methods onthe microbiological safety of home-dried meat jerkyrdquo Journal ofFood Protection vol 67 no 10 pp 2337ndash2341 2004

[16] M Hersleth V Lengard W Verbeke L Guerrero and TNaeligs ldquoConsumersrsquo acceptance of innovations in dry-cured hamimpact of reduced salt content prolonged aging time and neworiginrdquo Food Quality and Preference vol 22 no 1 pp 31ndash412011

[17] A Al-Nehlawi S Guri B Guamis and J Saldo ldquoSynergisticeffect of carbon dioxide atmospheres and high hydrostaticpressure to reduce spoilage bacteria on poultry sausagesrdquo LWT-Food Science and Technology vol 58 no 2 pp 404ndash411 2014

[18] Polish Committee for Standardization Polish Standard PN-ISO 14422000 Meat and Meat Products Determination ofMoisture ContentmdashReference Method Polish Committee forStandardization Warsaw Poland 2000

[19] Polish Committee for Standardization Polish StandardPN-A-040181975Az32002 Agricultural Food ProductsmdashDe-termination of Nitrogen by The Kjeldahl Method and Expressingas Protein Polish Committee for Standardization WarsawPoland 2002

[20] Polish Committee for Standardization Polish Standard PN-ISO 14442000 Meat and Meat Products Determination of FreeFat Content Polish Committee for Standardization WarsawPoland 2000

[21] Polish Committee for Standardization Polish Standard PN-ISO1841-22002Meat andMeat Products Determination of ChlorideContentmdashPart 2 Potentiometric Method Polish Committee forStandardization Warsaw Poland 2002

[22] F Shahidi ldquoThe 2-thiobarbituric acid (TBA) methodology forthe evaluation of warmed-over flavour and rancidity in meatproductsrdquo in Proceedings of the 36th ICoMST pp 1008ndash1015Havana Cuba 1990

[23] Polish Committee for Standardization Polish Standard PN-ISO4121 1998 SEnsory Analysis Methodology Evaluation of FoodProducts Using The Method of Scaling Polish Committee forStandardization Warsaw Poland 1998

[24] R Maddock ldquoUS Products-Dry Sausagerdquo in Handbook of Fer-mented Meat and Poultry pp 295ndash300 Wiley-Blackwell 2ndedition 2014

[25] J Arnau P Gou and J Comaposada ldquoEffect of the relativehumidity of drying air during the resting period on thecomposition and appearance of dry-cured ham surfacerdquo MeatScience vol 65 no 4 pp 1275ndash1280 2003

[26] H DeMaere I Fraeye E DeMey et al ldquoFormation of naturallyoccurring pigments during the production of nitrite-free dryfermented sausagesrdquoMeat Science vol 114 pp 1ndash7 2016

[27] A Olivares J L Navarro A Salvador and M Flores ldquoSensoryacceptability of slow fermented sausages based on fat contentand ripening timerdquo Meat Science vol 86 no 2 pp 251ndash2572010

Review ArticleHealth and Safety Considerations of Fermented Sausages

Askild Holck1 Lars Axelsson1 Anette McLeod1 Tone Mari Rode2 and Even Heir1

1Nofima AS Norwegian Institute of Food Fisheries and Aquaculture Research PO Box 210 1431 As Norway2Nofima AS Norwegian Institute of Food Fisheries and Aquaculture Research PO Box 8034 4068 Stavanger Norway

Correspondence should be addressed to Askild Holck askildholcknofimano

Received 2 March 2017 Accepted 20 April 2017 Published 28 May 2017

Academic Editor Marta Laranjo

Copyright copy 2017 Askild Holck et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fermented sausages are highly treasured traditional foods A large number of distinct sausages with different properties areproduced using widely different recipes and manufacturing processes Over the last years eating fermented sausages has beenassociated with potential health hazards due to their high contents of saturated fats high NaCl content presence of nitrite and itsdegradation products such as nitrosamines and use of smoking which can lead to formation of toxic compounds such as polycyclicaromatic hydrocarbons Here we review the recent literature regarding possible health effects of the ingredients used in fermentedsausages We also go through attempts to improve the sausages by lowering the content of saturated fats by replacing them withunsaturated fats reducing the NaCl concentration by partly replacing it with KCl and the use of selected starter cultures withdesirable properties In addition we review the food pathogenic microorganisms relevant for fermented sausages (Escherichia coliSalmonella enterica Staphylococcus aureus Listeria monocytogenes Clostridium botulinum and Toxoplasma gondii) and processingand postprocessing strategies to inhibit their growth and reduce their presence in the products

1 Introduction

Meat is especially rich in proteins vitamins and mineralsand is an important element in human diet [1] Due to itsperishable nature meat historically had to undergo differentmethods of conservation One strategy was mincing themeat with salt and spices and lowering the water contentby drying Fermented sausages were thus created and aretreasured traditional foods Nowadays a large number ofdifferent sausages are produced using widely different recipesand manufacturing processes In 1995 the production offermented sausages in the EU was estimated to be about750000 tons [2] Spain produces around 200000 tons peryear while France produces another 110000 tons [3] Theproduction figures for 2014 for Norway and Finland were7300 tons and 7000 tons respectively [4]

Traditionally fermented sausages were consideredhealthy and safe foods More recently eating fermentedsausages has been associated with health hazards caused bythe high contents of saturated fats and NaCl presence ofnitrite and degradation products such as nitrosamines anduse of smoking which can lead to toxic compounds such as

polycyclic aromatic hydrocarbons in the products Hazardscan also be both of directmicrobiological nature the sausagespotentially being contaminated with food pathogens andof indirect microbiological nature by metabolic activity ofmicroorganisms causing presence of biogenic amines andmycotoxins

Raw meat is an ideal medium for growth of manymicroorganisms due to its high moisture content (70ndash80)and its abundance of proteins peptides and amino acidsgrowth factors and minerals In addition it usually containsfermentable glycogen and has a pH favorable for manymicroorganisms This is why raw meat is a highly perishableproduct and should be preserved For fermented sausagesthis preservation consists of a number of strategies (hurdles)working together These include lowering of pH by ferment-ing sugars tomainly lactic acid lowering ofwater activity (119886

119908)

by salting drying by evaporating water inhibiting growthof aerobic bacteria by creating an anaerobic environmentinhibiting microbial growth by addition of nitrate or nitriteand inhibiting surface growth by smoking or by addition ofspecific molds Together these hurdles generally lead to ashelf-stable product However traditional fermented sausage



manufacturing processes do not ensure microbiologicallysafe products Several foodborne outbreaks attributed to dryor semidry fermented sausages (DFSs) (see references below)have demonstrated that actions must be taken to ensurethat these products are safe to consume In most cases thepathogen in question does not grow in the finished productsbut survive long enough in high enough numbers to causedisease

Here we give an overview of the literature pertainingto health issues and microbiological issues for fermentedsausages and strategies to produce healthier and microbio-logically safer sausages

2 Production of Fermented Sausages

The large variety of fermented sausages and fermentationprocesses that exist have been thoroughly described else-where [2 5 6] Most often fermented sausages are producedfrom two-thirds of lean meat from animals such as pork andbeef and one-third of fat nearly always pork backfat In shortmeat is cut and mixed with fat spices salt sugar sodiumnitrite (sometimes nitrate) and starter culture Generally thestarter culture is a single species of lactic acid bacteria (LAB)or a LAB mixed with other bacteria such as Staphylococcusxylosus or S carnosus The mix is stuffed into natural orartificial casings of varying diameters and subjected to afermentation procedure where the LAB grow and convertthe sugar to lactic acid which leads to a pH decrease fromaround 58 down to 53ndash46 depending on the amount ofavailable fermentable sugars and process conditions Thestaphylococci when present will contribute to flavor devel-opment and reduction of nitrite and nitrate Subsequently thesausages are dried until the desired 119886

119908is reached Fermenta-

tion and drying steps are performed in smoke chambers anddrying rooms with controlled temperature and humidity

Fermented sausages can be either dry or semidry [7]Generally DFSs have 119886

119908le 090 while for semidry sausages

119886119908ranges between 090 and 095 [8] American type dry

sausages such as Genoa salami dry salami and pepperonicontain 25ndash40 moisture are heavily spiced are not heatedabove 267∘C have a firm texture and are usually shelf-stableIn Europe these fermented sausages can be further dividedinto Northern and Mediterranean types [9] Northern typeproducts such as cervelatwurst Westphalian salami plock-wurst boerenmetworst and Belgian salami often containbeef and pork and are characterized by relatively shortripening periods of up to 3 weeks and involve clearly sep-arated fermentation and drying periods Rapid acidulationto final pH values below 5 and smoking ensure microbi-ological safety and shelf-life Mediterranean type sausagessuch as Spanish salchichon and chorizo and Italian salamiare predominately pork products and involve longer ripeningperiods up to several months often without clear separationbetween fermentation and drying Smoke is not applied andacidulation to final pH values above 5 is slower Insteadof smoking the sausages are often covered with specificmolds Semidry sausages such as summer sausage cervelatLebanon Bologna and Mettwurst are usually fermented athigher temperatures 325ndash381∘C for more than 18 h to a

final pH lt 47 They have a moisture content between 45 and50 are heavily smoked are lightly spiced and are usuallyheated to an internal endpoint temperature between 43 and65∘C

3 Sausage Ingredients Related to Health

31 Fat Consuming a healthy diet throughout the life coursehelps prevent malnutrition in all its forms as well as arange of noncommunicable diseases and conditions [10]Theincreased production of processed food rapid urbanizationand changing lifestyles have led to a shift in dietary patternsPeople are consuming more foods high in energy (fats andsugars) Energy intake (calories) should be in balance withenergy expenditure Evidence indicates that total fat shouldnot exceed 30 of total energy intake to avoid unhealthyweight gain with a shift in fat consumption away from satu-rated fats to unsaturated fats so that saturated fats contributeno more than 10 of the total energy intake [10] Regardingpolyunsaturated fatty acids controlled feeding and cohortstudies of eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA) intakes have demonstrated physiological benefitson blood pressure heart rate triglycerides and likely inflam-mation endothelial function and cardiac diastolic functionConsistent evidence for a reduced risk of fatal coronary heartdisease and sudden cardiac death at consumption of approx250mgday of EPA plus DHA was demonstrated [11] Inindustrialized countries approx 36ndash40 of the total caloriesin the food supply come from fat nearly half of which is frommeat intake [12 13]

A way to reduce the amount of fat in fermented sausagesis to simply add less backfat to the batter There are howeverlimitations as to how large such a reduction can be beforesensory and technological quality of the sausages are reducedsince fats contribute profoundly to taste texture and mouthfeeling In Norway one of the large producers of fermentedsausages has a commercial product called ldquoExtra Salamirdquowhich is produced with 20 less fat than in the standardsalami recipe An alternative strategy is to replace some ofthe pork backfat with more healthy unsaturated fats or oilsAgain several challenges are associated with substitution ofanimal fat for oils in comminutedmeat products Incorporat-ing hydrophobic oils can be difficult as meat contains approx75 water and is hydrophilic Also increasing the contentof unsaturated fatty acids increases the susceptibility to lipidoxidation which reduces shelf-life [14] By emulsifying orencapsulating the oil and by addition of antioxidants thisproblem can in many cases be mitigated

In a set of experiments with DFSs 25 of the porkbackfat was substituted for an emulsion with linseed oil [15]No oxidation problems were detected during the ripeningprocess in such sausages with butylhydroxytoluene andbutylhydroxyanisole added as antioxidants No substantialchanges in odor appearance flavor and oxidation status wereobserved In Dutch-style fermented sausages 15 or 30 ofthe backfat was replaced with pure commercial encapsulatedfish oil added either as such or as a preemulsified mixturewith soy protein isolate [16] Sausages with encapsulated fishoil appeared to retain the overall quality and no clear effects


were found in the different sensory attributes when using 15untrained assessors

Inmost experimentswhere oil partly replaced backfat theoil was added together with stabilizers In low fat fermentedsausages (total fat content 10) 20 of the fat was substitutedfor preemulsified olive oil and added 120580-carrageenan [17] Theapplication of vacuum packaging over the last two weeks ofripening improved the physicochemical characteristics of thesausages and resulted in sensory attributes equal to or betterthan the high fat control sausages with 30 backfat Likewise328 of the fat could be replaced by a linseed oil carrageenangelled emulsion without loss of sensory qualities [18] InPamplona-style chorizo both low sodium ion and low fat(20 less than standard recipe) sausages were produced [19]Here 58 of the NaCl was substituted for 20 KCl and 38CaCl2 and 50 of the backfat was replaced with an alginate

emulsion consisting of 64water and 30olive oil 5 inulinwas also added to sausages These sausages retained sensorynotes similar to those of the traditional control chorizo andachieved a good acceptability rating Fat can also be partlyreplaced with other compounds When 50 of the porkbackfat was replaced with konjac gel a low-calorie ingredientwith a high content of nondigestible fiber the sausages hadan overall acceptability similar to the control sausages [12]A ldquoSuper Salamirdquo with 45 less fat and with 10 canola oilencapsulated in alginate and guar gum is available on theNorwegian market The finished sausages contain 20 fatof which 25 is saturated fat 60 is monounsaturated and15 is polyunsaturated A review of approaches to healthierformulations of comminuted meat products in conjunctionwith fat and salt has been published by Bolger et al [14]

32 Salt Salt serves many important functions in fermentedsausages where it contributes to taste texture microbio-logical safety and overall acceptability High sodium ionconsumption (gt2 gNa+day equivalent to 5 g salt (NaCl)day)contributes to high blood pressure and increase of the riskof heart disease and stroke [20] Most people consume toomuch salt on average 9ndash12 grams per day or around twicethe recommended maximum level of intake The principalbenefit of lowering salt intake is a corresponding reductionin high blood pressure WHOMember States have agreed toreduce the global populationrsquos intake of salt by a relative 30by 2025 Reducing salt intake has been identified as one of themost cost-effective measures countries can take to improvepopulation health outcomes An estimated 25 million deathscould be prevented each year if global salt consumption werereduced to the recommended level Meat and meat productscontribute 21 to the sodium intake [21]

Fermented sausages contain high amounts of salt whichcontributes to the microbiological safety and shelf-life bybinding water andmaking it unavailable formicroorganismsSalt also has a profound impact on the technological prop-erties of the meat and thus on the sausage texture It facili-tates solubilisation of myofibrillar proteins increases bindingproperties of proteins to improve texture and increasesviscosity of meat batters [22] As the Na+ ions cause healthissues reducing the NaCl content andor replacing some ofit with other salts like KCl or CaCl

2has been investigated

Potassium ions can give a bitter taste which poses restrictionson to how much can be introduced in a product No changesin organoleptic characteristics of fermented sausages weredetected when KCl substitutions for NaCl were lower than40 [23] Corral et al observed the same for slow fermentingsausages fermented and dried at 10ndash12∘C for 57 days where16 of theNaCl was replacedwith KCl [24] Although a slightreduction in aroma development was detected the sausageswere judged to have the same overall quality as the controlswith 27 NaCl

Dos Santos et al produced fermented sausages with 50reduction of NaCl (125 gkg) sausages where 50 of theNaCl was substituted for KCl CaCl

2 or a 1 1 mixture of

KCl and CaCl2[25] A 50 NaCl reduction and a 50

substitution of the NaCl for KCl did not influence thefermentation and maturation process Sausages with CaCl

2

showed a decrease in pH an increase in 119886119908 and lower lactic

acid production Overall sensory acceptance decreased insausages with reduced sodium content However preferencemapping identified a group of consumers that existed forfermented sausages with 50 reduced NaCl substituted forKCl or a blend of KCl and CaCl

2 De Almeida et al produced

salami sausages with 60 reduction in NaCl and addingdifferent amounts of a 1 1 blend of KCl and CaCl

2[26] The

salt replacement mixtures did not affect the technologicalprocess but the sausages had lower acceptabilityThe authorssuggested to enhance the sensory perception by additionof spices and other flavor enhancers This strategy wassuccessfully used when sausages were produced with 25 or50 of their NaCl replaced with KCl and supplemented with2 yeast extract [27]The increased volatile compounds fromcatabolism of the yeast extract suppressed the sensory qualitydefects caused by KCl introduction KCl is considered safeand exhibits an antimicrobial activity similar to that of NaCl[28] Replacing some of the NaCl with KCl should thereforenot influence antimicrobial safety of the sausages Generalimplications of salt and sodium reduction on microbial foodsafety have been reviewed earlier [29]

33 Nitrite In addition to its important preservative effectnitrite is involved in development of the red curing colorformation and flavor development and acts as an antioxidant[30 31]

According to the Commission Regulation (EU) number11292011 nitrates (sodium nitrate E251 potassium nitrateE252) and nitrites (sodium nitrite E250 and potassiumnitrite E249) are listed as permitted food additives Max-imum dose authorized for use in cured meat products bythe EU is 300mgkg nitrate (for some products 250mgkgnitrate) and 150mgkg K-nitrite (or 150mgkg Na-nitrite)measured as ingoing amounts [32] Nitratemay be reduced byGram+ catalase+ cocci (GCC+) to nitrite in the meat Nitrateis less used nowadays and primarily employed in dry curedhams and dry sausages where long slow curing processesnecessitate a long-term reservoir for nitrite that is reducedto nitric oxide in several reactions which can then react withmyoglobin in themeat to give the red cured color [33ndash35] Fornitrite the residual amounts will vary with the formulationof the product especially if ascorbate (vitamin C) is added to


prevent oxidation and to improve the color of the productAccording to EFSA the ingoing amount of nitrite ratherthan the residual amount contributes to the inhibitory effectagainst microorganisms

Flavor is a complex stimulus involving taste odor textureand temperature The meat salt lactic acid and spices aremajor contributors to flavor Nitrite contributes to the curedmeat flavor Several experiments with bacon frankfurtersand hams produced with and without nitrite have beenreviewed [36]The results usually showed higher flavor scoresfor products produced with nitrite

The antioxidant properties of nitrite will inhibit develop-ment of rancid off-flavors [37]The antioxidant properties arecaused by nitrite being oxidized to nitrate by sequesteringoxygen which is then not available for oxidizing fatty acidsSimilarly nitrogen oxide can easily sequester oxygen andbe oxidized to NO

2[34] In addition the stable complexes

between nitrite-derived compounds and heme-bond ironinhibit the release of free Fe2+ which is therefore not availablefor initiation of lipid peroxidation [38] The antioxidantproperties of nitrites have also been partly explained by nitriteand dinitrogentrioxides reacting with unsaturated lipids toform nitro-nitroso derivatives and thus stabilizing the lipidsagainst peroxidation changes [39]

From a health perspective nitrates are relatively non-toxic but nitrites and nitrite metabolic compounds such asnitric oxide and N-nitroso compounds have raised concernsover potential adverse health effects [40] The InternationalAgency for Research on Cancer (IARC) has concluded thatnitrates and nitrites are probably carcinogenic to humansunder conditions favoring nitrosation where an NO groupis covalently bound to carbon sulphur oxygen or nitrogenatoms in an organic molecule During curing in acidicenvironment undissociated nitrous acid picks up a hydrogenion and splits off a water molecule The resulting positivelycharged nitrosonium ion may then react with amino groupsto form N-nitrosamines Some of these N-nitrosaminesare carcinogenic In meat the most relevant nitrosaminesare N-nitrosodimethylamine (NDMA) N-nitrosopiperidine(NPIP) and N-nitrosopyrrolidine (NPYR) Formation ofthese compounds is only possible when secondary amines arepresent pH must be lt55 and temperature must be gt130∘C(NPYR) or the productmust be stored for a long time at roomtemperature (NDMA NPYR) [38] N-nitrosamines can alsobe formed from biogenic amines In a survey of DFSs of bothNorth and South European types in BelgiumN-nitrosamineswere detected in 54 of 101 samples [41] The total amountremained below 55 120583gkg except in one sample with 14120583gkgNPIP was the most prevalent N-nitrosamine present abovelimit of detection in 28 of the sausages There was only alimited relation between N-nitrosamine content and residuallevel of NaNO

3and no relationship with NaNO

2level The

authors assumed that the amounts of N-nitrosamines werelow because the median concentrations of residual NaNO

2

and NaNO3levels were lower than 20mgkg in the screened

products EFSA refers to several surveys on residual levelsof nitrite in cured meat products [32] The range variedconsiderably but generally the average residue levels werelow For example in France 74 of raw dried cured meat

products tested were in the range 0ndash9mgkg In Germany116 samples of cured meat products were tested of which85 were below 20mgkg Some reduction of the total N-nitrosamine content in DFSs appeared to be possible throughthe addition of ascorbic acid [42] A large number of agricul-tural food products seafoods meat products vegetable oilssauces and seasonings contain N-nitrosamines in the range02 to a few 120583gkg [43] A benchmark dose methodology fordeveloping tolerable daily intakes (TDIs) has been developedbased on a large lifetime cancer dose-response study ofNDMA in drinking water given to rats [44] Taking intoaccount inter- and intraspecies differences a TDI range of 40to 93 ngkgday was calculated From these considerationsintake of NDMA from DFSs will generally be well below theTDI

Partly due to the health concerns in conjunction withnitrite there has been a growing popularity of cured meatsproduced as ldquonaturalrdquo and ldquoorganicrdquo without addition ofnitrate or nitrite [33 45 46]These ldquonatural curingrdquo processesconsisted of adding a natural source of nitrate along witha nitrate-reducing starter culture Most often the naturalsource was a concentrated vegetable extract of celery (Apiumgraveolens var dulce) with about 3 nitrate Sometimes theextracts are pretreated to convert the nitrate to nitrite beforeuse Others have been employing Swiss chard (Beta vulgarisvar cicla) powders This product contains 30 to 35 nitrateA benefit of this product comparedwith celery extracts is thatit contains no allergens

The World Health Organization estimates that the dailydietary intake of nitrate is usually between 40 and 172mg[47] A substantial amount of dietary nitrate comes viafruits and vegetables For example approximately 98 ofthe dietary intake of Swedish children originates from fruitsand vegetables and only 2 from cured meat products [48]In contrast dietary nitrite amounts to less than 20 ofthe daily nitrite exposure The remaining 80 results fromendogenous bioconversion of dietary nitrate to nitrite insaliva Humans generally consume 03 to 26mg nitrite eachday [47] Some reports estimate that cured meat contributes48 of the daily nitrite intake [49]

Nitric oxide is involved in regulation of blood pressureand in regulations of gastrointestinal respiratory and gen-itourinary tract functions and immunologic reactions [50]The basal level of nitrate in blood is around 2mgkg andthat of nitrite approx 100-fold lower [50] Lack of nitricoxide production can lead to a number of conditions likehypertension atherosclerosis and thrombosis and can beameliorated by dietary nitrite interventions [51] A numberof case control studies have been conducted worldwide todetermine if there is a link between gastric cancer and nitrateintake [49] No such link has been found Other studiestrying to link nitrates and nitrites consumption to brainesophageal and nasopharyngeal cancers have been inconclu-sive

In conclusion one might argue that the positive effects ofcuring are overwhelming against the small possibility of theformation of low doses of nitrosamines The intake of curingagents frommeat products is small in comparison with otherfoods [34]


34 Smoke Smoking is a traditional treatment of Northerntype fermented sausages and is part of the conservation toinhibit growth of molds and bacteria on the product surfaceIn addition smoking adds a desirable smoky flavor delayslipid oxidation and adds color from light lemon to darkbrown depending on the kind of smoldering wood and thetimetemperature regime of the process Smoke developsfrom the charring of wood usually beech oak alder hickoryor maple as well as fruit trees The wood is normally cutinto shavings or saw dust The thermal composition of thewood followed by oxidation generates hundreds of differ-ent compounds mainly H

2O CO CO

2 alcohols carbonyl

compounds carboxylic acids esters hydrocarbons nitrogenoxides and phenols [52 53] Most smoke compounds wouldnot be allowed by law to be added to foods in pure formhowever since the toxicity and concentration in the productsare very low smoking is generally regarded as safe Many ofthe phenols such as guaiacol and its derivatives cresol pyro-catechols and pyrogallol show high antimicrobial activityThe content and distribution of these compounds in smokedmeats are related to their solubility in lipid and water phasesof the products It is not yet possible to predict exactly theconcentration of smoke phenols that is necessary to inhibitbacteria The inhibitory concentration of smoke phenols forListeria monocytogenes is in the range of 10ndash100120583gg whichis in the same range as that found whenmini-salamis (20mmdiameter) were smoked with beech (35ndash75 120583gg) [54] Thedesirable smoky flavor is predominately from phenols suchas syringol 4-methylsyringol 4-allylsyringol guaiacol 4-methylguaiacol and trans-isoeugenol [52]

Some hydrocarbons formed in smoke are hazardous tohuman health namely the polycyclic aromatic hydrocarbons(PAHs)These are highly hydrophobic compounds consistingof two or more fused aromatic rings mainly of hydrogenand carbon atoms Compounds with four or more ringsare less volatile and adsorb on soot and other combustionparticles There are 15-16 PAHs that are considered by theIARC and the European Union due to their carcinogenic andmutagenic properties [55 56] They are classified as carcino-genic probably carcinogenic possibly carcinogenic and notclassifiable Benzo(a)pyrene (BaP) is the only compound inthe carcinogenic group Special attention has been given toa group of eight of the PAHs (PAH8) which were used inprevious cancer studies and in EFSAs risk evaluation [55]The PAH compounds convert to diol epoxides and bindcovalently to DNA and cause errors in replication mutationand tumor genesis BaP when administered by the oral routehas been reported to produce tumors of the gastrointestinaltract liver lungs and mammary glands of mice and rats andhas also been associated with several other cancers [57]

For nonsmokers the main source of PAH is foods Themedian dietary exposure across European countries wascalculated both for mean and for high dietary consumersand varied between 235 ngday (39 ngkg body weight (bw)per day) and 389 ngday (65 ngkg bw per day) respectivelyfor benzo(a)pyrene alone and 1168 ngday (195 ngkg bw perday) and 3078 ngday (513 ngkg bw per day) respectivelyfor PAH8 The two highest contributors to the dietaryexposure were cereals and cereal products and seafood and

seafood products A number of products contain PAHs withundetectable levels of BaPTheEFSA therefore concluded thatbenzo(a)pyrene is not a suitable indicator for the occurrenceof PAHs in food and one should rather use a specific groupof four (PAH4) or eight PAHs (PAH8) based on the availabledata relating to occurrence and toxicityThe EUCommissionhas in the Commission Regulation (EU) 8352011 establishedan upper limit of BaP and PAH4 for smoked meat andsmokedmeat products As of Sept 1 2014 the limit for BaP is2 120583gkg and the total amount of PAH4 is 12 120583gkg [58] Theaccumulation of PAHs in different smoked meat products isrelated very significantly to the parameters of smoking andthe kind of wood used for smoke generation and even on thelocation of the product in the kiln which affects the tempera-ture and the flow rate of the smoke [52] Codex AlimentariusCommission code of practiceCACRCP682009 specifies tenvariables that need to be controlled to minimize and preventPAH contamination of meat products during smoking [59]These variables are fuel type smoking or drying method(direct or indirect) smoke generation process (temperatureairflow friction versus smoldering liquid smoke) distancebetween the food and the heat source position of the food inrelation to the heat source fat content of the food durationof smoking and direct drying temperature during smokinganddirect drying cleanliness andmaintenance of equipmentand finally design of the smoking chamber and the equipmentused for smokeair mixture (which influences the smokedensity in the smoking chamber) The importance of thesefactors has been reviewed by Ledesma et al [53]

The content of PAHs in smoked meat products is usuallywell below the maximum level set by the EU Commission[52] The greatest amount of BaP is deposited on the meatproduct casing and only a minor fraction then migratesinto the product [53] The content of PAH in sausages willdepend on the type of casing used Both for dry fermentedPetrovska kolbasa sausages from Serbia and traditional DFSsfrom Portugal the PAH contamination level was lower whencollagen casings were used [60 61]

One option to reduce PAH in meat products is by usingliquid smoke This is an easier more rapid and repro-ducible process [53] Liquid smoke is produced by chillingand thereby condensing wood smoke The liquid smoke isthen refined and filtered to remove toxic and carcinogenicimpurities containing PAH Use of liquid smoke is thereforegenerally considered to be of less health concern thantraditional smoking

35 Starter Cultures In a traditional process for producingfermented sausages bacteria yeast and fungi contribute tovarious degrees to the final product However it is generallyaccepted that LAB play the most prominent role since theinitial acidification is essential both technologically and froma safety perspective [62] Low pH and organic acids willinhibit contaminant spoilage flora and potential pathogensand ensure preservation Acid conditions also aid in textureformation due to meat protein coagulation and in color for-mation through the reactions of nitrite and nitrogen monox-ide with myoglobin [62] Although LAB also contribute toaroma formation mainly through organic acid production


other bacterial groups appear to be more important Theseare the Gram-positive catalase-positive cocci (GCC+) inparticular the coagulase-negative staphylococci (CNS) CNSconvert amino acids and free fatty acids to potent aromacompounds essential for taste notes of fermented sausages Inaddition CNS also possess highly active nitrate reductase andcatalase which contribute to color formation by producingnitrite from nitrate [35] and the limitation of lipid oxidationthat may cause rancidity respectively [63 64] Traditionalproduction of fermented sausages is based on spontaneousfermentation that is endogenous microorganisms present inthe raw material will perform the microbial transformationof the material However it has long been known that betterreproducibility could be obtained by adding a small portionof a previous successful batch when starting a new the so-called ldquoback-sloppingrdquo technique [65] This is the forerunnerto the use of starter cultures that is the intentional additionof premade microbial cultures to a fermentation processeither single ormixed in order to control and standardize theprocess The first-generation starter cultures for fermentedsausages were developed in the 1940s in the USA Howeverthese were not based on the dominating microorganismsfound in spontaneous fermentation or even isolated frommeat but rather on their technological feasibility for exam-ple surviving freeze-drying and their fast acid productionrate These cultures primarily strains of the genera Pediococ-cus were useful for the particular products produced in theUSA that is ldquosummer sausagesrdquo with very short productionand maturation times [62] However they were less suitablefor products of the European tradition with longer fermen-tation and maturation times Research in the 1960s 1970sand 1980s also confirmed in many later studies revealed thatthese types of sausages were dominated by L sakei or therelated species L curvatus and to some degree L plantarum[62 66ndash68] The second-generation LAB starter culturesnow widely used are often based on these [69] Molecularcharacterization by for example genome sequencing andcomparative genomics has shown that strains of L sakeiisolated frommeat andmeat fermentation have evolved to beperfectly adapted to this particular environment [70ndash72] Lplantarum lacks this specific adaptation but is a fast-growinghighly flexible bacterium with the largest genome size ofthe lactobacilli Some specific nonstarter LAB (ldquohouse florardquo)strains of L plantarum have been shown to outcompete com-mercial starters based on L sakei or L curvatus in industrialsausage production [73] GCC+ strains were isolated fromfermented meat products in the early 1900s and their role inaroma formation and color stability was established in the1950s [2 69] They were subsequently suggested for use asstarter cultures for sausage production first as single culturesbut later mixed cultures were shown to be superior to both asingle GCC+ culture or a single LAB culture [2 63 64] Thesuccess of these mixed cultures is likely because they reflectthe course and dynamics of a spontaneous fermentationbetter than a single culture and thereby retain the aroma andtaste of the traditional products [63 74 75]TheGCC+ strainsmost often found in spontaneous fermentation and also usedas starters are CNS and belong to the species Staphylococcuscarnosus S xylosus and S saprophyticus [64 74]

Mold growth on the external surface of DFS is desirableon some types of fermented sausages in many Europeancountries especially around the Mediterranean but also infor example Hungary and BelgiumThe distinct grey-whitishappearance of these products is an attractive feature Inthe traditional manufacture of these products the processrelies on the fortuitous inoculation of the maturing sausagesby spores resident in the air The different factories havetheir own distinct ldquohouse florardquo which are adapted to theprocess and will eventually dominate the surface growthand ensure some reproducibility of the product quality Thesurface molds contribute to the taste and aroma of thesausages by lipolytic proteolytic and lactic acid oxidizingactivities enhance general quality parameters through oxy-gen consumption which counteract rancidity developmentand improve color The mold surface layer also modifies thedrying rate and thus prevents excessive drying of the sausages[76] The specific conditions prevailing on the sausagesurfaces for example temperatures from 10 to 20∘C andrelative humidity starting at 90ndash95 and decreasing duringthe ripening period select for certain genera of molds inparticular Penicillium and occasionally Aspergillus Commonspecies are P nalgiovense P chrysogenum and P nordicum[76ndash78] Mold starter cultures have been developed mostoften consisting of spores of P nalgiovense [78 79] The mainselection criteria for these cultures are their low potentialfor mycotoxin production (see below) and their ability tooutcompete the ldquohouse florardquo while retaining the ability toproduce sausages of acceptable taste aroma and appearance[76 78ndash80]

Fungal surface colonization of maturing sausages startswith salt and acid tolerant yeast species such asDebaryomyceshansenii However along with the decrease in 119886

119908 there

is generally a shift in the mycobiota towards molds [81]Although the role of yeasts in sausage fermentation is notequally well known as for bacteria or molds it can besignificant in some products [82 83] Lipolytic proteolyticand lactate oxidation activities account for this effect [81ndash83]Starter cultures containing D hansenii have been developedsometimes in combination with mold spores [81]

All starter cultures are by definition ldquofunctionalrdquo sincetheir activities contribute to the transformation of the rawmaterial and to the appearance and quality of the finalproduct However the description of a starter culture asldquofunctionalrdquo often pertains to one (or several) additionalfunction(s) beyond the normal properties of a starter cultureSeveral such additional functions have been described forexample properties that enhance food safety (see also below)or have a technological advantage [64] In recent years inaccordance with trends in consumer demands functionalityfor enhanced health properties has been studied Probioticstarter cultures have been one of the main themes in thisresearch [84] The term ldquoprobioticsrdquo was coined in the1950s as an antonym to ldquoantibioticsrdquo The term subsequentlydeveloped into a scientific concept and was defined asldquolive microorganisms that when administered in adequateamounts confer a health benefit on the hostrdquo by FAOWHOin 2001 This definition was later reinforced as adequateand sufficient [85] LAB especially bacteria belonging to


the genus Lactobacillus are recognized as common inhabi-tants of the human gastrointestinal tract and have receivedconsiderable attention in the last decades for their health-promoting properties and use as probiotics The use ofprobiotic strains in fermented products was first employedin the dairy industry and milk-based products are still themost common vehicles for delivery of probiotics [86] How-ever being products where LAB proliferate and dominatefermented sausages are also potential carriers for delivery ofprobiotic LAB strains [64 87 88] There are some significantchallenges in using fermented sausages as probiotic productsin comparison with dairy products The most important areas follows (i) the meat raw material is not sterilized orpasteurized before the fermentation process and a probioticbacterium must therefore be as competitive as any starterculture normally used for the fermentation to outcompetethe endogenous flora (ii) the mature sausage constitutesa harsh environment with low 119886

119908and containing salt and

nitrate thus survival of the probiotic after fermentationshould be validated (iii) the numbers of the probiotic aftermaturation and storage must be very high since the servingsize and daily consumption of fermented sausage product aregenerally less than a comparable dairy product and (iv) theprobiotic should produce an acceptable product with regardto taste and quality [89 90] There are two main alternativesin the research and development of probiotic fermentedsausages The first is to select strains based on their probioticproperties and subsequently investigate the suitability of thestrain(s) in the production of fermented sausages Usingthis strategy already commercial probiotic strains have beenstudiedThe perhaps most well-documented probiotic strainLactobacillus rhamnosusGG has been used in several studiesfor this purpose with varying success [91ndash94] Although theGG strain can perform the fermentation there seems to bea balance between inoculum size off-taste (due to excessiveacid) and enough survival in the finished product which isdifficult to achieve [94] Similar problems were encounteredusing another well-documented strain L plantarum 299v[95] A better outcomewas obtained with a new L rhamnosusstrain isolated from human intestine and with potentialprobiotic properties [95] The disadvantage of using such astrain is that it is not possible to use the wealth of previousdocumentation which a well-known strain might have inpromoting the product The second strategy that has beenused for developing probiotic meat products is to use strainsisolated from successful meat fermentation or even meatstarter cultures [73 96] Such strains have to be assessed forpotential probiotic properties but are usually well adaptedto the meat fermentation environment These strains willalso suffer from the fact that their probiotic propertieswill be poorly documented in comparison to well-knowndocumented strains There have been attempts to launchprobioticmeat products commercially in Germany and Japan[97] but the outcome in commercial terms is unclear Anobstacle in the development of probiotic products in generalis also that EFSA has so far rejected all health claims ofprobiotics using a very strict assessment in their approvalprocess [85 98]

4 Microbial Hazards Associated withFermented Sausages

Although historically considered as safe the characteristicsof DFSs can provide survival and even growth of certainpathogens in these products Surveys have shown the pre-sence of pathogenic Escherichia coli Salmonella Typhimu-rium Staphylococcus aureus and L monocytogenes in dryfermented sausages Clostridium botulinum and Toxoplasmagondii have also been reported as potential microbial risks forconsumers of DFSs

Pathogenic microorganisms can be introduced throughcontaminated raw materials or through cross-contaminationfrom equipment or personnel during processing or at retailConditions during sausage processing and pathogen char-acteristics determine the ability for pathogen growth andsurvival and also determine possible strategies for pathogenelimination to ensure product safety

41 E coli Pathogenic E coli belong to various pathotypeswith verocytotoxigenic E coli (VTEC) (synonymous toShigatoxigenic E coli (STEC)) predominantly associatedwithmeat VTEC strains produce Shiga-toxins 1 andor 2 Theymay carry different virulence factors responsible for varia-tions in clinical manifestations A subgroup of VTEC causingsevere infections of enterohemorrhagic colitis and possiblyhemolytic uremic syndrome (HUS) characterized by acuterenal failure and anemia is designated enterohemorrhagic Ecoli (EHEC)More than 150 different serotypes of VTEC havebeen associated with human diarrheal infections SerotypeO157H7 strains have been the most known disease causingVTECNon-O157 have emergedwith the serotypesO26O45O103 O111 O121 and O145 also known as the ldquobig sixrdquobeing most frequently associated with human disease [99]Rawmeat ingredients contaminated through the slaughteringprocess are regarded a primary source of VTEC in DFSsCattle are regarded a primary O157H7 VTEC reservoiralthough other animals such as sheep swine goat anddeer can also be carriers of VTEC In outbreaks caused bycontaminated DFSs VTEC serogroups of O157 O26 O111and O103 have been causative agents [6] Low cell numbers(10ndash1000) are sufficient to cause disease [100 101] and levelslower than 1 cell (EHEC O111NM) per 10 g were reportedin a salami outbreak from Australia Although growth ofpathogenic E coli during initial phases of fermented sausageproduction can occur combinations of low pH and 119886

119908inhibit

growth ofE coli in finished products [88] However extensivepathogen survival in finished products has been reported[102ndash105] Strategies for effective VTEC elimination in DFSsare a challenge for producers It has been suggested thatserotype O157H7 strains have enhanced tolerance to acidscompared to other serotypes and that this may have a rolein their capacity to cause outbreaks via low pH foods likeDFSs for example [106 107] However within this and otherserotypes strains variations in acid resistance exist The lowinfectious dose the serious outcome of EHEC infectionsand several reported outbreaks linked toVTECcontaminatedDFSs highlight VTEC as the most serious safety risk in DFSs


Effective strategies for VTEC reductionelimination duringthe whole farm to fork chain are therefore required

42 Salmonella Salmonella are important zoonotic patho-genswith high economic significance in animals andhumansAs foodborne pathogens the two S enterica serovars Epi-dermidis and Typhimurium are dominating among humancases Serovar Epidermidis is associated with eggs andpoultry while Typhimurium is linked to meat of pork andbovine origin [108] Most salmonellosis infections are self-limiting yet severe and life-threatening complications (egsepsis) can follow Infected animals are the primary sourceof Salmonella where transmission to environments andfoods likely occurs through fecal contamination and cross-contamination According to EFSA 28 of the samplestaken from minced meat and meat preparations from otherspecies than poultry intended to be eaten cooked testedpositive for Salmonella in the EU in 2010 [109] In foodssuch as minced meat and meat preparations intended to beeaten raw 18of sampleswere Salmonellapositive A coordi-nated approach has led to a significant reduction of humancases of salmonellosis in the EU in the last decade StillSalmonella were the most common causative agent of food-borne outbreaks reported in EU in 2013 [108] Salmonellahave been implicated in several outbreaks linked to con-sumption of DFSs where contaminated meat ingredients area common source Reported outbreaks seem to be dominatedby fermented sausages produced from pork meat contam-inated with 119878 Typhimurium although other serovars (egMontevideo Goldcoast) have also been causative agents [110ndash113] The infectious dose can be low where 10ndash1000 cellsare sufficient to cause disease [114] Studies have shownSalmonella to be more sensitive than E coli O157H7 and Lmonocytogenes to at least certain DFS manufacturing processparameters [103 115 116] As for reduction of other pathogensuse of starter cultures has a positive effect on Salmonellareductions for example [117 118] Reported differences inSalmonella reductions are influenced by variations in recipesprocesses and strains and direct comparisons between stud-ies are difficult At higher contamination levels completeelimination through traditional processing is difficult

43 S aureus S aureus is common on skin and mucosalmembranes of humans with estimates of 20ndash30 persistentand 60 for intermittent colonization [119] The bacteriumis also found on food animals S aureus produces a rangeof staphylococcal enterotoxins (SEs) of which some showemetic activity [120] SEs are amajor cause of food poisoningwhich typically occurs after ingestion of foods particularlymeat and dairy products that have been contaminated andstored at elevated temperatures where S aureus have grownand produced toxins Symptoms are of rapid onset due to thepreformed toxins in the food and include nausea and violentvomiting with or without diarrhea The disease is usuallyresolved within 24ndash48 hours Staphylococcal toxin SEA isthe most common cause of staphylococcal food poisoningworldwideThe SEs belong to a group of superantigen toxinswhich bypass conventional antigen recognition by interactionwith major histocompatibility complex class II molecules on

antigen presenting cells and with T-cell receptors on specificT-cells [121] SEs are also able to penetrate the gut liningand activate immune responses thereby leading to vomiting[122] The level of S aureus present in the foods causing dis-ease in an English survey ranged from no viable S aureusdetected to 15 times 1010 cfug with a median of 30 times 107 cfug[123]

S aureus does not compete well with the indigenousmicroorganisms in foods and will grow better in processedfoods where the competing flora has been destroyed forexample in products contaminated after a heat treatment orwhen the food process gives S aureus a selective advantageThis can be the case for cured meats since S aureus cantolerate high amounts of salt and grow down to 119886

119882= 086

S aureus is able to grow in a wide range of temperatures (7∘to 48∘C) with an optimum 37∘C and pH (4 to 10) with anoptimumof 6 to 7 [124]These characteristics enable S aureusto grow in a wide variety of foods

Although S aureus can tolerate high salt and low pHand is often implicated in meat outbreaks (ham pork andsausages) few incidences on food poisoning from fermentedsausages are reported [123 125ndash129] Outbreaks caused by Saureus are usually old of which some have been registered byCenter for Disease Control [130ndash134] S aureus is frequentlyfound in fermented sausages but generally at levels too lowto produce enterotoxin amounts sufficient to cause illnessAlthough S aureus can tolerate salt and nitrite it is a poorcompetitor under anaerobic conditions at low pH and lowtemperatures If sausages are fermented at no higher than25∘C for 2 to 3 days and the initial count of S aureus is below104 cfug the risk of enterotoxin formation is low [2] Forsemidry sausages fermentation up to 43∘C is common in theUS and a rapid pH drop during manufacture will ensureinhibition of S aureus Consequently the American MeatInstitute in 1982 specified themaximum time allowed to reachpH 53 [2] Apparently the use of appropriate process controlsand starter cultures has significantly reduced the incidenceof ldquosummer sausagesrdquo outbreaks of S aureus food poisoningsin the US [2] North Carolina State University Meat lab hasproposed in their HACCP program that to ensure safetyproducts should be fermented to pH 53 or below within 1200degree hours [135]

When chorizo was inoculated with S aureus and withouta starter culture and fermented at 30∘C the pathogen grewwell S aureus growth was however reduced by using starterculture lower fermentation temperature (20∘C) and higherconcentrations of spices nitrites nitrates and ascorbate[136] In addition no enterotoxin A was detected in the lattersausages after drying Both strategies using specific startercultures and starter cultures in combinationwith bacteriocinshave been shown to reduce the presence of S aureus [137ndash139] S aureus growth in Italian dry salami was affected by theinitial pH initial levels of S aureus lactic acid bacteria dayof fermentation and interactions between these parameters[140 141]

Other species of staphylococci (CNS) are frequentlyfound in foods Some are also used as starter cultures in DFSOf a set of 129 such different strains only one strain carriedan enterotoxin gene and 78 of the strains did not carry


decarboxylases for biogenic amine formation Although 78of the strains possessed at least one gene encoding antibioticresistance these CNS were considered to pose a low safetyhazard [142]

44 L monocytogenes Foods contaminated by L monocyto-genes can cause listeriosis infections varying from mild flu-like symptoms to life-threatening disease with a high fatalityrate in vulnerable populations Ready-to-eat (RTE) productsconsumed without prior heat treatments and containinghigher than 100 cellsg are considered to pose a direct risk tohuman health L monocytogenes is ubiquitous in nature [143]and contamination of DFSs can occur through contaminatedingredients preferably raw meat The important role ofcontaminated processing equipment and environments as asource ofListeria inDFSs has been indicated in several studies[144ndash147] Thus L monocytogenes are commonly found inDFSs with reported prevalence up to 40 [148] Prevalencein beef is usually in the range 0ndash10 but with generallyhigher prevalence reported on pork meat [149 150] Never-theless only one outbreak in Philadelphia USA in 19861987with possible epidemiological association to fermented meatis known Fermented sausages have been evaluated to beproducts of low to moderate risk associated with listeriosisThis is due to usually low levels of L monocytogenes inthese products and that a high minimum infectious dose(gt104 cells) is normally required for illness Some growthof L monocytogenes can occur in the initial phase of DFSprocessing but the combinations of low pH (53ndash46) and119886119908(le090) generally restrict growth of the bacterium in the

fermented sausage productsThe extent to whichDFSs can beconsidered safe is primarily dependent on the fermentationand drying process With the wide specter of fermentedsausages produced not all sausage recipes and processingconditions may ensure products where the levels of L mono-cytogenes are compliant with the microbial criterion of le100colony-forming units per gram [151] It is therefore importantfor the DFS producers to gather information on the safety oftheir products in terms of L monocytogenes contaminationand growth and implement processing parameters to assurefood safety

The effects of using starter cultures for increasedpathogen reductions have been shown in several studiesfor example [152ndash154] In general enhanced reductions wereobtained in products with low pH and low 119886

119908and stored

under ambient conditions [103 116 155] Reductions of Lmonocytogenes during fermentation and drying in fermentedsausages are dependent on many factors including straindifferences in their ability to tolerate and adapt to DFSconditions that are also dependent on recipe and processingconditions [147 156]

45 C botulinum C botulinum is a strictly anaerobic sporeforming bacterium Spores of C botulinum occur in thesoil and may enter the meat from contaminated hides Thebotulinum neurotoxins are produced in growing vegeta-tive cells after the spores have germinated The toxins cancause nausea vomiting fatigue dizziness dryness in mouthand throat paralysis of muscles double vision respiration

problems and death The toxins bind irreversibly to periph-eral nerve endings and block the release of nevrotransmittersAn overview over reported outbreaks associated with meatand fish has been given previously [157] The rapid alert sys-tem for food and feed (RASFF) for the years 2010ndash2015 doesnot report any outbreaks of C botulinum from fermentedsausages C botulinum that can affect man are often groupedinto proteolytic and nonproteolytic strains The proteolyticstrains are the most hardy ones and can grow down to a pHof 46 or at 10 NaCl and down to 119886

119908of 094 They also have

spores that can withstand boiling for extended periods Thecombination of low pH high NaCl and low 119886

119908ensures that

C botulinum will not grow in matured fermented sausage Inaddition nitrate or nitrite is added to the sausage batter toinhibit growth of C botulinum and other pathogens Nitrateis reduced by GCC+ in the batter to nitrite The mechanismbywhich nitrite inhibitsC botulinum is uncertain Nitrite hasbeen reported to inhibit the phosphoroclastic system of Cbotulinum [158] This could be of importance for inhibitingC botulinum the 2-3 initial days of sausage production wherethe water activity is high and before the fermenting lactic acidbacteria have lowered the pH

Hospital et al produced two types of Mediterraneanfermented sausages salchichon and fuet with final pH of 50and 52 respectively [159] 119886

119908was between 088 and 090

One batch contained the maximum ingoing dose allowed bythe EU 150mgkg NaNO

3and 150mgkg NaNO

2 They also

made sausages with 25 and 50 nitrate and nitrite reductionsand control sausages without nitratenitrite In no cases wastoxin production detected from spores added to the sausageseven though the conditions for growth of C botulinumremained acceptable for 8ndash12 days during manufacture Cellfree extracts from a meat isolate of Staphylococcus sciurihave been shown to inhibit C botulinum in vitro and mayshow some potential in inhibiting C botulinum in fermentedsausages [160]

The C botulinum concern in conjunction with curedproduct is more relevant for nonfermented products whichcould support growth than for fermented sausagesThe use ofnitrite in fermented sausages the conditions in the sausagesnot being able to support growth of the bacterium thenumber of C botulinum spores generally being very low ifpresent and the lack of registered outbreaks from fermentedsausages together point to a low risk of food poisoning fromthese products

46 Toxoplasma gondii T gondii is an obligate intracellularparasite which is widely distributed in the world Conven-tionally it is associated with handling cats and cat litterhowever Center for Disease Control and Prevention USAnow estimates that 50 of toxoplasmosis is foodborne andthat foodborne toxoplasmosis causes 327 deaths annuallyand is the leading cause of death from foodborne pathogensafter Salmonella in USA [161 162] Consuming undercookedmeat products has been considered the major risk factorHealthy adults generally have no symptoms whereas severeillness can occur in infected fetuses newborns immunocom-promised individuals and transplant patients Nitrite andnitrate spices low pH and cold storage have no effects on


the viability of T gondii cysts [163] The cysts do not survivefreezing for longer than 4 hours Using frozen meat for thesausage batterwill thus reduce the risk of infection RegardingDFS production duration of the fermentation is critical toT gondii survival Tissue cysts remain viable in fermentedsausages after 12 h of treatment even in presence of 2 curingsalt When fermented sausages were produced containingexperimentally contaminated goat meat no viable cysts weredetected in the final sausages after 12 days [164] These andother risk evaluations conclude that fermentation over longperiods reduces the risk of infection [163]

5 Other Microbiology Related Health andSafety Concerns

51 Biogenic Amines Biogenic amines (BAs) are basic non-volatile low-molecular weight nitrogenous compoundscommon in living organisms where they perform variousfunctions on for example the nervous gastric and intestinalsystems and on regulation of blood pressure [165] They areformed as a result of normal metabolic activities in humansanimals plants and microorganisms generally throughdecarboxylation of the corresponding amino acids BAs areof considerable food safety concern as they may be presentin various foods and when ingested in excessive amountsmay cause certain diseases or disease-like conditions dueto a disturbance of the normal physiological concentrationsSymptoms of intoxication include headaches flushes nau-sea cardiac palpitations and increased or decreased bloodpressure The most important BAs in foods are histamineputrescine cadaverine tyramine tryptamine phenylethy-lamine spermine and spermidine [166] Of these histamineand tyramine are themost toxic Presence of someof the otherBAs may enhance the effects of histamine or tyramine [166]Normal physiological concentrations of BAs are carefullyregulated in the human body For instance the amines canbe oxidized by monoamine oxidases (MAO) or diamineoxidases (DAO) Hypersensitivity for BAs in some humansmay be caused by decreased activity of these enzymes dueto deliberate inhibition (MAO inhibitor drugs) or geneticdisposition [167] Definitive toxicity levels or limits are there-fore difficult to determine [165] Amino acid decarboxylasesare the enzymes responsible for the formation of BAs Theseenzymes are widely present in spoilage microorganisms butalso ubiquitous in desirable microorganisms such as bacteriaimportant in fermented sausages that is LAB and CNS [168]

High levels of biogenic amines may occur in foods suchas fish fish products and fermented foods (meat dairysome vegetables beers and wines) Generally the potentialof BA formation increases with the protein content of theraw material as the breakdown of proteins provides theamino acid precursors for BAs Fish and cheese are the mostimplicated products in foodborne BA intoxication No casesof BA poisoning have implicated fermented sausages as thecause although measured amounts of BAs have in someinstances reached similar levels as in fish related outbreaks[165]

The most important BAs present in fermented sausagesof food safety concern are tyramine phenylethylamine and

histamine with tyramine usually being the most abundant[168] Contaminant Gram-negative enterobacteria andorpseudomonads present in the raw material are the mostimportant BA producers before the onset of the fermentationby LABHighBA content of food products is often consideredan indication of spoilage or hygiene failure in the handlingof the raw material [165 168] Good hygienic quality of themeat and a rapid pH reduction in the initial stage of thesausage production process are essential for inhibition andcontrol of BA production by these contaminants [169] Saltand nitrite tolerant Gram-positive bacteria such as LAB andCNS will initiate the fermentation and eventually dominatethe microflora Prominent tyramine producers among LABrelevant for sausage fermentation are L curvatus and manyenterococcal strains found in artisanal sausage manufacturein southern Europe [170] Histamine producers are veryrare among sausage LAB and histamine when present insausage is considered to be produced by mainly contami-nant enterobacteria [169] However specific strains of forexample L buchneri and L parabuchneri harbor the histidinedecarboxylase enzyme and are considered spoilage organismsin cheese [171 172] Although never dominating a sausage fer-mentation such lactobacilli may be present as contaminants[169] Other LAB relevant for sausage fermentation suchas L sakei and L plantarum are generally nonaminogenic[168 170 173] Amino acid decarboxylases are uncommonin the most common CNS relevant for sausage fermentationfor example Staphylococcus xylosus S saprophyticus and Sequorum [173] However occasional strains of S carnosus andS equorummay show BA production [142 173]

Different strategies have been investigated to controland minimize BA formation in fermented sausages Theaddition of specific inhibitory agents to the meat batter suchas wine [174] or plant essential oils [175] is an exampleSuch additions reduce the initial contaminating flora therebyreducing BA formation but may also change the producttaste and appearance Methods have been suggested for theremoval of BAs after their formation such as the use offermentative bacteria with amine oxidase activity [176] orthe use of gamma radiation [177] However such proceduresare considered inappropriate since it may disguise incidentsof hygienic malpractice andor spoilage [169] The generallyrecommended and most efficient way of reducing andorcontrolling BA formation in fermented sausages seems to bethe use of nonaminogenic starter cultures [165 168 175 178ndash182] The use of a LAB starter culture results in a morerapid pH decrease than a spontaneous fermentation therebyinhibiting contaminant Gram-negative bacteria and thusthe potential for BA formation at the initial stages of theprocess The dominance of nonaminogenic LAB during thefermentation ensures minimal BA production Nonamino-genic CNSwill contribute to the effectMixed cultures of bothnonaminogenic LAB and CNS have been shown to performbetter than single starters probably because each startercontrols and dominates different parts of the microflora [169178] To ensure dominance of the selected starters the useof so-called autochthonous starter cultures is recommended[168 173 183] These are bacterial strains isolated from theparticular products they subsequently should be used in


as starters Such starters are potentially better adapted toeach specific process than commercial cultures and will alsopreserve the quality and taste of the original product If com-mercial cultures remain the only option they should be testedfor performance since highly competitive nonstarter LABmay dominate the fermentation despite the use of starter cul-tures [73]

In conclusion the selection of starter cultures especiallyLAB for use in fermented sausage production should usethe absence of amino acid decarboxylase activity as a basiccriterion

52 Mycotoxins The surface colonization of dry fermentedsausages by fungi is nearly inevitableThe conditions are idealfor for example Penicillium species unless specific measuresare taken to minimize fungal growth such as mechanicalremoval or the use of dipping regimes with antifungalcompounds for example sorbate solutions Smoking mayalso inhibit the growth of fungi to some extent One ormore of these measures are often used in the NorthernEuropean especially Scandinavian tradition of fermentedsausage production where mold growth is undesirableHowever as mentioned mold growth on the surface is adesirable and characteristic feature of many products in somecountries A safety concern with regard to surface growth ofmolds on fermented sausages is mycotoxin production MostPenicillium species are capable of producing one or moremycotoxins [184 185] the most important being ochratoxinA (OTA) patulin citrinin cyclopiazonic acid and roquefor-tine In surveys of molds isolated from fermented sausagespotentially toxigenic Penicillium strains are commonly found[79 186] Actual production of mycotoxins in the productshas also been shown though to a lesser degree [79 80 187]P nalgiovense strains were early selected as starter culturesdue to their apparent low toxigenic potential and usefultechnological properties [78 188] This seems still to be thebest choice as more recent studies confirm low toxigenicpotential [76 79]

Fungal starter cultures alone may not always be able tooutcompete resident house flora which has adapted overlong time Other measures may be necessary to controlmycotoxin production OTA represents the most importantmycotoxin produced by different molds relevant for sausageproduction that isPenicillium strains [80]P verrucosum andP nordicum are capable of producing OTA when they growon the sausages surface during both ripening and storage[187] OTA is undesirable because it is classified by IARC intoldquoGroupBrdquo as amoleculewith possible carcinogenic activity inhumans [189] Ozonated air has been suggested as a methodfor preventing the growth of OTA producing molds [187]Protective yeast cultures (D hansenii and Saccharomycopsisfibuligera) were recently shown to inhibit OTA producingfungi in a fermented meat product [190] It is unclear ifthis technique can be applied to fermented sausages wherea mold coat is desired Another biocontrol approach is theuse of nontoxigenic molds producing small cysteine-richantifungal proteins (AFPs) These strains or the purifiedAFPs have been suggested as useful for controlling growthandmycotoxin production by toxigenic fungi on dry-ripened

foods [191 192] A more practical approach is to carefullychoose the environmental parameters during ripening espe-cially with regard to 119886

119908and temperature in order to favor

colonization of starter cultures against OTA producing fungi[193]

53 Antibiotic Resistance The growing level of resistance toantibiotics in bacteria presents a serious concern to humanand animal health and presents significant financial andsocietal costs Antibiotic resistance (AR) in food bacteria isof concern because they may act as reservoirs for AR genesEven if the relative amount of antibiotic resistant bacteriain a particular fermented food product may be low theabsolute number can nevertheless be significant because largeamounts of living bacteria are ingested when the food isconsumed Food bacteria may carry transferable AR whichcould be transferred to commensal or pathogenic bacteriain the gastrointestinal tract The presence of transmissibleAR genes should therefore be an important safety criterionin the selection of starter cultures [180] Enterococci aregenerally not used as starter cultures for fermented sausagesbut may be involved in spontaneous fermentation Entero-cocci have been thoroughly investigated with regard to ARbecause of their clinical significance AR is also frequentlydetected among food enterococci [194] Because enterococciharbor different gene transfer mechanisms (eg pheromone-responsive plasmids conjugative and nonconjugative plas-mids and transposons) theymay acquire these determinantsfromother enterococcal strains and transfer them to potentialpathogens [195] This represents a possible risk related to theuse of enterococci as probiotics or starter cultures [194 195]Thus no enterococcal strains are currently included in theQPS (qualified presumption of safety) list of EFSA (EuropeanFood Safety Authority) [196]

Lactobacilli have a long history of safe use in fermentedfood which supports their GRAS (generally recognized assafe) and QPS status granted by FDA (US Food and DrugAdministration) and EFSA respectively Many Lactobacillusspecies are intrinsically resistant to a number of antibioticsfor example streptomycin and vancomycin [180 197] How-ever transmissible AR has frequently been detected alsoin strains isolated from fermented sausages [180 198ndash200]Tetracycline resistance mediated by the tetM gene and theermB erythromycin resistance gene seem to be the mostcommon [180 199] In vitro experiments have shown thatAR determinants can be transferred from meat associatedLAB to other LAB and to pathogens [201 202] A similarpattern exists in CNS [180 203 204] showing that most ARgenes are shared in nearly all meat associated Gram-positivebacteria [180] This may reflect the (mis)use of antibiotics inanimal husbandry for decades leading to a large pool of ARgenes present in the microbial population spreading also tobacteria in the food chain [205] To minimize the potentialrisks associated with the intentional use of microorganismsin food (eg starter cultures andor probiotics) includingtransfer of AR EFSA has regulated the industrial use ofbacteria as starter cultures through the QPS system [196] Inaddition guidelines have been developed for assessing AR inrelevant strains [206]


6 Reduction of Microbial Hazards

Reported outbreaks anddisease history have shown thatmainmicrobial pathogens in DFSs include VTEC and SalmonellaAs a food safety hazard in DFS L monocytogenes is regardedless relevant although their presence throughout the man-ufacturing processes of DFS is well documented [207ndash210]Nevertheless L monocytogenes is a significant pathogenwhere its presence in ready-to-eat products is troublesomeIts elimination from DFS products is therefore importantStrategies for control and elimination of pathogens in DFSinclude optimization of recipe and process parameters andeventually use of postprocess treatments of finished sausagesto ensure safe products Several outbreaks caused by VTECcontaminated fermented sausages lead the US Food Safetyand Inspection Service to establish a lethality performancestandard requiring 5-log reduction of E coli during DFSprocessing In Canada a 5-log reduction is recommendedwhile in Australia the required reduction is 3-log units [211]

There are limitations in howmuch different parameters inrecipe and process can be varied without negatively affectingthe characteristics and sensory quality of these productsCombination of parameters in recipe and process accordingto the ldquohurdle conceptrdquo for optimal reduction of pathogenswhile maintaining the sensory quality of the products hasbeen one approach More recently the effects of more noveltechnologies for for example meat batter decontaminationand postprocess treatments of finalized DFS have beenevaluated [212]

An overview of reported processing and postprocessstrategies for elimination of pathogens inDFSwith particularfocus on VTEC is provided below

61 Reductions of Pathogens in Raw Meat Ingredients Con-taminated raw meat and possibly nonmeat ingredients canprovide important sources of VTEC and Salmonella Freezingof raw meat prior to be used in DFS production is notuncommon Bacteria in the meat can be damaged by afreezethaw process and this has been shown to provide anextra 05-1-log reduction of E coliO157H7 in the final salamiproduct [213] Another strategy commercially used in theUSA is heat treatments of raw meat ingredients by lacticacidndashhot water (80ndash90∘C) The process provided 36ndash39-log reductions of Salmonella and E coli O157 in final DFSthough with some negative sensory influences [214] Use ofhigh pressure processing (HPP) of meat trimmings for DFSaffected the physiochemical properties of the meat battersand had negative effect on the sensory properties of theDFS [215] Irradiation in the range 15ndash4 kGy of raw meatfatingredients prior to production of DFS delivered a 5-logreduction of E coliO157H7 but was less effective in reducingL monocytogenes [216 217] Irradiation resulted in productswith quality indicators closely resembling those of traditionaldry sausage [216 218]

62 Reductions of Pathogens through Changes in Recipe andProcess Parameters There are large variations in the reduc-tions of pathogenic E coli Salmonella and Listeria in dif-ferent processes and products of DFS This is expected due

to the broad range of DFS products varying in pH saltcontent 119886

119908 recipe and production process like fermentation

temperature and maturation time Parameters important forVTEC reductions have been reviewed previously [6 7]Reduction of VTEC in traditional production processes ofsalami pepperoni and some other types ofDFSwas generally1-2 log although some higher reductions were also reported[6] Comparable reductions are often reported for Salmonellawhile inactivation of Lmonocytogenes is generally lower typ-icallylt1 log [103 116 152 219ndash222] Reduced inactivation ofLmonocytogenes is probably due to their overall high toleranceto acid high salt and low 119886

119908environments [223] In several

studies ingredients or production parameters (ie nitriteconcentration fermentation temperature final pH degree ofdrying and ripening time) have been varied systematicallyto enhance the safety of DFS [6] Our group studied thepotential for VTEC reductions by combining recipe andprocess parameters within limits that would give acceptableproducts of two types of DFS salami and Morr [104 224]The factorial designed experiments showed that high levelsof salt and curing salt (NaCl and NaNO

2) and glucose

(lower final pH in the sausages) along with fermentation atelevated temperature provided enhanced VTEC reductionsHigh fat and large casing diameters gave the opposite effectThe importance of 119886

119908for VTEC reductions in DFS was

documented High and optimal fermentation temperaturewere important to ensure growth and activity of the starterculture with subsequent lactic acid production pH dropmoisture loss and 119886

119908reduction over time In line with

other studies approximately 3-log reductions were obtainedcompared to 15-log reductions for standard recipeDFS [104]Higher reductions have been reported but seem difficult toobtain within levels relevant to producing high quality DFS[6]

A meta-analysis of 44 separate studies investigated therelative effects of temperature pH and 119886

119908on the survival

of E coli during manufacture of fermented meats The studyindicated that temperature (fermentation maturation andstorage) accounted for 61 of the variability in the data whilepH and 119886

119908accounted for less than 8 [225] Similarly in

a meta-analysis including 13 studies on inactivation of Lmonocytogenes in fermented sausages temperature explained60 of the data variability while pH and 119886

119908explained only a

small part [226]The above studies show that elevated temperatures in

the range 25ndash47∘C although not lethal to E coli and Lmonocytogenes per se would be effective for pathogen inac-tivation in the processing of DFS under conditions wherethe bacteria are unable to grow Increased inactivation ofrelevant pathogens including VTEC L monocytogenes andSalmonella with increasing temperatures has been shownin several studies [102 103 222 225ndash227] For effectiveinactivation of pathogens it is crucial to obtain conditionspreventing pathogen growth (low pH 119886

119908) but once these

conditions have been reached it is the factors of time andtemperature that most dramatically improve the microbialsafety of the product Overall optimal combinations ofhurdles and control strategies during DFS processing couldenhance the safety of DFS but finished products could still


contain surviving pathogens No single parameter appearsto enhance VTEC reduction enough to entirely eliminatepathogens Consequently application of several measures toreduce risk should be taken

Changes in recipe or process parameters do not neces-sarily lead to enhanced reduction of pathogens For examplewhen semidry reduced fat (20 less than control) Italiansalami was spiked with E coli S Typhimurium and Lmonocytogenes the reductions during manufacturing weresimilar to those of other typical Italian salami [228]

Application of novel technologies combined with tra-ditional hurdles (eg low pH 119886

119908 and temperature) in

the production process of DFS also presents an interestingvenue for enhancing the quality and safety of fermentedmeat products [212] For optimal combinations of controlstrategies it is important to consider bacterial stress toleranceand cross-protection scenarios ranging frompossible antago-nistic to additive to synergistic effects that can be obtained bycombining different treatments and hurdles see for exampleGayan et al [229]

Overall optimal combinations of hurdles and controlstrategies during DFS processing could enhance the safety ofDFS however finished products may still contain survivingpathogens

63 Importance of Starter Cultures for Safety The importanceof using starter cultures for effective reduction and inactiva-tion of pathogens of E coli Salmonella and Listeria in DFS iswell documented [7 64 105 230] Different starter culturesmay vary in their abilities to reduce these pathogens [64153 231 232] Combinations of starters may give increasedreduction in E coli during sausage production [233 234]Theperformance of Lactobacillus sakei in sausage fermentationwas shown to be improved by heat cold and salt stressprior to inoculation [235] Selection criteria for lactic acidbacteria used as starter cultures in fermented sausage werereviewed by Ammor andMayo [236]The growing interest inartisanal products of fermented sausages has also identified aneed for the isolation and use of appropriate starter culturesthat could provide increased food safety and maintain thecharacteristics of such products These sausages are oftenproduced following traditional practice in small processingunits with no use of starter cultures and less control oftemperature and humidity during fermentation and ripeningcompared to industrial production [237 238]

The main preservative effect of starter cultures for fer-mented sausages is production of organic acids mainly lacticacid by LAB [239] It has long been recognized that LABmay produce additional antimicrobial compounds [240 241]Of these the bacteriocins have received the most attentionBacteriocins are antibacterial peptides or proteins that kill orinhibit the growth of closely related bacteria For many LABbacteriocins the inhibitory spectrum includes only otherLAB likely to be present in the same ecological niche thus giv-ing the bacteriocin producer a competitive advantage [242ndash244] However some LAB bacteriocins have a somewhatlarger spectrum of inhibition and may be active towardsa broader panel of Gram-positive bacteria including food-borne pathogens such as L monocytogenes Bacillus cereus

S aureus and different clostridia The use of bacteriocin-producing LAB as starters for fermented sausages thereforeshows potential for natural enhanced safety of these products[64 87 242] The so-called class IIa bacteriocins sometimesreferred to as ldquopediocin-likerdquo (after the first discovery of thisclass pediocin PA-1) are particularly potent against Listeriaspecies including L monocytogenes [245] Class IIa bacteri-ocins are relatively small amphiphilic peptides of 35ndash5 kDaand the mode of action is permeabilization of the cell mem-brane of susceptible cells mediated via a membrane-locatedreceptor protein [246] Production of class IIa bacteriocinsis a relatively common trait among LAB species relevant forfermented sausages that is L curvatus and L sakei [245 247ndash251] Bacteriocinogenic strains of these species have thereforebeen tested as starter cultures in several fermented sausageexperiments and their antilisterial effect has been evaluated[239 252ndash259] Generally bacteriocinogenic L curvatus andL sakei starters could reduce the L monocytogenes numbersto some degree in the finished product compared to con-trols with nonbacteriocinogenic cultures However the effectvaried between barely significant to a 2-log cfug reductiondepending on strain and recipe This rather modest effectcompared to the promising inhibitory potential as measuredin in vitro experiments can be explained by interaction ofthe bacteriocin with the sausage matrix for example fatadsorption or proteolytic degradation [260] Moreover thepotential for bacteriocin production by the producer strainmay be inhibited to some degree in the sausage environment[64] The most common LAB bacteriocins used for sausagessuch as those of class IIa also have some general drawbacksThey have no activity whatsoever on some of the mainpathogens relevant for the product Salmonella and EHEC[239] In addition L monocytogenes strains may developresistance to some bacteriocins especially class IIa at rela-tively high frequencies in vitro [241]Whether this occurs in afood product is currently unclear In conclusion bacteriocin-producing starters may enhance food safety to some degreebut can never replace good manufacturing practices [64]

64 Preservatives for Enhanced Safety The addition of vari-ous compounds with antibacterial effects has been evaluatedas ingredients inDFS for improved safetyMicroencapsulatedallyl isothiocyanate (AIT) at 500 ppm gave 475-log reduc-tions ofE coliO157H7 inDFS 28 days after processinggt3 logmore than control DFS [261] Deodorized mustard powdercontaining AIT as an antimicrobial ingredient provided 5-log reduction of E coli O157H7 28 days after processingwhen used at 4 in DFS [262ndash266] However mustard levelsneeded to cause the required inhibition of E coli O157H7reduced consumer acceptability of the sausages [265] Otheringredients tested include the use of lactoferrin [234] anddiacetyl [267] The former was shown to provide mainlynonlethal injury of E coli O157H7 while an extra 1-logreduction was obtained by addition of 300 ppm diacetyl tothe sausage batter The antibacterial activity of essential oilsfrom herbs and spices were recently demonstrated in DFS Atconcentrations of 0005 and 005 decreases of Salmonellaand L monocytogenes were gt2 log and significantly higherthan in control sausages However the sensory impact of


essential oils is a factor limiting their application in DFS[268]

65 Postprocessing Treatments Storage of DFS at elevatedtemperatures (ge20ndash25∘C) short-term heat treatments andfreezingthawing regimes are the most widely applied post-process measures In the review of Holck et al reductionsrates of E coliO157H7 showed large variations but generallyincreased with lower pH lower 119886

119908 and higher storage tem-

peratures [6] Storage at low temperatures (4∘C) for up to twomonths usually gives marginal reductions [105 227] whereasstorage at 20ndash25∘C may result in considerable reductions

Including a storage step at ambient temperatures inaddition to the production process itself may not be enoughto achieve the 5-log reduction required in some countriesHeat treatments may be effective to reduce the numbers ofpathogens in sausages also taking into account the fact thatE coli O157H7 show reduced tolerance to heat in low pHmeat products compared to higher pH meat products [269ndash271] Total reductions of gt5 log were obtained for severalcombinations of products and storagemild heat treatmentregimes More recent data from our group have shown heattreatments of 43∘C 24 h to provide gt5-log total reductionsfor 11 E coli strains including different VTEC serotypesSimilar reductions were obtained by freezing at minus20∘C for24 h combined with 1 month of storage at 20∘C [272] Higherresistance to heat has been observed for L monocytogenescompared to E coli and Salmonella in DFS [221] Others havereported that heat treatments providing gt5-log reductionsof E coli in Lebanon Bologna were sufficient for similarreductions of L monocytogenes [273] The studies illustratethat inactivation of L monocytogenes is dependent on thesame parameters as inactivation of E coli and Salmonella butthat lethal effects on pathogens are product dependent

Different freezingthawing and storagemild heat treat-ment regimes of DFS showed negligible sensory effects ontreated DFS [274] Other studies have reported variablequality and sensory effects ranging from unacceptable toimproved sensory scores due to heat treatments at highertemperatures (geapprox 50∘C) [105 221 275 276] Combina-tions of high temperature and reduced treatment times maybe regarded as most feasible in industrial production Opti-mal treatment regimes are likely to differ between productswith different characteristics [102 221 273 274 276]

High pressure processing (HPP) has been employed inmany areas of food production [277] In DFS products HPPhas potential for postprocess reduction or elimination ofL monocytogenes in the final products in compliance withthe requirements (9 CFR part 430 the Listeria Rule) forL monocytogenes control of such RTE products as issuedby FSIS HPP is recognized by the FDA as a method forachieving the 5-log VTEC reduction in DFS processing thatare required in USA [278] and Canada [279] DFS productshaving a texture that is less susceptible to changes duringHPPcompared to raw meat products are suitable for HPP TheDFS color is barely affected even at very high pressure levelsand the in-package pasteurization by pressure is an advantageas possible recontamination is avoided As a postprocessingmethod it also has the advantage that it can be performed

at low temperatures Several consumer trials have revealedthat the sensory quality of HPP treated RTE products ismaintained after a storage period [280ndash282] However therecan be some differences betweenHPP treated and nontreatedDFS during the storage period Raw meat ingredients areless suited for HPP treatments Omer et al found that theorganoleptic properties of DFS made fromHPP treated meattrimmings changed substantially and were less favored after2 weeks of storage compared with the nontreated ones [215]When frozen rawmaterials were used the sensory differencesbetween treated and nontreated samples were reduced

Very high pressure levels up to 600MPa are often usedfor DFS Several studies have shown high initial reductions ofmicroorganisms after HPP [280 282] Gill and Ramaswamyshowed that the E coliO157 numbers were reduced by greaterthan 4-log cfug by HPP (600MPa 3min) and remainedstatic after processing in Hungarian salami but increasedin All Beef salami during storage at 15∘C [280] They alsoshowed that increasing the holding time to up to 9mindid not give additional reductions In a HPP study ofNorwegian type DFS treatment at 600MPa for 10min gavereductions of 29-log cfug of E coliO103H25 and treatmentin cycles (600MPa for 200 s 3 cycles) gave a somewhat higherreduction of 33-log cfug [282] The same study showedthat elevated levels of dextrose NaCl and nitrite gave lowerreduction (27-log cfug) compared with the standard recipePorto-Fett et al tested treatments of DFS added pathogenswith several pressure levels between 483 and 600MPa for1ndash12min [220]The reduction varied from 16 to 58-log cfugdepending on pressure conditions and bacteria (Listeria Ecoli and Salmonella) During storage additional reductionswere observed for all bacteria tested

Differences in pathogen reductions obtained in the var-ious studies of pressurizing DFS can be related to variationin the recipe fermentation regime and water activity levelThe production process of DFS is shown to give a reductionof about 2-log cfug of VTEC [282] With the additionalreduction of 3-log cfug due to HPP this will provide thedesired 5-log reduction that is often required

7 Mathematical Models for PredictingSurvival of Pathogens in DFS

Predictive modeling has developed as an adjunct to tradi-tional microbiological techniques Essentially the survivalandor growth of an organism of concern may be pre-dicted on the basis of a mathematical relationship betweenmicrobial growth rate and environmental conditions [283]A large number of mathematical models to predict thepopulation kinetics of E coli and other bacteria in foodsare publically available such as the ComBase Predictor(CP) [284] the Pathogen Modelling Program (PMP) [285]and Meat and Livestock Australia (MLA) E coli inactiva-tion model in fermented meat [286] These models havelimitations as they primarily focus on the static effect of119886119908 NaNO

2 pH and temperature The MLA model con-

siders dynamic changes however only those related totemperature in the sausage environment during production[225 286] A simpler version of the MLA model calculates


the reduction of E coli as a function of temperature andtime during fermentation and maturation available athttpwwwfoodsafetycentrecomaufermenterphp Specifi-cally the inactivation of E coli O157H7 has been modeledas a function of pH and 119886

119908in Soudjouk-style fermented sau-

sages during the process of fermentation and drying avail-able at httpspmperrcarsusdagovPMPOnlineaspx [222]The software THERM predicts growth of E coli O157H7Salmonella and S aureus as a function of the time-temperature history of raw meat products [287]

A dynamic model to predict VTEC concentrationthroughout manufacturing and storage of fermented rawmeat sausages has been developed by Quinto et al [288]Themodel is implemented in a tool called E coli SafeFerment(EcSF) available at httpwwwifracuksafetyEcoliSafeFer-ment EcSF integrates growth probability of growth andthermal and nonthermal inactivation models to give thepredictions of VTEC concentration under constant or fluctu-ating environmental conditions The tool can be applied forthe evaluation of the impact of modifications interventionsor unexpected events during the manufacturing processandor storage period on VTEC survival Recently Gunviget al developed three models for predicting survival ofVTEC L monocytogenes and Salmonella taking into accountthe dynamics of the sausage environment and maturationof fermented sausages [289] Based on challenge experi-ments under production conditions of dried and semidriedsausages themodels covered dynamic changes related to var-ious pH decreases weight losses during maturation NaNO

2

concentrations and 119886119908 Their ldquoConFermrdquo tool is available in

a user-friendly interface at httpdmripredictdk Predictivemodels can be useful for estimating pathogen reductionhowever for processes within the ranges of the variables usedfor the development of the specific model They also needto be interpreted with caution due to their wide confidenceintervals of the fitted equations which corresponds to anuncertainty in predictions

8 Concluding Remarks

Fermented meats are unique products often with elementsof culinary heritage and identity The preservation role ofthe nutritious meat has become largely obsolete after theintroduction of the cold chain Yet fermented sausagesremain very popular and are produced in large amounts inan immense variety Fermented sausages comprise a relativelysmall fraction of the total meat consumption For examplein Germany the annual per capita consumption of fermentedsausages has been estimated to 45 kg which is 7 of thetotal meat consumption [2] Due to their high fat salt nitriteand smoke content health considerations are still relevantWe have discussed several health and microbiological issuesrelated to consumption of fermented sausages Additionalinformationmay be found in the book FermentedMeat Prod-ucts Health Aspects which considers the safety of fermentedmeat products through a whole food chain approach [290]

A topic not covered in the present review is the suspectedconnection between meat in itself and cancer A workinggroup of the IARC recently classified processed meat as

ldquocarcinogenic to humansrdquo and redmeat as ldquoprobably carcino-genic to humansrdquo for colorectal cancer appealing to criticallyconsider the future role of meat in a healthy diet Consid-erations around meat and cancer and possible mitigationstrategies have been summarized previously [291] Groupsof consumers claim personal health motives for reducingor banning the consumption of meat [292] A response tonegative perception related to meat products embraces aninnovation agenda [293] However the borderline betweeninnovation and tradition appears complex since traditionalproducts tend to be perceived more basic and natural [294]The benefits and risks associated with red and processedmeatconsumption should not necessarily cause dilemmas if thesemeats are produced to ensure optimal microbial safety andconsumed in moderate amounts as part of balanced diets[291]



Acknowledgments

The preparation of this paper was funded by grants financedby the Norwegian Research Council (Project 221663) and theResearch Levy on Agricultural Products (Project 262306)

References

[1] P M D C C Pereira and A F D R B Vicente ldquoMeat nutri-tional composition and nutritive role in the human dietrdquoMeatScience vol 93 no 3 pp 586ndash592 2013

[2] F K Lucke ldquoFermented sausagesrdquo inMicrobiology of FermentedFoods B J B Wood Ed vol 2 pp 441ndash483 Blackie AcademicProfessional London UK 1998

[3] H Safa P Gatellier A Lebert L Picgirard and P-S MiradeldquoEffect of combined salt and animal fat reductions on physico-chemical and biochemical changes during the manufacture ofdry-fermented sausagesrdquo Food and Bioprocess Technology vol8 no 10 pp 2109ndash2122 2015

[4] A Holck E Heir T Johannessen and L Axelsson ldquoNorthEuropean productsrdquo in Handbook of Fermented Meat andPoultry F Toldra Ed pp 313ndash320 Wiley Blackwell WestSussex UK 2nd edition 2015

[5] F Toldra Y H Hui I Astiasaran J G Sebranek and R TalonHandbook of FermentedMeat and Poultry Second edition 2014

[6] A L Holck L Axelsson T M Rode et al ldquoReduction ofverotoxigenic Escherichia coli in production of fermented sau-sagesrdquoMeat Science vol 89 no 3 pp 286ndash295 2011

[7] K J K Getty R K Phebus J L Marsden D Y C Fung andC L Kastner ldquoEscherichia coli O157H7 and fermented sau-sages a reviewrdquo Journal of Rapid Methods and Automation inMicrobiology vol 8 no 3 pp 141ndash170 2000

[8] F K Lucke ldquoFermented meatsrdquo in The Microbiological Safetyand Quality of Food B M Lund A C Baird-Parker and GV Gould Eds vol 1 pp 420ndash444 Aspen Publishers IncGaithersburg Md USA 2000

[9] D Demeyer M Raemaekers A Rizzo et al ldquoControl ofbioflavour and safety in fermented sausages first results of a


European projectrdquo Food Research International vol 33 no 3-4 pp 171ndash180 2000

[10] WHO 2015 Healthy diet Fact sheet N∘394 httpwwwwhointmediacentrefactsheetsfs394en

[11] FAO ldquoFats and fatty acids in human nutrition report of anexpert consultationrdquo in FAO Food and Nutrition Paper vol 1-180 Food and Agricultural Organisation 2008

[12] C Ruiz-Capillas M Triki A M Herrero L Rodriguez-Salas and F Jimenez-Colmenero ldquoKonjac gel as pork backfatreplacer in dry fermented sausages processing and qualitycharacteristicsrdquoMeat Science vol 92 no 2 pp 144ndash150 2012

[13] P R Sheard J D Wood G R Nute and R C Ball ldquoEffects ofgrilling to 80∘C on the chemical composition of pork loin chopsand some observations on theUKnational food survey estimateof fat consumptionrdquo Meat Science vol 49 no 2 pp 193ndash2041998

[14] Z Bolger N P Brunton J G Lyng and F J Monahan ldquoCom-minuted meat productsmdashconsumption composition andapproaches to healthier formulationsrdquo Food Reviews Interna-tional vol 33 no 2 pp 143ndash166 2016

[15] D Ansorena and I Astiasaran ldquoThe use of linseed oil improvesnutritional quality of the lipid fraction of dry-fermentedsausagesrdquo Food Chemistry vol 87 no 1 pp 69ndash74 2004

[16] N M Josquin J P H Linssen and J H Houben ldquoQuality cha-racteristics of Dutch-style fermented sausages manufacturedwith partial replacement of pork back-fat with pure pre-emulsified or encapsulated fish oilrdquo Meat Science vol 90 no1 pp 81ndash86 2012

[17] D A Koutsopoulos G E Koutsimanis and J G BloukasldquoEffect of carrageenan level and packaging during ripeningon processing and quality characteristics of low-fat fermentedsausages produced with olive oilrdquo Meat Science vol 79 no 1pp 188ndash197 2008

[18] M Alejandre C Poyato D Ansorena and I AstiasaranldquoLinseed oil gelled emulsion a successful fat replacer in dryfermented sausagesrdquoMeat Science vol 121 pp 107ndash113 2016

[19] M J Beriain I Gomez E Petri K Insausti and M V SarriesldquoThe effects of olive oil emulsified alginate on the physico-chemical sensory microbial and fatty acid profiles of low-saltinulin-enriched sausagesrdquo Meat Science vol 88 no 1 pp 189ndash197 2011

[20] WHO 2016 Salt reduction fact sheet httpwwwwhointmediacentrefactsheetsfs393en

[21] E Desmond ldquoReducing salt A challenge for themeat industryrdquoMeat Science vol 74 no 1 pp 188ndash196 2006

[22] R N Terrell ldquoReducing the sodium content of processedmeatsrdquo Food Technology vol 37 pp 66ndash71 1983

[23] J Gelabert P Gou L Guerrero and J Arnau ldquoEffect of sodiumchloride replacement on some characteristics of fermentedsausagesrdquoMeat Science vol 65 no 2 pp 833ndash839 2003

[24] S Corral A Salvador and M Flores ldquoSalt reduction in slowfermented sausages affects the generation of aroma activecompoundsrdquoMeat Science vol 93 no 3 pp 776ndash785 2013

[25] B A Dos Santos P C B Campagnol A G da Cruz M AMorgano RWagner andMA R Pollonio ldquoIs there a potentialconsumermarket for low-sodium fermented sausagesrdquo Journalof Food Science vol 80 no 5 pp S1093ndashS1099 2015

[26] M A De Almeida N D M Villanueva J S D S Pinto ESaldana and C J Contreras-Castillo ldquoSensory and physico-chemical characteristics of low sodium salamirdquo Scientia Agri-cola vol 73 no 4 pp 347ndash355 2016

[27] P C B Campagnol B A dos Santos R Wagner N N Terraand M A R Pollonio ldquoThe effect of yeast extract additionon quality of fermented sausages at low NaCl contentrdquo MeatScience vol 87 no 3 pp 290ndash298 2011

[28] E Bidlas and R J W Lambert ldquoComparing the antimicrobialeffectiveness of NaCl and KCl with a view to saltsodiumreplacementrdquo International Journal of Food Microbiology vol124 no 1 pp 98ndash102 2008

[29] P J Taormina ldquoImplications of salt and sodium reduction onmicrobial food safetyrdquo Critical Reviews in Food Science andNutrition vol 50 no 3 pp 209ndash227 2010

[30] M H Fooladi A M Pearson T H Coleman and R AMerkelldquoThe role of nitrite in preventing development of warmed-overflavourrdquo Food Chemistry vol 4 no 4 pp 283ndash292 1979


[32] EFSA Panel on Biological Hazards (BIOHAZ) ldquoOpinion of theScientific Panel on biological hazards (BIOHAZ) related to theeffects of NitritesNitrates on theMicrobiological Safety ofMeatProductsrdquo EFSA Journal vol 2 no 3 p 14 2004

[33] J G Sebranek and J N Bacus ldquoCured meat products withoutdirect addition of nitrate or nitrite what are the issuesrdquo MeatScience vol 77 no 1 pp 136ndash147 2007

[34] K-O Honikel ldquoThe use and control of nitrate and nitrite for theprocessing of meat productsrdquoMeat Science vol 78 no 1-2 pp68ndash76 2008

[35] W P Hammes ldquoMetabolism of nitrate in fermented meats thecharacteristic feature of a specific group of fermented foodsrdquoFood Microbiology vol 29 no 2 pp 151ndash156 2012

[36] J I Gray B MacDonald A M Pearson and I D MortonldquoRole of nitrite in cured meat flavor a reviewrdquo Journal of FoodProtection vol 44 no 4 pp 302ndash312 1981

[37] D P Cornforth ldquoRole of nitric oxide in treatment of foodsrdquo inNitric Oxide Principles andActions J R Lancaster Ed pp 259ndash287 Academic Press San Diego Calif USA 1996

[38] S AndreeW Jira K-H Schwind HWagner and F SchwageleldquoChemical safety of meat andmeat productsrdquoMeat Science vol86 no 1 pp 38ndash48 2010

[39] L A Freybler J I Gray AAsghar AM BoorenAM Pearsonand D J Buckley ldquoNitrite stabilization of lipids in cured porkrdquoMeat Science vol 33 no 1 pp 85ndash96 1993

[40] M Govari and A Pexara ldquoNitrates and nitrites in meatproductsrdquo Journal of theHellenic VeterinaryMedical Society vol66 no 3 pp 127ndash140 2015

[41] E De Mey K De Klerck H De Maere et al ldquoThe occurrenceof N-nitrosamines residual nitrite and biogenic amines incommercial dry fermented sausages and evaluation of theiroccasional relationrdquo Meat Science vol 96 no 1 pp 821ndash8282014

[42] L Li J Shao X Zhu G Zhou and X Xu ldquoEffect of plant poly-phenols and ascorbic acid on lipid oxidation residual nitrite andN-nitrosamines formation in dry-cured sausagerdquo InternationalJournal of Food Science and Technology vol 48 no 6 pp 1157ndash1164 2013

[43] J-E Park J-E Seo J-Y Lee and H Kwon ldquoDistribution ofseven N-nitrosamines in foodrdquo Toxicological Research vol 31no 3 pp 279ndash288 2015

[44] D J Fitzgerald and N I Robinson ldquoDevelopment of a tolerabledaily intake for N-nitrosodimethylamine using a modified


benchmark dose methodologyrdquo Journal of Toxicology and Envi-ronmental Health - Part A Current Issues vol 70 no 19 pp1670ndash1678 2007

[45] J G Sebranek A L Jackson-Davis K L Myers and N ALavieri ldquoBeyond celery and starter culture advances in naturalorganic curing processes in theUnited StatesrdquoMeat Science vol92 no 3 pp 267ndash273 2012

[46] A U Alahakoon D D Jayasena S Ramachandra and C JoldquoAlternatives to nitrite in processed meat up to daterdquo Trends inFood Science and Technology vol 45 no 1 pp 37ndash49 2015

[47] WHO Nitate and nitrite in drinking water Background docu-ment for development of WHO Guidelines for Drinking-waterQuality World Health Organisation Geneva Switzerland 2011

[48] K Larsson P O Darnerud N-G Ilback and L Merino ldquoEsti-mated dietary intake of nitrite and nitrate in Swedish childrenrdquoFood Additives and Contaminants - Part A Chemistry AnalysisControl Exposure and Risk Assessment vol 28 no 5 pp 659ndash666 2011

[49] D L Archer ldquoEvidence that ingested nitrate and nitrite arebeneficial to healthrdquo Journal of Food Protection vol 65 no 5pp 872ndash875 2002

[50] S Moncada and A Higgs ldquoThe L-arginine-nitric oxide path-wayrdquoThe New England Journal of Medicine vol 329 no 27 pp2002ndash2012 1993

[51] D K Parthasarathy andN S Bryan ldquoSodium nitrite the ldquocurerdquofor nitric oxide insufficiencyrdquo Meat Science vol 92 no 3 pp274ndash279 2012

[52] Z E Sikorski and I Sinkiewicz ldquoPrinciples of smokingrdquo inHandbook of FermentedMeat and Poultry F Toldra Ed pp 39ndash45 Wiley Blackwell West Sussex UK 2015

[53] E Ledesma M Rendueles and M Dıaz ldquoContamination ofmeat products during smoking by polycyclic aromatic hydro-carbons processes and preventionrdquo Food Control vol 60 pp64ndash87 2016

[54] A Hitzel M Pohlmann F Schwagele K Speer and W JiraldquoPolycyclic aromatic hydrocarbons (PAH) and phenolic sub-stances in cold smoked sausages depending on smoking condi-tions using smouldering smokerdquo Journal of Food Research vol1 pp 45ndash59 2012

[55] EFSA ldquoPolycyclic Aromatic Hydrocarbons in Food - ScientificOpinion of the Panel on Contaminants in the Food ChainrdquoEFSA Journal vol 6 no 8 p 724 2008

[56] L Singh J G Varshney and T Agarwal ldquoPolycyclic aromatichydrocarbonsrsquo formation and occurrence in processed foodrdquoFood Chemistry vol 199 pp 768ndash781 2016

[57] C R Daniel K L Schwartz J S Colt et al ldquoMeat-cookingmutagens and risk of renal cell carcinomardquo British Journal ofCancer vol 105 no 7 pp 1096ndash1104 2011

[58] EU Commission ldquoCommission regulation (EU) No 8352011of 19 August 2011 amending Regulation (EC) No 18812006 asregards maximum levels for polycyclic aromatic hydrocarbonsin foodstuffsrdquo Official Journal of the European Union p L 2152011

[59] CODEX Alimentarius Commission Code of Practice for TheReduction of Contamination of Food with Polycyclic AromaticHydrocarbons (PAH) from Smoking and Direct drying ProcessesCACRCP 68-2009 Codex Alimentarius Commission (CAC)2009

[60] S Skaljac L Petrovic T Tasic et al ldquoInfluence of smokingin traditional and industrial conditions on polycyclic aromatichydrocarbons content in dry fermented sausages (Petrovskaklobasa) from Serbiardquo Food Control vol 40 pp 12ndash18 2014

[61] A Gomes C Santos J Almeida M Elias and L C RoseiroldquoEffect of fat content casing type and smoking procedures onPAHs contents of Portuguese traditional dry fermented sau-sagesrdquo Food and Chemical Toxicology vol 58 pp 369ndash374 2013



[64] F Leroy J Verluyten and L de Vuyst ldquoFunctional meat startercultures for improved sausage fermentationrdquo International Jour-nal of Food Microbiology vol 106 no 3 pp 270ndash285 2006

[65] E Puolanne and E Petaja-kanninen ldquoPrinciples of meat fer-mentationrdquo in Handbook of Fermented Meat and Poultry FToldra Ed pp 13ndash17 West Sussex UK Wiley Blackwell 2ndedition 2015

[66] L Cocolin P Dolci K Rantsiou R Urso C Cantoni and GComi ldquoLactic acid bacteria ecology of three traditional fer-mented sausages produced in the North of Italy as determinedby molecular methodsrdquoMeat Science vol 82 no 1 pp 125ndash1322009

[67] T Aymerich B Martın M Garriga and M Hugas ldquoMicrobialquality and direct PCR identification of lactic acid bacteria andnonpathogenic staphylococci from artisanal low-acid sausagesrdquoApplied and Environmental Microbiology vol 69 no 8 pp4583ndash4594 2003

[68] C Fontana P S Cocconcelli and G Vignolo ldquoMonitoringthe bacterial population dynamics during fermentation of arti-sanal Argentinean sausagesrdquo International Journal of FoodMicrobiology vol 103 no 2 pp 131ndash142 2005

[69] P S Cocconcelli and C Fontana ldquoCharacteristics and appli-cations of microbial starters in meat fermentationsrdquo in MeatBiotechnology F Toldra Ed pp 129ndash148 Springer Sci BusinessMedia Berlin Germany 2008


[71] V G H Eijsink and L Axelsson ldquoBacterial lessons in sausagemakingrdquo Nature Biotechnology vol 23 no 12 pp 1494-14952005

[72] O L Nyquist A McLeod D A Brede L Snipen A Aakraand I F Nes ldquoComparative genomics of Lactobacillus sakeiwithemphasis on strains from meatrdquo Molecular Genetics and Geno-mics vol 285 no 4 pp 297ndash311 2011


[74] G Blaiotta C Pennacchia F Villani A Ricciardi R Tofalo andE Parente ldquoDiversity and dynamics of communities of coagu-lase-negative staphylococci in traditional fermented sausagesrdquoJournal of AppliedMicrobiology vol 97 no 2 pp 271ndash284 2004

[75] E Parente S Grieco and M A Crudele ldquoPhenotypic diver-sity of lactic acid bacteria isolated from fermented sausagesproduced in Basilicata (Southern Italy)rdquo Journal of AppliedMicrobiology vol 90 no 6 pp 943ndash952 2001

[76] E Berni ldquoMoldsrdquo inHandbook of Fermented Meat and PoultryF Toldra Ed pp 147ndash153 Wiley Blackwell West Sussex UK2nd edition 2014


[77] S J Andersen ldquoCompositional changes in surface mycofloraduring ripening of naturally fermented sausagesrdquo Journal ofFood Protection vol 58 no 4 pp 426ndash429 1995

[78] L Leistner ldquoMould-fermented foods Recent developmentsrdquoFood Biotechnology vol 4 no 1 pp 433ndash441 1990


[80] L Iacumin L M Chiesa D Boscolo et al ldquoMoulds and ochra-toxin A on surfaces of artisanal and industrial dry sausagesrdquoFood Microbiology vol 26 no 1 pp 65ndash70 2009

[81] M Selgas and M Garcia ldquoYeastsrdquo in Handbook of FermentedMeat and Poultry F Toldra Ed pp 139ndash146 Wiley BlackwellWest Sussex UK 2nd edition 2015

[82] J-P Encinas T-M Lopez-Dıaz M-L Garcıa-Lopez A Oteroand B Moreno ldquoYeast populations on Spanish fermented sau-sagesrdquoMeat Science vol 54 no 3 pp 203ndash208 2000



[85] C Hill F Guarner G Reid et al ldquoExpert consensus documentthe International Scientific Association for Probiotics and Pre-biotics consensus statement on the scope and appropriate useof the term probioticrdquo Nature Reviews Gastroenterology ampHepatology vol 11 no 8 pp 506ndash514 2014

[86] M Fernandez J A Hudson R Korpela and C G De LosReyes-Gavilan ldquoImpact on human health of microorganismspresent in fermented dairy products an overviewrdquo BioMedResearch International vol 2015 Article ID 412714 2015


[88] K Incze ldquoDry fermented sausagesrdquoMeat Science vol 49 no 1pp S169ndashS177 1998

[89] L de Vuyst G Falony and F Leroy ldquoProbiotics in fermentedsausagesrdquoMeat Science vol 80 no 1 pp 75ndash78 2008


[91] S Erkkila E Petaja S Eerola L Lilleberg TMattila-Sandholmand M-L Suihko ldquoFlavour profiles of dry sausages fermentedby selected novel meat starter culturesrdquo Meat Science vol 58no 2 pp 111ndash116 2001

[92] S Erkkila M-L Suihko S Eerola E Petaja and T Mattila-Sandholm ldquoDry sausage fermented by Lactobacillus rhamnosusstrainsrdquo International Journal of Food Microbiology vol 64 no1-2 pp 205ndash210 2001

[93] R Rubio A Jofre T Aymerich M D Guardia andM GarrigaldquoNutritionally enhanced fermented sausages as a vehicle forpotential probiotic lactobacilli deliveryrdquo Meat Science vol 96no 2 pp 937ndash942 2014

[94] R Rubio T Aymerich S Bover-Cid M D Guardia J Arnauand M Garriga ldquoProbiotic strains Lactobacillus plantarum299V and Lactobacillus rhamnosus GG as starter cultures forfermented sausagesrdquo LWT - Food Science and Technology vol54 no 1 pp 51ndash56 2013

[95] R Rubio B Martın T Aymerich and M Garriga ldquoThe poten-tial probiotic Lactobacillus rhamnosus CTC1679 survives thepassage through the gastrointestinal tract and its use asstarter culture results in safe nutritionally enhanced fermentedsausagesrdquo International Journal of Food Microbiology vol 186pp 55ndash60 2014

[96] S Erkkila and E Petaja ldquoScreening of commercial meat startercultures at low pH and in the presence of bile salts for potentialprobiotic userdquoMeat Science vol 55 no 3 pp 297ndash300 2000

[97] K Arihara ldquoStrategies for designing novel functional meatproductsrdquoMeat Science vol 74 no 1 pp 219ndash229 2006

[98] J Glanville S King F Guarner C Hill and M E Sanders ldquoAreview of the systematic review process and its applicability foruse in evaluating evidence for health claims on probiotic foodsin the European Unionrdquo Nutrition Journal vol 14 no 1 articleno 16 2015

[99] FSIS ldquoShiga toxin-producingEscherichia coli in certain raw beefproductsrdquo 2011 Federal Register Dept of Agriculture USA 7658157

[100] N J C Strachan M P Doyle F Kasuga O Rotariu and ID Ogden ldquoDose response modelling of Escherichia coli O157incorporating data from foodborne and environmental out-breaksrdquo International Journal of Food Microbiology vol 103 no1 pp 35ndash47 2005

[101] J Tilden Jr W Young A-M McNamara et al ldquoA new route oftransmission for Escherichia coli infection from dry fermentedsalamirdquo American Journal of Public Health vol 86 no 8 I pp1142ndash1145 1996

[102] R Lindqvist and M Lindblad ldquoInactivation of Escherichia coliListeria monocytogenes and Yersinia enterocolitica in fermentedsausages during maturationstoragerdquo International Journal ofFood Microbiology vol 129 no 1 pp 59ndash67 2009

[103] H Nissen and A Holck ldquoSurvival of Escherichia coli O157H7Listeria monocytogenes and Salmonella Kentucky in Norwegianfermented dry sausagerdquo Food Microbiology vol 15 no 3 pp273ndash279 1998

[104] E Heir A L Holck M K Omer et al ldquoReduction of verotoxi-genic Escherichia coli by process and recipe optimisation in dry-fermented sausagesrdquo International Journal of FoodMicrobiologyvol 141 no 3 pp 195ndash202 2010

[105] M Calicioglu N G Faith D R Buege and J B LuchanskyldquoViability of Escherichia coli O157H7 during manufacturingand storage of a fermented semidry soudjouk-style sausagerdquoJournal of Food Protection vol 65 no 10 pp 1541ndash1544 2002

[106] K W Arnold and C W Kaspar ldquoStarvation-induced and sta-tionary-phase-induced acid tolerance in Escherichia-coli O157H7rdquo Applied and Environmental Microbiology vol 61 pp 2037ndash2039 1995

[107] T M Bergholz and T S Whittam ldquoVariation in acid resistanceamong enterohaemorrhagic Escherichia coli in a simulatedgastric environmentrdquo Journal of Applied Microbiology vol 102no 2 pp 352ndash362 2007

[108] EFSA ldquoThe European Union summary report on trends andsources of zoonoses zoonotic agents and food-borne outbreaksin 2013rdquo EFSA Journal vol 13 no 1 p 3991 2015

[109] EFSA and ECDC ldquoThe European union summary report ontrends and sources of zoonoses zoonotic agents and food-borneoutbreaks in 2010rdquo EFSA Journal vol 10 no 3 p 2597 2012

[110] C Pierre ldquoFoodborne outbreaksrdquo in Handbook of FermentedMeat and Poultry F Toldra Ed pp 435ndash439 Wiley BlackwellWest Sussex UK 2015


[111] K G Kuhn M Torpdahl C Frank K Sigsgaard and SEthelberg ldquoAn outbreak of Salmonella Typhimurium tracedback to salami Denmark April to June 2010rdquo Eurosurveillancevol 16 no 19 pp 13ndash16 2011

[112] V Bremer K Leitmeyer E Jensen et al ldquoOutbreak ofSalmonella Goldcoast infections linked to consumption offermented sausage Germany 2001rdquo Epidemiology and Infectionvol 132 no 5 pp 881ndash887 2004

[113] C M Gossner D van Cauteren S le Hello et al ldquoNation-wide outbreak of Salmonella enterica serotype 412I- infectionassociated with consumption of dried pork sausage FranceNovember toDecember 2011rdquoEurosurveillance vol 17 no 5 pp19ndash22 2012

[114] P F M Teunis F Kasuga A Fazil I D Ogden O Rotariu andN J C Strachan ldquoDose-responsemodeling of Salmonella usingoutbreak datardquo International Journal of Food Microbiology vol144 no 2 pp 243ndash249 2010

[115] N Chikthimmah and S J Knabel ldquoSurvival of Escherichia coliO157H7 SalmonellaTyphimurium and Listeriamonocytogenesin and on vacuum packaged Lebanon Bologna stored at 36 and130∘Crdquo Journal of Food Protection vol 64 no 7 pp 958ndash9632001

[116] K K Nightingale H Thippareddi R K Phebus J L MarsdenandA LNutsch ldquoValidation of a traditional Italian-style salamimanufacturing process for control of Salmonella and Listeriamonocytogenesrdquo Journal of Food Protection vol 69 no 4 pp794ndash800 2006

[117] L Coroller S Jeuge O Couvert S Christieans andM EllouzeldquoExtending the gamma concept to non-thermal inactivationa dynamic model to predict the fate of Salmonella during thedried sausages processrdquo FoodMicrobiology vol 45 pp 266ndash2752015

[118] B T Cenci-Goga P V Rossitto P Sechi S Parmegiani VCambiotti and J S Cullor ldquoEffect of selected dairy startercultures on microbiological chemical and sensory characteris-tics of swine and venison (Dama dama) nitrite-free dry-curedsausagesrdquoMeat Science vol 90 no 3 pp 599ndash606 2012

[119] J A Kluytmans and H F Wertheim ldquoNasal carriage of Staphy-lococcus aureus and prevention of nosocomial infectionsrdquo Infec-tion vol 33 no 1 pp 3ndash8 2005

[120] M A Argudın M C Mendoza and M R Rodicio ldquoFoodpoisoning and Staphylococcus aureus enterotoxinsrdquo Toxins vol2 no 7 pp 1751ndash1773 2010

[121] N Balaban andA Rasooly ldquoStaphylococcal enterotoxinsrdquo Inter-national Journal of Food Microbiology vol 61 no 1 pp 1ndash102000

[122] J W Shupp M Jett and C H Pontzer ldquoIdentification of atranscytosis epitope on staphylococcal enterotoxinsrdquo Infectionand Immunity vol 70 no 4 pp 2178ndash2186 2002

[123] A A Wieneke D Roberts and R J Gilbert ldquoStaphylococcalfood poisoning in theUnited Kingdom 1969ndash90rdquo Epidemiologyand Infection vol 110 no 3 pp 519ndash531 1993

[124] S R Tatini ldquoThermal stability of enterotoxins in foodrdquo Journalof Milk and Food Technology vol 39 no 6 pp 432ndash438 1976

[125] E P Casman ldquoStaphyloccal enterotoxinrdquo Annals of the NewYork Academy of Sciences vol 128 no 1 pp 124ndash131 1965

[126] F L Bryan ldquoRisks associated with vehicles of foodborne patho-gens and toxinsrdquo Journal of Food Protection vol 51 no 6 pp498ndash508 1988

[127] A Kerouanton J A Hennekinne C Letertre et al ldquoCharac-terization of Staphylococcus aureus strains associated with food

poisoning outbreaks in Francerdquo International Journal of FoodMicrobiology vol 115 no 3 pp 369ndash375 2007

[128] EFSA ldquoThe European union summary report on trends andsources of zoonoses zoonotic agents and food-borne outbreaksin 2009rdquo EFSA Journal vol 9 no 3 p 2090 2011

[129] J-A Hennekinne M-L De Buyser and S Dragacci ldquoStaphy-lococcus aureus and its food poisoning toxins characterizationand outbreak investigationrdquo FEMS Microbiology Reviews vol36 no 4 pp 815ndash836 2012

[130] Center for Disease Control ldquoGastroenteritis associated withsalamirdquoMorbidity andMortalityWeekly Report vol 20 pp 253ndash258 1971

[131] Center for Disease Control ldquoGastroenteritis associated withGenoa salamirdquoMorbidity and Mortality Weekly Report vol 20pp 261ndash266 1971

[132] Center for Disease Control ldquoStaphylococcal food poisoningassociated with Italian dry salamirdquo Morbidity and MortalityWeekly Report vol 24 pp 374ndash379 1975

[133] Center for Disease Control An Unusual Outbreak of Staphylo-coccal Food Poisoning Associated with Fermented Salami-UnitedStates Veterinary Public Health Notes CDC Atlanta Ga USA

[134] Center for Disease Control ldquoStaphylococcal food poisoningassociated with Genoa and hard salami United StatesrdquoMorbid-ity and Mortality Weekly Report vol 29 pp 179-180 1979

[135] NCSU Meat Lab Not Heat-Treated Shelf-Stable HACCP Pro-gram North Carolina State University Raleigh NC USA 2015

[136] M E Gonzalez-Fandos M Sierra M L Garcıa-Lopez M CGarcıa-Fernandez andAOtero ldquoThe influence ofmanufactur-ing and drying conditions on the survival and toxinogenesis ofStaphylococcus aureus in two Spanish dry sausages (chorizo andsalchichon)rdquoMeat Science vol 52 no 4 pp 411ndash419 1999

[137] G Kaban and M Kaya ldquoEffect of starter culture on growth ofStaphylococcus aureus in sucukrdquo Food Control vol 17 no 10 pp797ndash801 2006

[138] S Ananou M Maqueda M Martınez-Bueno A Galvez andE Valdivia ldquoControl of Staphylococcus aureus in sausages byenterocin AS-48rdquoMeat Science vol 71 no 3 pp 549ndash556 2005

[139] H Hampikyan ldquoEfficacy of nisin against Staphylococcus aureusin experimentally contaminated sucuk a Turkish-type fer-mented sausagerdquo Journal of Food Protection vol 72 no 8 pp1739ndash1743 2009

[140] J Metaxopoulos C Genigeorgis M J Fanelli C Franti andE Cosma ldquoProduction of Italian dry salami effect of starterculture and chemical acidulation on staphylococcal growth insalami under commercial manufacturing conditionsrdquo Appliedand Environmental Microbiology vol 42 pp 863ndash871 1981

[141] J Metaxopoulos C Genigeorgis M J Fanelli C Franti and ECosma ldquoProduction of Italian dry salami I initiation of staphy-lococcal growth in salami under commercial manufacturingconditionsrdquo Journal of Food Protection vol 44 no 5 pp 347ndash352 1981


[143] R B Tompkin ldquoControl of Listeria monocytogenes in the food-processing environmentrdquo Journal of Food Protection vol 65 no4 pp 709ndash725 2002

[144] E Giaouris E Heir M Hebraud et al ldquoAttachment and biofilmformation by foodborne bacteria in meat processing environ-ments causes implications role of bacterial interactions and


control by alternative novel methodsrdquoMeat Science vol 97 no3 pp 289ndash309 2014

[145] A S Gounadaki P N Skandamis E H Drosinos and G-J E Nychas ldquoMicrobial ecology of food contact surfaces andproducts of small-scale facilities producing traditional sausa-gesrdquo Food Microbiology vol 25 no 2 pp 313ndash323 2008

[146] V Ferreira J Barbosa M Stasiewicz et al ldquoDiverse geno-and phenotypes of persistent Listeria monocytogenes isolatesfrom fermented meat sausage production facilities in PortugalrdquoApplied and EnvironmentalMicrobiology vol 77 no 8 pp 2701ndash2715 2011

[147] D Thevenot M L Delignette-Muller S Christieans and CVernozy-Rozand ldquoPrevalence of Listeria monocytogenes in 13dried sausage processing plants and their productsrdquo Interna-tional Journal of Food Microbiology vol 102 no 1 pp 85ndash942005

[148] P Skandamis and G-J E Nychas ldquoPathogens risks and con-trolrdquo inHandbook of FermentedMeat and Poultry F Toldra Edpp 389ndash412 Wiley Blackwell West Sussex UK 2015

[149] D Thevenot A Dernburg and C Vernozy-Rozand ldquoAnupdated review of Listeria monocytogenes in the pork meatindustry and its productsrdquo Journal of Applied Microbiology vol101 no 1 pp 7ndash17 2006

[150] J R Rhoades G Duffy and K Koutsoumanis ldquoPrevalence andconcentration of verocytotoxigenic Escherichia coli Salmonellaenterica and Listeria monocytogenes in the beef productionchain A reviewrdquo Food Microbiology vol 26 no 4 pp 357ndash3762009

[151] EU Commission ldquoCommission regulation (EC) No 20732005of 15 November 2005 onmicrobiological criteria for foodstuffsrdquoOfficial Journal of the European Union no L 338 pp 1ndash26 2005

[152] K A Glass and M P Doyle ldquoFate and thermal inactivationof Listeria monocytogenes in beaker sausage and pepperonirdquoJournal of Food Protection vol 52 pp 226ndash231 1989

[153] E Lahti T Johansson T Honkanen-Buzalski P Hill and ENurmi ldquoSurvival and detection of Escherichia coliO157H7 andListeria monocytogenes during the manufacture of dry sausageusing two different starter culturesrdquo Food Microbiology vol 18no 1 pp 75ndash85 2001

[154] N Zdolec M Hadziosmanovic L Kozacinski Z Cvrtila andI Filipovic ldquoInfluence of protective cultures on Listeria mono-cytogenes in fermented sausages a reviewrdquo Archiv fur Lebens-mittelhygiene vol 59 pp 60ndash64 2008

[155] S C Ingham D R Buege B K Dropp and J A LosinskildquoSurvival of Listeria monocytogenes during storage of ready-to-eat meat products processed by drying fermentation andorsmokingrdquo Journal of Food Protection vol 67 no 12 pp 2698ndash2702 2004

[156] D Thevenot M L Delignette-Muller S Christieans and CVernozy-Rozand ldquoFate of Listeria monocytogenes in experi-mentally contaminated French sausagesrdquo International Journalof Food Microbiology vol 101 no 1-2 pp 189ndash200 2005

[157] A H W Hauschild ldquoEpidemiology of human foodborne botu-lismrdquo in Clostridium botulinum Ecology and Control in FoodsA H W Hauschild and K L Dodds Eds pp 69ndash104 MarcelDekker Inc New York NY USA 1993

[158] L F J Woods and J M Wood ldquoA note on the effect ofnitrite inhibition on the metabolism of Clostridium botulinumrdquoJournal of Applied Bacteriology vol 52 no 1 pp 109-110 1982

[159] X F Hospital E Hierro S Stringer and M Fernandez ldquoAstudy on the toxigenesis byClostridium botulinum in nitrate and

nitrite-reduced dry fermented sausagesrdquo International Journalof Food Microbiology vol 218 pp 66ndash70 2016

[160] M Sanchez Mainar R Xhaferi S Samapundo F Devlieghereand F Leroy ldquoOpportunities and limitations for the productionof safe fermented meats without nitrate and nitrite using anantibacterial Staphylococcus sciuri starter culturerdquo Food Controlvol 69 pp 267ndash274 2016

[161] E Scallan RMHoekstra F J Angulo et al ldquoFoodborne illnessacquired in the United Statesmdashmajor pathogensrdquo EmergingInfectious Diseases vol 17 no 1 pp 7ndash15 2011

[162] M B Batz S Hoffmann and J GlennMorrisRanking the RisksThe 10 Pathogen-Food Combinations with the Greatest Burden onPublic Health University of Florida Gainesville Fla USA 2011

[163] M Guo R L Buchanan J P Dubey et al ldquoQualitative assess-ment for Toxoplasma gondii exposure risk associated with meatproducts in the United Statesrdquo Journal of Food Protection vol78 no 12 pp 2207ndash2219 2015

[164] H Neumayerova J Jurankova A Salakova L Gallas KKovarcık and B Koudela ldquoSurvival of experimentally inducedToxoplasma gondii tissue cysts in vacuum packed goat meat anddry fermented goat meat sausagesrdquo Food Microbiology vol 39pp 47ndash52 2014

[165] EFSA Panel on Biological Hazards (BIOHAZ) ldquoScientific opin-ion on risk based control of biogenic amine formation infermented foodsrdquo EFSA Journal vol 9 no 10 article 2393 2011

[166] A R Shalaby ldquoSignificance of biogenic amines to food safetyand human healthrdquo Food Research International vol 29 no 7pp 675ndash690 1996

[167] G Spano P Russo A Lonvaud-Funel et al ldquoBiogenic aminesin fermented foodsrdquo European Journal of Clinical Nutrition vol64 pp 95ndash100 2010


[169] M Carmen Vidal-Carou M Teresa Veciana-Nogues M LuzLatorre-Moratalla and S Bover-Cid ldquoBiogenic amines risksand controlrdquo in Handbook of Fermented Meat and Poultry FToldra Ed pp 413ndash428Wiley BlackwellWest Sussex UK 2ndedition 2014

[170] S Bover-Cid M Hugas M Izquierdo-Pulido andM C Vidal-Carou ldquoAmino acid-decarboxylase activity of bacteria isolatedfrom fermented pork sausagesrdquo International Journal of FoodMicrobiology vol 66 no 3 pp 185ndash189 2001

[171] D Molenaar J S Bosscher B Ten Brink A J M Driessenand W N Konings ldquoGeneration of a proton motive force byhistidine decarboxylation and electrogenic histidinehistamineantiport in Lactobacillus buchnerirdquo Journal of Bacteriology vol175 no 10 pp 2864ndash2870 1993

[172] D Wuthrich H Berthoud D Wechsler E Eugster S IrmlerandR Bruggmann ldquoThehistidine decarboxylase gene cluster ofLactobacillus parabuchneriwas gained by horizontal gene trans-fer and is mobile within the speciesrdquo Frontiers in Microbiologyvol 8 article 218 pp 1ndash12 2017


[174] F Coloretti G Tabanelli C Chiavari et al ldquoEffect of wine addi-tion on microbiological characteristics volatile molecule pro-files and biogenic amine contents in fermented sausagesrdquoMeatScience vol 96 no 3 pp 1395ndash1402 2014


[175] S Lu H Ji Q Wang et al ldquoThe effects of starter cultures andplant extracts on the biogenic amine accumulation in tradi-tional Chinese smoked horsemeat sausagesrdquo Food Control vol50 pp 869ndash875 2015

[176] F Gardini M Martuscelli M A Crudele A Paparella and GSuzzi ldquoUse of Staphylococcus xylosus as a starter culture in driedsausages effect on the biogenic amine contentrdquo Meat Sciencevol 61 no 3 pp 275ndash283 2002

[177] J-H Kim H-J Ahn J-W Lee et al ldquoEffects of gamma irradia-tion on the biogenic amines in pepperoni with different pack-aging conditionsrdquo Food Chemistry vol 89 no 2 pp 199ndash2052005

[178] S Bover-Cid M Izquierdo-Pulido and M C Vidal-CarouldquoMixed starter cultures to control biogenic amine productionin dry fermented sausagesrdquo Journal of Food Protection vol 63no 11 pp 1556ndash1562 2000

[179] G Suzzi and F Gardini ldquoBiogenic amines in dry fermentedsausages a reviewrdquo International Journal of Food Microbiologyvol 88 no 1 pp 41ndash54 2003

[180] R Talon and S Leroy ldquoDiversity and safety hazards of bacteriainvolved in meat fermentationsrdquoMeat Science vol 89 no 3 pp303ndash309 2011

[181] X Wang H Ren W Wang and Z J Xie ldquoEffects of a starterculture on histamine reduction nitrite depletion and oxidativestability of fermented sausagesrdquo Journal of Food Safety vol 36no 2 pp 195ndash202 2016

[182] C Xie H-H Wang X-K Nie L Chen S-L Deng and X-LXu ldquoReduction of biogenic amine concentration in fermentedsausage by selected starter culturesrdquoCYTA - Journal of Food vol13 no 4 pp 491ndash497 2015

[183] N Z P Prpich M P Castro M E Cayre O A Garro and GM Vignolo ldquoAutochthonous starter culture selection to keeptraditions in the manufacture of dry sausages aliverdquo Annals ofMicrobiology vol 65 pp 1709ndash1719 2014

[184] J I Pitt and L Leistner ldquoToxigenicPenicillium speciesrdquo inMyco-toxins and Animal Foods J E Smith and R S Henderson Edspp 91ndash99 CRC Press Boca Raton Fla USA 1991

[185] M J Sweeney and A D W Dobson ldquoMycotoxin productionby Aspergillus Fusarium and Penicillium speciesrdquo InternationalJournal of Food Microbiology vol 43 no 3 pp 141ndash158 1998

[186] T-M Lopez-Dıaz J-A Santos M-L Garcıa-Lopez and AOtero ldquoSurface mycoflora of a Spanish fermented meat sausageand toxigenicity of Penicillium isolatesrdquo International Journal ofFood Microbiology vol 68 no 1-2 pp 69ndash74 2001

[187] L Iacumin S Milesi S Pirani G Comi and L M ChiesaldquoOchratoxigenic mold and ochratoxin a in fermented sausagesfrom different areas in northern italy occurrence reduction orprevention with ozonated airrdquo Journal of Food Safety vol 31 no4 pp 538ndash545 2011

[188] H Mintzlaff and L Leistner ldquoUntersuchungen zur Selektioneines technologisch geeigneten und toxikologisch unbeden-klichen Schimmelpilz-Stammes fur die Rohwurst-HerstellungrdquoZentralblatt fur Veterinarmedizin Reihe B vol 19 no 4 pp 291ndash300 1972

[189] IARC ldquoOchratoxin Ardquo in Some Naturally Occurring SubstancesFood Items and Constituents Heterocyclic Aromatic Amines andMycotoxinsMonographs on the Evaluation of Carcinogenic Risksto Humans pp 489ndash521 International Agency for Research onCancer Geneva Switzerland 1993

[190] L Iacumin M Manzano D Andyanto and G Comi ldquoBiocon-trol of ochratoxigenic moulds (Aspergillus ochraceus and Peni-cillium nordicum) by Debaryomyces hansenii and Saccharomy-copsis fibuligera during speck productionrdquo Food Microbiologyvol 62 pp 188ndash195 2017

[191] J Delgado R Acosta A Rodrıguez-Martın E Bermudez FNunez and M A Asensio ldquoGrowth inhibition and stability ofPgAFP from Penicillium chrysogenum against fungi commonon dry-ripened meat productsrdquo International Journal of FoodMicrobiology vol 205 pp 23ndash29 2015

[192] J Delgado R A Owens S Doyle M A Asensio and F NunezldquoAntifungal proteins frommoulds analytical tools and potentialapplication to dry-ripened foodsrdquo Applied Microbiology andBiotechnology vol 100 no 16 pp 6991ndash7000 2016

[193] A Rodrıguez D Capela A Medina J J Cordoba and NMagan ldquoRelationship between ecophysiological factors growthand ochratoxin A contamination of dry-cured sausage basedmatricesrdquo International Journal of Food Microbiology vol 194pp 71ndash77 2015

[194] J-C Ogier and P Serror ldquoSafety assessment of dairy microor-ganisms the Enterococcus genusrdquo International Journal of FoodMicrobiology vol 126 no 3 pp 291ndash301 2008

[195] C M A P Franz M E Stiles K H Schleifer and W HHolzapfel ldquoEnterococci in foods a conundrum for food safetyrdquoInternational Journal of Food Microbiology vol 88 no 2-3 pp105ndash122 2003

[196] EFSA Panel on Biological Hazards (BIOHAZ) ldquoScientific opin-ion on the maintenance of the list of QPS biological agentsintentionally added to food and feed (2013 update)rdquo The EFSAJournal vol 11 no 11 pp 3449ndash3555 2013

[197] J M Korhonen M Danielsen B Mayo et al ldquoAntimicrobialsusceptibility and proposed microbiological cut-off values ofLactobacilli by phenotypic determinationrdquo International Jour-nal of Probiotics and Prebiotics vol 3 no 4 pp 257ndash268 2008

[198] D Gevers L Masco L Baert G Huys J Debevere and JSwings ldquoPrevalence and diversity of tetracycline resistant lacticacid bacteria and their tet genes along the process line offermented dry sausagesrdquo Systematic and Applied Microbiologyvol 26 no 2 pp 277ndash283 2003


[200] C Devirgiliis P Zinno and G Perozzi ldquoUpdate on antibioticresistance in foodborne Lactobacillus and Lactococcus speciesrdquoFrontiers in Microbiology vol 4 article 301 pp 1ndash13 2013

[201] M Jahan and R A Holley ldquoTransfer of antibiotic resistancefrom Enterococcus faecium of fermented meat origin to Listeriamonocytogenes and Listeria innocuardquo Letters in Applied Micro-biology vol 62 no 4 pp 304ndash310 2016


[203] N Zdolec I Racic A Vujnovic et al ldquoAntimicrobial resis-tance of coagulase-negative staphylococci isolated from spon-taneously fermented sausagesrdquo Food Technology and Biotechnol-ogy vol 51 pp 240ndash246 2013

[204] E Marty C Bodenmann J Buchs et al ldquoPrevalence ofantibiotic resistance in coagulase-negative staphylococci fromspontaneously fermented meat products and safety assessment


for new startersrdquo International Journal of FoodMicrobiology vol159 no 2 pp 74ndash83 2012

[205] M J Fraqueza ldquoAntibiotic resistance of lactic acid bacteria iso-lated from dry-fermented sausagesrdquo International Journal ofFood Microbiology vol 212 pp 76ndash88 2015

[206] EFSA Panel on Additives Products or Substances Used inAnimal Feed (FEEDAP) ldquoGuidance on the assessment of bac-terial susceptibility to antimicrobials of human and veterinaryimportancerdquo EFSA Journal vol 10 no 6 article 2740 2012

[207] S Buncic L Paunovic and D Radisic ldquoThe fate of Listeriamonocytogenes in fermented sausages and in vacuum-packagedfrankfurtersrdquo Journal of Food Protection vol 54 no 6 pp 413ndash417 1991

[208] C M Hew M N Hajmeer T B Farver J M Glover and DO Cliver ldquoSurvival of Listeria monocytogenes in experimentalchorizosrdquo Journal of Food Protection vol 68 no 2 pp 324ndash3302005

[209] J Samelis and J Metaxopoulos ldquoIncidence and principalsources of Listeria spp and Listeria monocytogenes contami-nation in processed meats and a meat processing plantrdquo FoodMicrobiology vol 16 no 5 pp 465ndash477 1999

[210] A De Cesare R Mioni and G Manfreda ldquoPrevalence ofListeria monocytogenes in fresh and fermented Italian sausagesand ribotyping of contaminating strainsrdquo International Journalof Food Microbiology vol 120 no 1-2 pp 124ndash130 2007

[211] ldquoReview of processing requirements for uncooked comminutedfermented meat (UCFM) productsrdquo Food Standards AustraliaNew Zealand Canberra Australia pp 1ndash10 2002

[212] K S Ojha J P Kerry G Duffy T Beresford and B K TiwarildquoTechnological advances for enhancing quality and safety offermented meat productsrdquo Trends in Food Science and Techno-logy vol 44 no 1 pp 105ndash116 2015

[213] N G Faith N Parniere T Larson T D Lorang C W Kasparand J B Luchansky ldquoViability of Escherichia coli O157H7in salami following conditioning of batter fermentation anddrying of sticks and storage of slicesrdquo Journal of Food Protectionvol 61 no 4 pp 377ndash382 1998

[214] B Blagojevic D Antic B Adzic T Tasic P Ikonic and SBuncic ldquoDecontamination of incoming beef trimmings withhot lactic acid solution to improve microbial safety of resultingdry fermented sausages a pilot studyrdquo Food Control vol 54 pp144ndash149 2015

[215] M K Omer B Prieto E Rendueles et al ldquoMicrobiologicalphysicochemical and sensory parameters of dry fermentedsausages manufactured with high hydrostatic pressure pro-cessed raw meatrdquoMeat Science vol 108 pp 115ndash119 2015

[216] S C Johnson J G Sebranek D G Olson and B R WiegandldquoIrradiation in contrast to thermal processing of pepperoni forcontrol of pathogens effects on quality indicatorsrdquo Journal ofFood Science vol 65 no 7 pp 1260ndash1265 2000

[217] J Samelis A Kakouri I N Savvaidis K Riganakos and M GKontominas ldquoUse of ionizing radiation doses of 2 and 4 kGyto control Listeria spp and Escherichia coli O157H7 on frozenmeat trimmings used for dry fermented sausage productionrdquoMeat Science vol 70 no 1 pp 189ndash195 2005

[218] I Chouliara J Samelis A Kakouri et al ldquoEffect of irradiationof frozen meatfat trimmings on microbiological and physic-ochemical quality attributes of dry fermented sausagesrdquo MeatScience vol 74 no 2 pp 303ndash311 2006

[219] A C S Porto-Fett C-A Hwang J E Call et al ldquoViabilityof multi-strain mixtures of Listeria monocytogenes Salmonella

Typhimurium or Escherichia coli O157H7 inoculated into thebatter or onto the surface of a soudjouk-style fermented semi-dry sausagerdquo Food Microbiology vol 25 no 6 pp 793ndash8012008

[220] A C S Porto-Fett J E Call B E Shoyer et al ldquoEvaluationof fermentation drying andor high pressure processing onviability of Listeria monocytogenes Escherichia coli O157H7Salmonella spp and Trichinella spiralis in raw pork and Genoasalamirdquo International Journal of Food Microbiology vol 140 no1 pp 61ndash75 2010

[221] M Ducic N Klisara S Markov B Blagojevic A Vidakovicand S Buncic ldquoThe fate and pasteurization-based inactivationof Escherichia coli O157 Salmonella Typhimurium and Listeriamonocytogenes in dry fermented sausagesrdquo Food Control vol59 pp 400ndash406 2016

[222] C-A Hwang A C S Porto-Fett V K Juneja S C Ing-ham B H Ingham and J B Luchansky ldquoModeling the sur-vival of Escherichia coli O157H7 Listeria monocytogenes andSalmonella Typhimurium during fermentation drying andstorage of soudjouk-style fermented sausagerdquo InternationalJournal of Food Microbiology vol 129 no 3 pp 244ndash252 2009

[223] M Gandhi and M L Chikindas ldquoListeria a foodborne patho-gen that knows how to surviverdquo International Journal of FoodMicrobiology vol 113 no 1 pp 1ndash15 2007

[224] A McLeod I Mage E Heir L Axelsson and A L HolckldquoEffect of relevant environmental stresses on survival of entero-hemorrhagic Escherichia coli in dry-fermented sausagerdquo Inter-national Journal of Food Microbiology vol 229 pp 15ndash23 2016

[225] O J McQuestin C T Shadbolt and T Ross ldquoQuantificationof the relative effects of temperature pH and water activityon inactivation of Escherichia coli in fermented meat by meta-analysisrdquo Applied and Environmental Microbiology vol 75 no22 pp 6963ndash6972 2009

[226] M Mataragas K Rantsiou V Alessandria and L CocolinldquoEstimating the non-thermal inactivation of Listeria monocy-togenes in fermented sausages relative to temperature pH andwater activityrdquoMeat Science vol 100 pp 171ndash178 2015

[227] N G Faith N Parniere T Larson T D Lorang and J BLuchansky ldquoViability of Escherichia coli O157H7 in pepperoniduring the manufacture of sticks and the subsequent storage ofslices at 21 4 and - 20∘C under air vacuum and CO2rdquo Inter-national Journal of Food Microbiology vol 37 no 1 pp 47ndash541997

[228] E Dalzini E Cosciani-Cunico V Bernini et al ldquoBehaviour ofEscherichia coli O157 (VTEC) Salmonella Typhimurium andListeria monocytogenes during the manufacture ripening andshelf life of low fat salamirdquo Food Control vol 47 pp 306ndash3112015

[229] EGayan S KGovers CWMichiels andAAertsen ldquoSeverelyheat injured survivors of E coli O157H7 ATCC 43888 displayvariable and heterogeneous stress resistance behaviorrdquo Frontiersin Microbiology vol 7 article 1845 pp 1ndash8 2016


[231] S Fadda C Lopez and G Vignolo ldquoRole of lactic acid bacteriaduringmeat conditioning and fermentation peptides generatedas sensorial and hygienic biomarkersrdquoMeat Science vol 86 no1 pp 66ndash79 2010


[232] M Garriga B Marcos B Martın et al ldquoStarter cultures andhigh-pressure processing to improve the hygiene and safety ofslightly fermented sausagesrdquo Journal of Food Protection vol 68no 11 pp 2341ndash2348 2005

[233] K Pidcock G M Heard and A Henriksson ldquoApplication ofnontraditional meat starter cultures in production of Hungar-ian salamirdquo International Journal of Food Microbiology vol 76no 1-2 pp 75ndash81 2002

[234] P Muthukumarasamy and R A Holley ldquoSurvival of Escherichiacoli O157H7 in dry fermented sausages containing micro-encapsulated probiotic lactic acid bacteriardquo Food Microbiologyvol 24 no 1 pp 82ndash88 2007

[235] E Hufner and C Hertel ldquoImprovement of raw sausage fer-mentation by stress-conditioning of the starter organism Lacto-bacillus sakeirdquo Current Microbiology vol 57 no 5 pp 490ndash4962008


[237] O R D Santa R E F DeMacedo H S D Santa CM ZanetteR J D S Freitas andNN Tera ldquoUse of starter cultures isolatedfrom nativemicrobiota of artisanal sausage in the production ofItalian Sausagerdquo Food Science and Technology vol 34 no 4 pp780ndash786 2015

[238] A Roccato M Uyttendaele F Barrucci et al ldquoArtisanal Italiansalami and soppresse identification of control strategies tomanagemicrobiological hazardsrdquo FoodMicrobiol vol 61 pp 5ndash13 2017

[239] F-K Lucke ldquoUtilization of microbes to process and preservemeatrdquoMeat Science vol 56 no 2 pp 105ndash115 2000

[240] S E Lindgren and W J Dobrogosz ldquoAntagonistic activitiesof lactic acid bacteria in food and feed fermentationsrdquo FEMSMicrobiology Letters vol 87 no 1-2 pp 149ndash163 1991

[241] I F Nes M Kjos and D Diep ldquoAntimicrobial componentsof lactic acid bacteriardquo in Lactic Acid Bacteria Microbial andFunctional Aspects I F Nes M KJOS D Diep and A VonWright Eds pp 285ndash329 CRC Press Boca Raton Fla USA4th edition 2011

[242] L De Vuyst and E J Vandamme Bacteriocins of Lactic AcidBacteria Blackie Academic and Professional London UK1994

[243] D B Diep and I F Nes ldquoRibosomally synthesized antibacterialpeptides in gram positive bacteriardquoCurrent Drug Targets vol 3no 2 pp 107ndash122 2002

[244] V G H Eijsink L Axelsson D B Diep L S Havarstein HHolo and I F Nes ldquoProduction of class II bacteriocins by lacticacid bacteria an example of biological warfare and commu-nicationrdquo Antonie van Leeuwenhoek International Journal ofGeneral and Molecular Microbiology vol 81 no 1-4 pp 639ndash654 2002

[245] Y Cui C Zhang YWang et al ldquoClass IIa bacteriocins diversityand new developmentsrdquo International Journal of MolecularSciences vol 13 no 12 pp 16668ndash16707 2012

[246] D B Diep M Skaugen Z Salehian H Holo and I F NesldquoCommonmechanisms of target cell recognition and immunityfor class II bacteriocinsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 104 no 7 pp 2384ndash2389 2007

[247] A L Holck L Axelsson K Huhne and L Krockel ldquoPurifica-tion and cloning of sakacin 674 a bacteriocin fromLactobacillus

sake Lb674rdquo FEMS Microbiology Letters vol 115 no 2-3 pp143ndash149 1994

[248] F Leroy and L De Vuyst ldquoSakacinsrdquo in Natural Food Antimi-crobial Systems A S Naidu Ed pp 589ndash610 CRC Press BocaRaton Fla USA 2000

[249] F P Rivas M P Castro M Vallejo E Marguet and C ACampos ldquoSakacin Q produced by Lactobacillus curvatus ACU-1 functionality characterization and antilisterial activity oncooked meat surfacerdquoMeat Science vol 97 no 4 pp 475ndash4792014

[250] P S Tichaczek J Nissen-Meyer I F Nes R F Vogel and WP Hammes ldquoCharacterization of the bacteriocins curvacin Afrom Lactobacillus curvatus LTH1174 and Sakacin P from L sakeLTH673rdquo Systematic and AppliedMicrobiology vol 15 no 3 pp460ndash468 1992

[251] R Urso K Rantsiou C Cantoni G Comi and L CocolinldquoSequencing and expression analysis of the sakacin P bacte-riocin produced by a Lactobacillus sakei strain isolated fromnaturally fermented sausagesrdquo Applied Microbiology and Bio-technology vol 71 no 4 pp 480ndash485 2006

[252] M Hugas M Garriga M T Aymerich and J M MonfortldquoInhibition of listeria in dry fermented sausages by the bac-teriocinogenic lactobacillus sake CTC494rdquo Journal of AppliedBacteriology vol 79 no 3 pp 322ndash330 1995

[253] M Hugas B Neumeyer F Pages M Garriga and W P Ham-mes ldquoAntimicrobial activity of bacteriocin-producing culturesin meat products 2 comparison of the antilisterial potentialof bacteriocin-producing lactobacilli in fermenting sausagesrdquoFleischwirtschaft vol 76 no 6 pp 649ndash652 1996

[254] M Hugas F Pages M Garriga and J MMonfort ldquoApplicationof the bacteriocinogenic Lactobacillus sakei CTC494 to preventgrowth of Listeria in fresh and cooked meat products packedwith different atmospheresrdquo Food Microbiology vol 15 no 6pp 639ndash650 1998

[255] U Schillinger M Kaya and F-K Lucke ldquoBehaviour of Lis-teria monocytogenes in meat and its control by a bacterio-cin-producing strain of Lactobacillus sakerdquo Journal of AppliedBacteriology vol 70 no 6 pp 473ndash478 1991

[256] F Ravyts S Barbuti M A Frustoli et al ldquoCompetitivenessand antibacterial potential of bacteriocin-producing starter cul-tures in different types of fermented sausagesrdquo Journal of FoodProtection vol 71 no 9 pp 1817ndash1827 2008


[258] M de Souza Barbosa S D Todorov I Ivanova J-M ChobertT Haertle and B D G de Melo Franco ldquoImproving safetyof salami by application of bacteriocins produced by an auto-chthonous Lactobacillus curvatus isolaterdquo Food Microbiologyvol 46 pp 254ndash262 2015

[259] E H Drosinos M Mataragas S Veskovic-Moracanin JGasparik-Reichardt M Hadziosmanovic and D AlagicldquoQuantifying nonthermal inactivation of Listeria monocyto-genes in European fermented sausages using bacteriocinogeniclactic acid bacteria or their bacteriocins a case study for riskassessmentrdquo Journal of Food Protection vol 69 no 11 pp2648ndash2663 2006

[260] I M Aasen S Markussen T Moslashretroslash T Katla L Axelssonand K Naterstad ldquoInteractions of the bacteriocins sakacin P


and nisin with food constituentsrdquo International Journal of FoodMicrobiology vol 87 no 1-2 pp 35ndash43 2003

[261] P A Chacon PMuthukumarasamy andRAHolley ldquoElimina-tion of Escherichia coliO157H7 from fermented dry sausages atan organoleptically acceptable level of microencapsulated allylisothiocyanaterdquo Applied and Environmental Microbiology vol72 no 5 pp 3096ndash3102 2006

[262] R P Cordeiro F B Luciano and R A Holley ldquoEvaluationof deodorized yellow mustard concentrations for control ofEscherichia coli O157 H7 viability in dry fermented sausagerdquoFood Control vol 33 no 1 pp 20ndash24 2013

[263] R P Cordeiro C Wu and R A Holley ldquoContribution of endo-genous plantmyrosinase to the antimicrobial activity of deodor-ized mustard against Escherichia coliO157H7 in fermented drysausagerdquo International Journal of FoodMicrobiology vol 189 pp132ndash138 2014

[264] GHGraumann andRAHolley ldquoInhibition ofEscherichia coliO157H7 in ripening dry fermented sausage by ground yellowmustardrdquo Journal of Food Protection vol 71 no 3 pp 486ndash4932008

[265] S Li M Aliani and R A Holley ldquoSensory evaluation ofdry-fermented sausage containing ground deodorized yellowmustardrdquo Journal of Food Science vol 78 no 10 pp S1595ndashS1601 2013

[266] F B Luciano J Belland andRAHolley ldquoMicrobial and chemi-cal origins of the bactericidal activity of thermally treated yellowmustard powder toward Escherichia coli O157H7 during drysausage ripeningrdquo International Journal of Food Microbiologyvol 145 no 1 pp 69ndash76 2011

[267] D-H Kang and D Y C Fung ldquoEffect of diacetyl on controllingEscherichia coli O157H7 and Salmonella Typhimurium in thepresence of starter culture in a laboratory medium and duringmeat fermentationrdquo Journal of Food Protection vol 62 no 9 pp975ndash979 1999

[268] J Garcıa-Dıez J Alheiro A L Pinto et al ldquoBehaviour of food-borne pathogens ondry cured sausagemanufacturedwith herbsand spices essential oils and their sensorial acceptabilityrdquo FoodControl vol 59 pp 262ndash270 2015

[269] K R Ellajosyula S Doores E W Mills R A Wilson R CAnantheswaran and S J Knabel ldquoDestruction of EscherichiacoliO157H7 and SalmonellaTyphimurium in LebanonBolognaby interaction of fermentation pH heating temperature andtimerdquo Journal of Food Protection vol 61 no 2 pp 152ndash157 1998

[270] J Bacus ldquoProcessing procedures to control Salmonella and Ecoli in fermented sausage productsrdquo Food Australia vol 49 no11 pp 543ndash547 1997

[271] M Calicioglu N G Faith D R Buege and J B LuchanskyldquoViability of Escherichia coli O157H7 in fermented semidrylow-temperature-cooked beef summer sausagerdquo Journal of FoodProtection vol 60 no 10 pp 1158ndash1162 1997

[272] T M Rode A Holck L Axelsson M Hoslashy and E Heir ldquoShigatoxigenic Escherichia coli show strain dependent reductionsunder dry-fermented sausage production and post-processingconditionsrdquo International Journal of FoodMicrobiology vol 155no 3 pp 227ndash233 2012

[273] N Chikthimmah R B Guyer and S J Knabel ldquoValidation of a5-log10 reduction ofListeriamonocytogenes following simulatedcommercial processing of Lebanon bologna in amodel systemrdquoJournal of Food Protection vol 64 no 6 pp 873ndash876 2001

[274] E Heir A L Holck M K Omer et al ldquoEffects of post-proc-essing treatments on sensory quality and Shiga toxigenic

Escherichia coli reductions in dry-fermented sausagesrdquo MeatScience vol 94 no 1 pp 47ndash54 2013

[275] G Duffy D C R Riordan J J Sheridan et al ldquoDifferences inthermotolerance of various Escherichia coliO157H7 strains in asalami matrixrdquo FoodMicrobiology vol 16 no 1 pp 83ndash91 1999

[276] D C R Riordan G Duffy J J Sheridan R C Whiting I SBlair and D A McDowell ldquoEffects of acid adaptation productpH and heating on survival of Escherichia coli O157H7 inpepperonirdquo Applied and Environmental Microbiology vol 66no 4 pp 1726ndash1729 2000

[277] M Campus ldquoHigh pressure processing of meat meat productsand seafoodrdquo Food Engineering Reviews vol 2 no 4 pp 256ndash273 2010

[278] C ReedChallenge study Escherichia coliO157H7 in fermentedsausage Letter to plantmanagers 28April 1995 FSISUSDepart-ment of Agriculture Washington DC USA 1995

[279] Interim guidelines for the control of verotoxinogenicEscherichia coli including E coli O157H7 in ready to eatfermented sausages containing beef or a beef product as aningredient 2000 Guideline no 12 Food Directorate HealthProtection Branch Health Canada

[280] A O Gill and H S Ramaswamy ldquoApplication of high pressureprocessing to kill Escherichia coli O157 in ready-to-eat meatsrdquoJournal of Food Protection vol 71 no 11 pp 2182ndash2189 2008

[281] M M Hayman I Baxter P J OrsquoRiordan and C M StewartldquoEffects of high-pressure processing on the safety quality andshelf life of ready-to-eat meatsrdquo Journal of Food Protection vol67 no 8 pp 1709ndash1718 2004

[282] M K Omer O Alvseike A Holck et al ldquoApplication of highpressure processing to reduce verotoxigenic E coli in two typesof dry-fermented sausagerdquoMeat Science vol 86 no 4 pp 1005ndash1009 2010

[283] T A McMeekin J N Olley T Ross and D A Ratkowsky Pre-dictive Microbiology Theory and Application Research StudiesPress Somerset UK 1993

[284] J Baranyi and M L Tamplin ldquoComBase a common databaseon microbial responses to food environmentsrdquo Journal of FoodProtection vol 67 no 9 pp 1967ndash1971 2004

[285] Pathogen Modeling Program Version 70 Eastern RegionalResearch Center Wyndmoo Pa USA 2003

[286] T Ross L Mcqueen and P Vanderlinde PRMS021a PredictiveModel for the Reduction of E coli in Uncooked ComminutedFermented Meat Products Food Safety Meat amp LivestockAustralia North Sydney Australia 2004

[287] S C Ingham M A Fanslau G M Burnham B H Ingham JP Norback and D W Schaffner ldquoPredicting pathogen growthduring short-term temperature abuse of raw pork beef andpoultry products Use of an isothermal-based predictive toolrdquoJournal of Food Protection vol 70 no 6 pp 1446ndash1456 2007

[288] E J Quinto P Arinder L Axelsson et al ldquoPredicting the con-centration of verotoxin-producing Escherichia coli bacteriaduring processing and storage of fermented raw-meat sausagesrdquoApplied and Environmental Microbiology vol 80 no 9 pp2715ndash2727 2014

[289] A Gunvig C Borggaard F Hansen T B Hansen and SAabo ldquoConFerm a tool to predict the reduction of pathogensduring the production of fermented and matured sausagesrdquoFood Control vol 67 pp 9ndash17 2016

[290] N ZdolecFermentedMeat Products HealthAspects CRCPressBoca Raton Fla USA 2016


[291] S De Smet and E Vossen ldquoMeatmdashthe balance between nutri-tion and health a reviewrdquoMeat Science 2016

[292] C J S De Backer and L Hudders ldquoMeat morals Relationshipbetweenmeat consumption consumer attitudes towards humanand animal welfare and moral behaviorrdquo Meat Science vol 99pp 68ndash74 2015

[293] D J Troy and J P Kerry ldquoConsumer perception and the roleof science in the meat industryrdquoMeat Science vol 86 no 1 pp214ndash226 2010

[294] L Guerrero M D Guardia J Xicola et al ldquoConsumer-drivendefinition of traditional food products and innovation in tradi-tional foods A qualitative cross-cultural studyrdquoAppetite vol 52no 2 pp 345ndash354 2009

Research ArticleEffects of Ozone Treatments on the PhysicochemicalChanges of Myofibrillar Proteins from Silver Carp(Hypophthalmichthys molitrix) during Frozen Storage

Rongrong Zhang12 Shanbai Xiong134 Juan You13 Yang Hu13 Ru Liu13 and Tao Yin13

1College of Food Science and Technology Huazhong Agricultural University Wuhan Hubei Province 430070 China2School of Agriculture and Food Sciences Faculty of Science University of Queensland Brisbane QLD 4072 Australia3National RampD Branch Center for Conventional Freshwater Fish Processing Wuhan 430070 China4Collaborative Innovation Center for Efficient and Health Production of Fisheries Changde Hunan Province 415000 China

Correspondence should be addressed to Tao Yin yintaomailhzaueducn

Received 12 January 2017 Revised 24 February 2017 Accepted 16 March 2017 Published 13 April 2017


Copyright copy 2017 Rongrong Zhang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Physicochemical changes of myofibrillar proteins from silver carp surimi during frozen storage as affected by twomanners of ozonetreatments were investigated For preparation of surimi treated with ozone ozone water (8mgL) was used in either the first (To1)or the second (To2) cycle of rinsing As compared with control samples (Tc) (rinsing two cycles with water) myofibrillar proteinsfrom To1 surimi showed slightly lower free sulfhydryl contents and higher surface hydrophobicity throughout frozen storage andlower Ca2+-ATPase activities after 30 d To2 did not significantly (119875 gt 005) affect these physicochemical properties indicating thatmyofibrillar proteins structure was well maintained Consequently To1 significantly (119875 gt 005) decreased breaking force of surimigels while To2 did not significantly (119875 gt 005) affect gel breaking force In addition the whiteness of surimi gels was increasedmoreobviously by To2 than by To1 The results indicate that To2 could be used as a mild oxidation treatment for improving white colorof silver carp surimi without negatively affecting gel texture

1 Introduction

Silver carp (Hypophthalmichthys molitrix) is one of the mainfreshwater fish species farmed in China In 2014 totalproduction output was 423 million t [1] Utilization ofsilver carp has increased in recent years due to massiveoverexploitation of sea-water fish and the resulting shortageof raw material for frozen surimi The output of silver carpsurimi was estimated at about 30000 t in 2013 [2] and hasrapidly grown reaching over 40000 t in 2015 [3] Howeversilver carp surimi possesses an earthy-musty off-odour [4]which is generally thought to be associated with geosmin(GEO) and 2-methylisoborneol (MIB) [5] Furthermorewhiteness of silver carp surimi is inferior to that of fish speciesthat are traditionally used for high-quality surimi products[6] Consequently these defects negatively affect consumerperception of surimi products made from silver carp

Ozone which has regulatory approval and is generallyrecognized as environmentally friendly has been broadlyused in water treatment sanitization cleaning and disin-fection of equipment in off-odour removal and for pro-cessing various food products [7] Ozone also shows greatpotential for improving the quality of aquatic products withregard to shelf life sensory and so forth [8] Zhang et al[9] reported that about 42ndash6919 GEO in bighead carp(Hypophthalmichthys nobilis) meat was removed by ozonewater (33ndash76mgL) rinsing for 5ndash20min According to thestudy by Wang et al [4] the muddy flavours of silver carpsurimi were effectively eliminated after washing for 20minusing ozone water with an initial concentration of 096mgLIn addition ozone is an oxidant that possesses a bleachingeffect which helps to increase the whiteness of surimi andother aquatic products [10 11] Accordingly the defects of



silver carp surimi in sensory quality and color may bealleviated by applying appropriate ozone treatment

Myofibrillar proteins are the major components of surimiand are responsible for the formation of gel texture uponheating During the rinsing process of surimi productionoxidation of myofibrillar proteins by ozone treatment maycause the formation of intra- andor intermolecular disulfidebonds peptide bond cleavage amino acid residue modifica-tion unfolding of protein molecules and alternation in pro-tein functionality [7] These changes to protein functionalityultimately affect the textural properties of surimi gels

Recently researchers have begun to investigate the effectsof ozone treatment on the properties of surimi and surimiproducts [4 9 12 13] Zhang et al [9] reported that ozonewater treatment was a mild oxidation protocol to enhancethe functionality of myofibrillar proteins from bighead carpOzone treatment was found to significantly (119875 lt 005)increase salt solubility Ca2+-ATPase activity carbonyl con-tent sulfhydryl content and gel textural values of proteinswithout increasing peroxide values too much Howeverdeformation of mackerel surimi gels with ozone treatmentwas found to be significantly (119875 lt 005) lower than thatof control samples (without ozone treatment) and graduallydecreased with increased ozonation time [14] Xie et al [12]also found that textural values of silver carp surimi gelssignificantly (119875 lt 005) decreased after rinsing with ozonewater Textural properties of surimi gels as affected by ozonewater treatment therefore may be influenced by differencesbetween fish species andor ozonation conditions

Studies on the properties of fish myofibrillar proteinsor mince as affected by ozone water rinsing are limited Inthe majority of published literature fish myofibrillar proteinswere immediately heated to form a gel after being washedwith ozone water and then subjected to penetration test forevaluation of the ozone treatment on gel texture [4 9 12]Ozone treatment enhances unfolding of fish myofibrillarproteins which may contribute to stronger gel formationduring the heating step [11] However surimi is an interme-diate product that is typically mixed with cryoprotectantsand then subjected to a period of frozen storage priorto being manufactured into different products Thereforepartially unfolding proteins with ozone treatment beforefreezing may result in promoting protein aggregation duringstorage which ultimately results in a weaker surimi gel beingformed in the finished product [26] The impacts of ozone-induced oxidation on the physicochemical changes of fishmyofibrillar proteins during frozen storage however havenot been reported

Currently in the production of silver carp surimi it iscommon to use water mince ratios of 5 1 to 3 1 with tworinsing cycles The majority of the water-soluble proteinsprimarily sarcoplasmic protein and lipids are removed afterthe first rinsing cycle [15] Oxidization of fish myofibrillarproteins may be influenced by the presence or absenceof sarcoplasmic proteins and lipids [16 17] In order todetermine appropriate application of ozone treatment forimproving silver carp surimi quality this study investigatedthe application of ozone water in the first or second cycle of

rinsing on the physicochemical changes of fish myofibrillarproteins during frozen storage and subsequent gelation prop-erties


21 Materials Silver carp (Hypophthalmichthys molitrix)approximate 15 kg was obtained from a local fish farm(Wuhan China) Reagents used for SDS-PAGE were pur-chased from Bio-Rad (Hercules CA USA) Adenosine tri-phosphate(ATP) 5 5-dithiobis (2-nitrobenzoic acid) (DTNB)and 1-anilino-8-napthalenesulfonate (ANS) were purchasedfrom Sigma-Aldrich Trading Co Ltd (Shanghai China)Sugar and sodium tripolyphosphate were purchased fromGuangshengyuan Food Co Ltd (Wuhan China) and XingfaGroup Co Ltd (Wuhan China) respectively All otherchemicals were of analytical grade

22 Preparation of Surimi TreatedwithOzone Silver carpwasheaded gutted and thoroughly cleaned prior to deboningthe carcass by a roll-type fish meat separator (YBYM-6004-B Yingbo Food Machinery Co Ltd Xiamen China) Theobtained fish mince was subjected to two rinsing cycles witha water mince ratio and rinsing time at 4 1 and 10minrespectively A total of 3 rinsing treatments were conducted(1) two washing cycles using ice water only (Tc) (2) 1st and2nd cycle using ice water containing 8mgL ozone and icewater respectively (To1) (3) 1st and 2nd cycle using ice waterand ice water containing 8mgL ozone respectively (To2)Ozone water containing an initial concentration of 8mgLwas prepared according to the method by Zhang et al [11]using a corona discharge ozone generator (SY-SB40 ShengYa Co Ltd Xuzhou China) After rinsing fish mince waswrapped in cheesecloth and centrifuged (SS-300 RunxinMachinery Works Zhangjiagang China) at 15000 rpm toremove excess water The concentrated myofibrillar proteinswere mixed with cryoprotectants (6 sucrose and 03tripolyphosphate) vacuum packaged (sim600 g each bag) andstored in a freezer (minus18∘C) until used (0 7 15 30 60 and 90days) Room temperature during all of the aforementionedoperations was maintained below 10∘C

23 Extraction of Myofibrillar Proteins Myofibrillar proteinswere extracted from surimi according to the method ofPoowakanjana and Park [18] with slight modification Briefly1 g surimi was add to 29mL buffer (06M KCl 20mMTris-HCl and pH 70) and homogenized (FJ-200 ShanghaiSpecimen and Models Factory China) at 8000 rpm for1min The homogenate was centrifuged at 15000timesg (J-26XP Beckman Coulter Inc Fullerton CA USA) at 4∘Cfor 30min After centrifugation the supernatant was filteredand used for analyzing free sulfhydryl content Ca2+-ATPaseactivity and surface hydrophobicity as detailed below Proteinconcentration of the supernatant was measured using theLowry method [19]

24 Determination of Free Sulfhydryl Content Free sulfhydrylcontent was determined according to the method of Jianget al [13] using Ellmanrsquos reagent (DTNB) with some


modifications Protein concentration of the myofibrillarprotein sample as described above was diluted to 05mgproteinmL using 06M KCl in 20mM Tris-HCl buffer (pH70) The diluted sample (05mL) was mixed with 2mL of8M urea in 02mM Tris-HCl buffer (pH 70) and 50 120583L of01M sodium phosphate buffer (pH 70) containing 10mMDTNB and 02mM EDTA The mixture was incubated at40∘C for 15min before measuring absorbance at 412 nm(722 s Shanghai Precision and Scientific InstrumentCo LtdChina) Free sulfhydryl content was calculated using theextinction coefficient of 13600Mminus1 cmminus1 and expressed asmol per 105 g protein

25 Determination of Ca2+-ATPase Activity Determinationof Ca2+-ATPase activity was performed according to themethod of Benjakul et al [20] with some modifications Themyofibrillar protein sample (1mL) was mixed with 05mL of05M Tris-maleate buffer (pH 70) and 05mL of 01MCaCl

2

Deionized water was added to a total volume of 95mLSubsequently 05mL of 20mM ATP was added to initiatethe reaction The mixture was incubated at 25∘C for 8minand then terminated by adding 5mL of chilled trichloroaceticacid (15 g100mL) The reaction mixture was centrifuged at3500timesg for 5min and filtered Inorganic phosphate liberatedin the filtrate was measured by the method of Fiske and Sub-barow [21] Specific activity was expressed as 120583M inorganicphosphate (Pi) releasedmg proteinmin

26 Determination of Surface Hydrophobicity Surface hydro-phobicity was measured using ANS probe according to themethod of Poowakanjana and Park [18] with slight modifica-tion Protein concentration of the myofibrillar proteins wasdiluted to 01 02 03 and 05mg proteinmL using 06MKCl in 20mMTris-HCl buffer (pH 70)Then 4mL of samplewith different protein concentrationswasmixedwith 20120583L of01M phosphate buffer (pH 74) containing 8mM ANS andleft at room temperature for 10min Fluorescence intensitywas immediately measured using a spectrofluorometer (RF-1501 Shimadzu Kyoto Japan) with excitation and emissionwavelengths of 390 nm and 470 nm respectively The surfacehydrophobicity was calculated from the initial slope of the netrelative fluorescence intensity versus themyofibrillar proteinsconcentration

27 Protein Patterns The protein pattern of myofibrillarproteins at different storage times (0 7 15 30 60 and 90 days)was revealed using SDS-PAGE according to Laemmli [22]with some modifications The sample was homogenized (IkaT18 Cole-Parmer Co Ltd Shanghai China) at 10000 rpmfor 1min and solubilized using 5 sodium dodecyl sulfatesolution (90∘C) Solubilized proteins were centrifuged at17000timesg for 20min at room temperature Protein contentof the supernatant was measured using the Lowry method[19] Protein sample (25mgmL) was dissolved in Laemmli5x sample buffer with or without 120573-mercaptoethanol andfollowed by heating at 100∘C for 3min 120573-ME as a reducingagent was used to cleave the RSminusSR bonds of proteins in theSDS-PAGE analysis Stacking and separating gels were made

using 5 (wv) and 12 (wv) acrylamide respectively Eachlane was loaded with 10 120583g protein After running gels werefixed and stained with 0125 Coomassie brilliant blue R-250and destained in DI water containing 50methanol and 10acetic acid

28 Preparation of Surimi Gel Vacuum-packaged frozensurimi was removed at the respective storage time (0 7 1530 60 or 90 days) and partially thawed at room temperaturefor 40min before being cut into approximately 2 cm times 2 cm times4 cm cubesThe cubes were comminuted using a silent cutter(Multiquick 3 Braun Germany) at speed 3 for 30 sec Sodiumchloride (2) was added to extract myofibrillar proteinsMoisture content was adjusted to 78 using ice water (0∘C)The mixture was blended and ground in a stainless steelmortar using twin pestles (CA 1 Kinn ShangHoo IronWorksTaiwan) at an agitation speed of 45 rpm for 30min Finaltemperature of the paste was below 10∘C The paste was thenstuffed into a polyethylene sausage casing (25 cm diameter)using a sausage stuffer (Tre-mss7kh Trs Spade Italy) Bothends were sealed with U-shaped aluminum wire clips usinga clipper (Hk12 Hakanson Sweden) The sample was thenheated at 90∘C for 30min Cooked gels were immediatelysubmerged in ice water and then stored overnight in arefrigerator (4∘C)

29 Texture Analysis Gel strength of surimi gels was deter-mined by themethod described by Yin and Park [23] Chilledsurimi gels were equilibrated at room temperature (sim25∘C)for 2 h Samples were then cut into 25 cm cylinders andsubjected to the penetration test using a TA-XT textureanalyzer (Stable Micro Systems Surrey UK) equipped witha spherical probe (diameter 50mm and crosshead speed of1mms)

210 Color Measurement Color parameters Llowast (lightness)alowast (redness to greenness) and blowast (yellowness to blueness) ofthe surimi gels were measured using a CR-400 colorimeter(Konica Minolta Osaka Japan) Whiteness was calculatedaccording to the equation (L-3blowast) developed by Park [24] forsurimi gel

211 Statistical Analysis Analysis of variance (ANOVA) wasconducted using the SAS program (V8 SAS Institute IncCarry NC USA) Differences among mean values wereevaluated by the Duncan multiple range test (DMRT) using a95 confidence interval

3 Result and Discussion

31 Free Sulfhydryl Content Conversion of sulfhydryl groups(R-SH) into disulfide covalent bonds (RS-SR) and otheroxidized species through oxidation of sulfhydryl groupsor disulfide interchanges is generally considered a goodindicator for analyzing the radical-mediated oxidation ofproteins [7] As shown in Figure 1 the free sulfhydryl contentsof silver carp myofibrillar proteins with and without ozonetreatments decreased significantly (119875 lt 005) after storing


A

AA A

A A

A

A A A

A A

A

A A A

A A

TcTo1To2

7 15 30 60 900Storage time (day)

0

2

4

6

8

10

12Fr

ee su

lfhyd

ryl c

onte

nt (m

ol105g

pro)

Figure 1 Changes in free sulfhydryl content of myofibrillar proteinsextracted from silver carp surimi during frozen storage Light greytwo washing cycles using ice water only (Tc) dark grey first andsecond washing cycle using ice water containing 8mgL ozone andice water respectively (To1) black first and second washing cycleusing ice water and ice water containing 8mgL ozone respectively(To2) Different letters indicate significant difference among samples(Tc To1 and To2)

(minus18∘C) for 7 d The sulfhydryl contents remained constant(119875 gt 005) during frozen storage from day 7 to day 30 andthen continued to decrease (119875 lt 005) at day 60 Reductionof free sulfhydryl content resulted from the formation ofdisulfide covalent bonds (RS-SR) as evidenced by changesof the protein patterns (Figure 4(a)) Myofibrillar proteinsexhibited a reduction of about 23 24 24 and 62respectively in the sulfhydryl contents after 7 d 15 d 30 d and60 d of storage

Changes in the sulfhydryl content of silver carp pro-teins during frozen storage were similar to that of croakerthreadfin bream and bigeyes snapper as reported by Benjakulet al [20] Myofibrillar proteins are mainly composed ofmyosin (sim55) and actin (sim20) which contain about 42and 12 sulfhydryl groups respectively [25] The sulfhydrylgroups include active sulfhydryl groups on the surface and thehidden sulfhydryl groups in the protein interior Oxidizationof the active sulfhydryl groups on the surface reducedsulfhydryl content during early storage (lt7 d) Subsequentlymyofibrillar proteins unfolded during extended storage thussome of the original hidden sulfhydryl groups were exposedto the surface These exposed sulfhydryl groups were thenactivated which caused sulfhydryl content to decrease fur-ther (gt30 d)The decrease in the sulfhydryl content coincidedwith an increase in surface hydrophobicity (Figure 3) whichrepresents change in the tertiary structure of the proteinSurprisingly the surface hydrophobicity significantly (119875 lt005) increased (Figure 3) at day 90 while the sulfhydrylcontent was not significantly (119875 gt 005) changed This obser-vation may be due to the masking of sulfhydryl groups byaggregation of partially unfolded myofibrillar proteins [20]

Although cryoprotectants are mixed into surimi to maintainprotein structure during frozen storage fish myofibrillarproteins continue to gradually unfold and subsequentlyaggregate during frozen storage [26]

Before frozen storage (0 d) the free sulfhydryl contentof the samples in the descending order was TC To2 andTo1 respectively But the differential was not significant (119875 gt005) However Zhang et al [11] reported that the free sulf-hydryl content of myofibrillar proteins recovered from big-head carp decreased about 12 after rinsing with 76mgLozone water The reason might be due to a much longerrinsing time (20min) used in their study This order of thethree samples (Tc To2 To1) with regard to free sulfhydrylcontent wasmaintained throughout frozen storage Free sulf-hydryl content of To1 was lower than that of To2 howeverthe difference was not significant (119875 gt 005) It might berelated to the formation of lipid radicals and peroxide duringthe ozone water rinsing which enhanced the oxidation ofthe myofibrillar proteins [16 17] It has been reported thatperoxyl radicals from lipids abstracted hydrogen atoms frommolecules of protein leading to a radical-mediated chainreaction similar to that of lipid oxidation [27] And Lund etal [28] reported that oxidation of lipids in meat systems tookplace faster than that of myofibrillar proteins and hence itwas more likely that lipid derivatives (radicals and peroxides)promoted proteins oxidation than the other way round Forthe ozone treatment the To1 sample was rinsed in ozonewater with the presence of a high concentration of lipids Asfor the To2 sample lipids were majorly removed prior to thesecond cycle of rinsing using ozone water

32 Ca2+-ATPase Activity Ca2+-ATPase activity is widelyused as an index of the denaturation of fish myofibrillarproteins during storage and processing [29] As shown inFigure 2 Ca2+-ATPase activity of silver carpmyofibrillar pro-teins with and without ozone treatment gradually declined(119875 lt 005) during frozen storage These results wereconsistent with the report by Cao et al [30] that Ca2+-ATPase activity of silver carp surimi with various types ofcryoprotectants decreased with frozen storage (minus80∘C) up to90 d The oxidation of sulfhydryl groups especially in thehead region (SH1 and SH2) caused Ca2+-ATPase activity todecline Moreover inter- andor intramolecular interactionsof myofibrillar proteins during frozen storage could alsocontribute to decreased Ca2+-ATPase activity [26]

Within 15 d of frozen storage Ca2+-ATPase activity of To1was higher (119875 lt 005) than the control (Tc) (Figure 2) Theresults generally coincided with the findings of Zhang et al[9 11] in which the Ca2+-ATPase activity of bighead carpproteins increased after rinsing with 51mgL ozone water for20min followed by rinsing with distilled water A change inthe tertiary structure of themyosin head region or an increasein its flexibility owing to a light extent of denaturation mayresult in increased Ca2+-ATPase activity [31] The results ofZhang et al [9] also indicated that the tertiary structure of themyosin head region was slightly influenced by ozone waterrinsing Rinsing with ozone exposed the globular myosin


AB

B B AB

B

AA A C

C C

B A A B AA

TcTo1To2


00

02

04

06

08

10

12

14

16Ca2+

-ATP

ase (

umol

Pim

g pr

om

in)

Figure 2 Changes in Ca2+-ATPase activity of myofibrillar proteinsextracted from silver carp surimi during frozen storage Light greytwo washing cycles using ice water only (Tc) dark grey first andsecond washing cycle using ice water containing 8mgL ozone andice water respectively (To1) black first and second washing cycleusing ice water and ice water containing 8mgL ozone respectively(To2) Different letters indicate significant difference among samples(Tc To1 and To2)

head which is typically buried within the protein structureoutside the tertiary structure [9]

After 30 d of frozen storage our results showed the Ca2+-ATPase activity of the To1 sample was lower (119875 lt 005) thanthat of Tc (Figure 2) As storage duration extended negativeeffects (sulfhydryl oxidation andor protein interactions)dominated and Ca2+-ATPase activity continued to declineBefore frozen storage Ca2+-ATPase activity of To2 was lower(119875 lt 005) than To1 (Figure 2) However after 30 d of frozenstorage theCa2+-ATPase activity of To2was higher (119875 lt 005)than To1 (Figure 2) This result might be due to the lesserextent of oxidation in To2 which minimally influenced thephysiological activity of myosin

33 Surface Hydrophobicity The changes in surface hydro-phobicity of silver carp myofibrillar proteins with differentozone treatments during frozen storage are illustrated inFigure 3 The surface hydrophobicity of the three samples(Tc To1 and To2) increased significantly (119875 lt 005) afterstoring for 7 d remained unchanged (119875 gt 005) for up to30 d and then subsequently increased (119875 lt 005) for upto 90 d After storing for 90 d the surface hydrophobicity ofthe control sample increased by approximately 87 Similartendencies were also found in the surface hydrophobicity ofcroaker threadfin bream and bigeye snapper myofibrillarproteins during frozen storage [16] The increase of surfacehydrophobicity during extended frozen storage is connectedto the exposure of the hydrophobic bonds of myofibrillarproteins which are located in the interior of the proteinstructure [20] Frozen storage directly altered the tertiarystructure of protein molecules which results in functionalityloss as observed by a decline in gelling ability (Figure 5)

AA A A

A

A

A

AA A

A

A

AA A A

A

A

0

10

20

30

40

50

60

Surfa

ce h

ydro

phob

icity

TcTo1To2


Figure 3 Changes in surface hydrophobicity of myofibrillar pro-teins extracted from silver carp surimi during frozen storage Lightgrey two washing cycles using ice water only (Tc) dark greyfirst and second washing cycle using ice water containing 8mgLozone and ice water respectively (To1) black first and secondwashing cycle using icewater and icewater containing 8mgL ozonerespectively (To2) Different letters indicate significant differenceamong samples (Tc To1 and To2)

Surface hydrophobicity is an effective indicator forreflecting the conformational change of protein from itsnative structure [26] Surface hydrophobicity of To1 washigher than that of Tc but not significantly (119875 gt 005)during 90 d of frozen storage (Figure 3) In addition thesurface hydrophobicity of To2 was comparable to Tc Resultsdemonstrated that the oxidization in this study is mildChanges of the surface hydrophobicity could be used toexplain changes of sulfhydryl content (Figure 1) and Ca2+-ATPase activity (Figure 2) as affected by ozone water rinsingand frozen storage

34 Protein Patterns SDS-PAGE was performed to monitorpolymerization or degradation of the myofibrillar proteinsas affected by ozone oxidation and frozen storage Bands ofmyosin heavy chain (MHC 200 kDa) and actin (AC 45 kDa)with high densities were clearly visible on all SDS-PAGEgels (Figure 4) In addition bands assigned to troponin-T (TN T 35 kDa) myosin light chain 1 (MLC 1 21 kDa)tropomyosin (TM 40 kDa) and several other proteins werealso observed but with relatively lower densities In theabsence of 120573-mercaptoethanol bands with molecular weightabove 200 kDa were noticed on the SDS-PAGE gel (Fig-ure 4(a)) However those bands (gt200 kDa) seemed todisappear from SDS-PAGE gel when 120573-mercaptoethanol waspresent (Figure 4(b))120573-mercaptoethanol is a reducing agent that possesses the

ability to cleave disulfide covalent bonds (RSminusSR) of proteinsTherefore the obvious difference in the protein patternscaused by 120573-mercaptoethanol could be mainly attributedto myosin heave chain (MHC) polymer formation through


MHCXLMHC

ACTMTN T

MLC 1

STD TcTc To1 To2 To2To1 Tc To1 To2STD Tc To2Tc To1 To2 To2To1 Tc To1250KD150KD100 KD75KD50KD

37KD

25 KD

20 KD

90 d60 d30 d15 d7 d0 d(a)

MHC

AC

STD Tc To2Tc To1 To2 To2To1 Tc To1STD Tc To2Tc To1 To2 To2To1 Tc To1

TMTN T

MLC 1

250KD150KD100 KD75KD50KD

37KD

25 KD

20 KD

90 d60 d30 d15 d7 d0 d(b)

Figure 4 Change in protein patterns of silver carp surimi during frozen storage Tc two washing cycles using ice water only To1 first andsecond washing cycle using ice water containing 8mgL ozone and ice water respectively To2 first and second washing cycle using icewater and ice water containing 8mgL ozone respectively (a) Protein sample was dissolved in loading buffer without 120573-mercaptoethanol (b)protein sample was dissolved in loading buffer with 120573-mercaptoethanol STD kaleidoscope protein standard MHCXL cross-links of myosinheavy chain MHC myosin heavy chain AC actin TM tropomyosin TN T troponin-T MLC 1 myosin light chain 1

disulfide covalent bonds (RS-SR) Myosin contains threekinds of active sulfhydryls including SH1 SH2 and SHaSH1 and SH2 are located in the globular myosin head andare closely related to Ca2+-ATPase activity SHa is distributedin the light meromyosin chain (LMM) and is related to theoxidation of the myosin heavy chain (MHC) and polymerformation [11] The number of bands (gt200 kDa) increasedwith frozen storage time up to 90 d (Figure 4(b)) Resultsconfirmed the formation of disulfide covalent bonds duringfrozen storage which also coincided with the reduction offree sulfhydryl content (Figure 1)

Regardless of 120573-mercaptoethanol there was no consider-able difference among samples rinsedwith andwithout ozonewater when compared at the same frozen storage periodThisindicates that the ozone treatments used in this study did notinduce detectable polypeptide chain breakage or RS-SR cross-linking Zhang et al [9] compared the effects of two mannersof ozone treatments (washing with ozonized water and

ozone-flotation) and various treatment times on myofibrillarproteins from bighead carpThey found that protein patternsamong all samples did not behave differently under ozonewater rinsing of different time However densities of bandswith molecular weights between 80 and 200 kDa clearlyincreased with ozone-flotation time Results in this studyconfirmed once again that rinsing silver carp myofibrillarproteins with 8mgL ozone water for 10min was a mildoxidation process

35 Gel Texture Gel-forming ability is an important indexfor surimi quality The integrity of myofibrillar proteins isessential to form a strong gel Breaking force and penetrationdistance of silver carp surimi gels with and without ozonetreatments decreased (119875 lt 005) gradually during frozenstorage (Figure 5) After 90 d of frozen storage breakingforce and penetration distance significantly (119875 lt 005)declined by 7ndash18 and 13ndash21 respectively The decrease


AA A A

AB ABB B B B A

BB

A ABA A

0

50

100

150

200

250

300

350Br

eaki

ng fo

rce (

g)

TcTo1To2


(a)

AA A A

BA

AB AB A A

A

B AA A

B A

0

2

4

6

8

10

12

14

Pene

trat

ion

dist

ance

(mm

)

TcTo1To2


(b)

Figure 5 Changes in breaking force (a) and penetration distance (b) of silver carp surimi gels during frozen storage Light grey two washingcycles using ice water only (Tc) dark grey first and second washing cycle using ice water containing 8mgL ozone and ice water respectively(To1) black first and secondwashing cycle using ice water and ice water containing 8mgL ozone respectively (To2) Different letters indicatesignificant difference among samples (Tc To1 and To2)

in textural values was in accordance with decreased Ca2+-ATPase activity (Figure 2) which can be used as an indicatorfor the integrity of the myosin molecules

Before storage breaking force and penetration distance ofsamples with ozone rinsing (To1 and To2) were significantlylower (119875 lt 005) than Tc which was consistent with resultsreported by Xie et al [12] Myofibrillar proteins unfoldand then aggregate to form three-dimensional gel networksthrough intermolecular interactions (hydrophobic interac-tions disulfide covalent bonds ionic bonds etc) of exposedfunctional groups Conversion of sulfhydryl groups intodisulfide covalent bonds before the myofibrillar proteins arewell unfolded may result in a weak gel [12] Breaking force ofTo1 was significantly lower (119875 lt 005) than that of Tc duringfrozen storage However breaking force of To2 after 15 d wasnot significantly (119875 gt 005) different from that of Tc

36 Whiteness Whiteness is an important factor affectingcostumer acceptability of the end surimi products As shownin Figure 6 whiteness of surimi rinsed with ozone water (To1and To2) was significantly higher than that of Tc which couldbe attributed to the bleaching function of ozone Duringozone water rinsing the porphyrin structure of the hemepigment is destroyed and consequently discolored [26] To2showed a better effect on increasing whiteness than To1In To2 lipids and heme pigments (mainly myoglobin andhemoglobin) were partially removed after the first cycle ofrinsing Thus ozone more effectively discolored the reducedamount of remaining pigments

The whiteness of all samples (Tc To1 and To2) continu-ously increased as frozen storage time increasedThedecreaseof gel-forming ability during frozen storage (Figure 5) mightcontribute to increased free water contained in the surimigels which led to increased reflectivity on the surface of

C C C B

B B

BB B

AA

BA A A AA

A

54

56

58

60

62

64

66

68

70

Whi

tene

ss

TcTo1To2


Figure 6 Changes in whiteness of silver carp surimi gel duringfrozen storage Light grey two washing cycles using ice wateronly (Tc) dark grey first and second washing cycle using icewater containing 8mgL ozone and ice water respectively (To1)black first and second washing cycle using ice water and icewater containing 8mgL ozone respectively (To2) Different lettersindicate significant difference among samples (Tc To1 and To2)

cooked gels and resulted in ldquowhiteningrdquo of the proteinsHowever Benjakul et al [16] reported that whiteness ofsurimi made from four kinds of fish species harvested inThailand gradually decreased with increased frozen storagetime This might be due to different surimi processingmethods In their study whole fish were subjected to differentperiods of frozen storage prior to being manufactured intosurimi Denaturation of heme proteins during frozen storage


can result in their irreversible binding tomyofibrillar proteinsand thus decreased whiteness of surimi [26]

4 Conclusion

The results demonstrated that physicochemical properties ofmyofibrillar proteins from silver carp surimi during frozenstorage were affected by the ozone treatment protocol (TcTo1 or To2) As compared to only water rinsing (Tc) additionof 8mgL ozone in the first cycle of rinsing (To1) enhancedoxidation and denaturation of myofibrillar proteins duringfrozen storage resulting in a gel with lower breaking forceAddition of ozone of the same concentration in the secondcycle of rinsing (To2)minimally affected the physicochemicalproperties of myofibrillar proteins including free sulfhydrylcontent Ca2+-ATPase activity surface hydrophobicity andgel textural values In addition To2 treatment significantlyincreased whiteness of the surimi gel Addition of ozone inthe second rinse cycle is therefore a promising technologyto upgrade freshwater fish surimi in color without negativelyaffecting gelation properties or gel texture

Additional Points

Practical Applications Ozone has regulatory approval andis recognized as being environmentally friendly thereforethere is great potential to use ozone in aquatic process-ing industries The present results indicated that applyingozone treatment (8mgL and 10min) in the second cycle ofrinsing minimally affected the physicochemical propertiesof myofibrillar proteins during frozen storage This studyprovides scientific evidence for using ozone treatment as amild oxidation treatment to improve the white color of silvercarp surimi without negatively affecting gel texture



Acknowledgments

Authors gratefully acknowledge financial support from TheNational Natural Science Foundation of China (31501517)and China Agriculture Research System (CARS-46-23) Aspecial thank you is extended toOSUSenior Faculty ResearchAssistant Ms Angela Hunt for her help in revising this articlein English

References

[1] China Fishery Ministry Fishery Yearbook China AgriculturePress Beijing China 2015

[2] P Gueneeugues and J Ianelli ldquoSurimi resources and marketrdquoin Surimi and Surimi Seafood J W Park Ed pp 25ndash62 CRCPress Boca Raton Fla USA 2014

[3] S S Chen ldquoSurimi industry in Chinardquo in Proceedings of the 2ndSurimi School China Xiamen China May 2016

[4] Y Wang L Liu S Liu X N Li and L Z Liu ldquoEffects of ozoneon deodorization process and gel strength of surimi from silvercarprdquo Journal ofWuhan Polytechnic University vol 32 pp 15ndash192013

[5] P Howgate ldquoTainting of farmed fish by geosmin and 2-methyl-iso-borneol a review of sensory aspects and ofuptakedepurationrdquo Aquaculture vol 234 no 1ndash4 pp 155ndash1812004

[6] Y K Luo R Kuwahara M Kaneniwa Y Murata and MYokoyama ldquoComparison of gel properties of surimi fromAlaska pollock and three freshwater fish species effects ofthermal processing and protein concentrationrdquo Journal of FoodScience vol 66 no 4 pp 548ndash554 2001

[7] C OrsquoDonnell B K Tiwari P J Cullen and R G RiceldquoStatus and trends of ozone in food processingrdquo in Ozone inFood Processing C OrsquoDonnell Ed pp 1ndash18 Wiley-BlackwellOxford UK 2012

[8] C O R Okpala ldquoInvestigation of quality attributes of ice-stored Pacific white shrimp (Litopenaeus vannamei) as affectedby sequential minimal ozone treatmentrdquo LWTmdashFood Scienceand Technology vol 57 no 2 pp 538ndash547 2014

[9] T Zhang Y Xue Z J Li Y M Wang W Yang and CH Xue ldquoEffects of ozone on the removal of geosmin andthe physicochemical properties of fish meat from bigheadcarp (Hypophthalmichthys nobilis)rdquo Innovative Food Science andEmerging Technologies vol 34 pp 16ndash23 2016

[10] L Feng T Jiang Y Wang and J Li ldquoEffects of tea polyphenolcoating combined with ozone water washing on the storagequality of black sea bream (Sparus macrocephalus)rdquo FoodChemistry vol 135 no 4 pp 2915ndash2921 2012

[11] T Zhang Y Xue Z J Li YMWangWG Yang andCH XueldquoEffects of ozone-induced oxidation on the physicochemicalproperties of myofibrillar proteins recovered from bighead carp(Hypophthalmichthys nobilis)rdquo Food and Bioprocess Technologyvol 8 no 1 pp 181ndash190 2014

[12] S D Xie L H Chen Y Zhang and B D Zheng ldquoEffects ofozone on the quality of fish-ball made from silver carprdquo Journalof Fujian Agriculture and Forestry University vol 38 pp 552ndash557 2009

[13] W X Jiang Y F He S B Xiong et al ldquoEffect of mild ozone oxi-dation on structural changes of silver carp (Hypophthalmichthysmolitrix) myosinrdquo Food and Bioprocess Technology vol 10 no 2pp 370ndash378 2017

[14] S T Jiang M L Ho S H Jiang L Lo and H C Chen ldquoEffectsof ozone on the quality of fish-ball made from silver carprdquoJournal of Fujian Agriculture and Forestry University vol 63 pp652ndash655 1998

[15] C A M Dewitt J T M Lin and A Ismond ldquoWaste manage-ment utilization and challengesrdquo in Surimi and Surimi SeafoodJ W Park Ed pp 314ndash335 CRC Press Boca Raton Fla USA2014

[16] S Benjakul W Visessanguan C Thongkaew and M TanakaldquoEffect of frozen storage on chemical and gel-forming proper-ties of fish commonly used for surimi production in ThailandrdquoFood Hydrocolloids vol 19 no 2 pp 197ndash207 2005

[17] S Saeed and N K Howell ldquoEffect of lipid oxidation and frozenstorage on muscle proteins of Atlantic mackerel (Scomberscombrus)rdquo Journal of the Science of Food and Agriculture vol82 no 5 pp 579ndash586 2002

[18] S Poowakanjana and J W Park ldquoBiochemical characterisationof Alaska pollock Pacific whiting and threadfin bream surimi


as affected by comminution conditionsrdquo Food Chemistry vol138 no 1 pp 200ndash207 2013

[19] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] S Benjakul W Visessanguan C Thongkaew and M TanakaldquoComparative study on physicochemical changes of muscleproteins from some tropical fish during frozen storagerdquo FoodResearch International vol 36 no 8 pp 787ndash795 2003

[21] C H Fiske and Y Subbarow ldquoThe colorimetric determinationof phosphorusrdquoThe Journal of Biological Chemistry vol 66 pp375ndash400 1925

[22] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[23] T Yin and J W Park ldquoEffects of nano-scaled fish bone on thegelation properties of Alaska pollock surimirdquo Food Chemistryvol 150 pp 463ndash468 2014

[24] J W Park ldquoFunctional protein additives in surimi gelsrdquo Journalof Food Science vol 59 no 3 pp 525ndash527 1994

[25] K Hofmann and R Hamm ldquoSulfhydryl and disulfide groups inmeatsrdquo Advances in Food Research vol 24 pp 1ndash111 1978

[26] T C Lanier J Yongsawatdigul and P Carvajal-RondanellildquoSurimi gelation chemistryrdquo in Surimi and Surimi Seafood J WPark Ed pp 101ndash131 CRC Press Boca Raton Fla USA 2014

[27] E R Stadtman and R L Levine ldquoFree radical-mediated oxida-tion of free amino acids and amino acid residues in proteinsrdquoAmino Acids vol 25 no 3-4 pp 207ndash218 2003

[28] MN LundMHeinonen C P Baron andM Estevez ldquoProteinoxidation in muscle foods a reviewrdquo Molecular Nutrition andFood Research vol 55 no 1 pp 83ndash95 2011

[29] G A M Donald and T C Lanier ldquoActomyosin stabilization tofreeze-thaw and heat denaturation by lactate saltsrdquo Journal ofFood Science vol 59 no 1 pp 101ndash105 1994

[30] L Cao Y An S Xiong S Li and R Liu ldquoConformationalchanges and kinetic study of actomyosin from silver carp surimiwith modified starch-sucrose mixtures during frozen storagerdquoJournal of Food Quality vol 39 no 1 pp 54ndash63 2016

[31] T Watanabe N Kitabatake and E Dol ldquoProtective effectsof non-ionic surfactantsagainst denaturation of rabbit skeletalmyosin by freezing and thawingrdquo Agricultural and BiologicalChemistry vol 52 no 10 pp 2517ndash2523 1988

Research ArticleEffects of Micron Fish Bone with Different Particle Size onthe Properties of Silver Carp (Hypophthalmichthys molitrix)Surimi Gels

Tao Yin123 Jae W Park34 and Shanbai Xiong12

1College of Food Science and Technology Huazhong Agricultural University Wuhan Hubei Province 430070 China2National RampD Branch Center for Conventional Freshwater Fish Processing Wuhan 430070 China3Oregon State University Seafood Research and Education Center 2001 Marine Drive Room 253 Astoria OR 97103 USA4Department of Food Bioscience and Technology Korea University 145 Anam-ro Seongbuk-gu Seoul Republic of Korea

Correspondence should be addressed to Shanbai Xiong xiongsbmailhzaueducn

Received 30 November 2016 Accepted 10 January 2017 Published 28 February 2017

Academic Editor Andrea Laukova

Copyright copy 2017 Tao Yin et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Qualities of silver carp surimi (SCS) gels incorporated withmicron fish bone of different particle size (22 to 012 120583m)were evaluatedTextural values whiteness and water holding capacity of the SCS gels with setting significantly increased (119875 lt 005) as the micronfish bone particle size decreased As the particle size decreased more calcium ion was apparently released from the fish bone(119875 lt 005) Consequently the released calcium ion increased the activity of endogenous transglutaminase (TGase) and resultedin the formation of more myosin heavy chain (MHC) cross-links in the SCS gel with setting Fish bone with particle size below048 120583m was steadily trapped in the three-dimensional SCS gel network without disrupting the matrices Results indicated thatsize reduction of the incorporated micron fish bone improved qualities of the SCS gel with setting by the means of releasing morecalcium ion and maintaining better gel matrices

1 Introduction

Fish bone in some cuisines or processed products is tradi-tionally eaten and regarded as an important calcium sourcesin Southeast Asia [1] Fish bone is rich in calcium (234 gkgdry bone) which is mainly in the form of hydroxyapatite(HA) and calcium carbonate [2] However so far fish bonegenerated from production process of numerous aquaticproducts (fillet and surimi etc) is conventionally used forfishmeal and fertilizer production or directly discarded intothe sea river and estuaries resulting in environmental pol-lutionThe fish bone can be converted into nutritive foods oradditives for humans by reducing its particle size Accordingto the reports micron fish bone powders with a minimumaverage particle size of 765 and 175 120583m were prepared usingsuperfine grinding and dry media milling respectively [3 4]Particle size of fish bone was further reduced to submicronrange (1 to 01 120583m) using high-energy wet media milling[5] Consequently there was about an eightfold increase in

calcium release as the particle size decreased from micron tosubmicron range [5 6] Size reduction improved propertiesof fish bone particle in solubility water holding capacity[3] calcium bioavailability [7] and sensory quality (iegrittiness) as well Recently researchers have paid attentionto developing calcium-fortified food products using thedownsized fish bone [8 9]

Surimi is an intermediate product of the concentratedmyofibrillar proteins produced through several times ofwashing and dewatering which inevitably remove the major-ity of minerals contained in the original fish flesh Gellingof the myofibrillar proteins is a vital process of formingdesired texture for surimi products During gelling processfunctional groups imbedded inside of the protein molec-ular are exposed which subsequently form intra andorintermolecular bonds resulting in a three-dimensional gelnetwork [10] Addition of calcium compounds has beenreported to improve gel functionality of surimi [11ndash13] Gen-erally calcium ion released from those compounds induces



endogenous transglutaminase (TGase) which catalyzes theformation of 120576-(120574-glutamyl) lysine cross-links (isopeptidecovalent bonds) betweenmyofibrillar proteins during settingand thus improves the texture of surimi gel [10] Furthermorecalcium ion enhances the unfolding of myosin and formsldquocalcium bridgerdquo among the negatively charged myofibrillarproteins [10 14] which may contribute to the improvementof gel texture as well

Silver carp is one of the main aquacultured freshwaterfish in China with a total production of 423million t in 2014[15] In recent years surimi production from marine fishin China has been limited possibly due to overfishing TheChinese surimi producers have compensated for the shortageof marine fish by using silver carp The production of surimifrom silver carp was estimated at around 30000 t in 2013[16] and has grown fast reaching over 40000 t in 2015 [17]Calcium compounds from fish bone with a particle size of028120583m have been reported to improve gel texture of Alaskapollock surimi [18] According to the reports impacts ofadding calcium compounds on gel properties vary to fishspecies used for preparing surimi [13 19] Micron fish bonewith an appropriate particle size may be used for textureenhancement of silver carp surimi products while providingadditional dietary minerals However there have been nostudies investigating the effect of micron fish bone on thegelation properties of silver carp surimiThe production costproperties and applications of fish bone products highlydepend on its particle size Therefore the objective of thisstudy is to investigate the effects of micron fish bone withdifferent particle size on the qualities of silver carp surimigels

2 Material and Methods

21 Materials Silver carp (Hypophthalmichthys molitrix)surimi (AAA grade) with cryoprotectants (6 sucrose and03 sodium tripolyphosphate) was obtained from a localsurimi plant (Jingli Aquatic Product Co Ltd HonghuChina) Frozen surimi was cut into about 800 g blocksvacuum-packaged and stored in a freezer (minus18∘C) through-out the experiments Silver carp backbone was collected afterthe deboning process of surimi production It was cleanedand stored in a freezer (minus18∘C) before use

NN1015840-Dimethylated casein (DMC) monodansylcadav-erine (MDC) and glutaraldehyde used for TGase activitytest were purchased from Sigma Chemical Company (StLouis MO USA) Reagents used for gel electrophoresiswere obtained from Bio-Rad (Hercules CA USA) All otherchemicals were of analytical grade

22 Preparation of Micron Fish Bone (MFB) Frozen fishbone was thawed with running tap water and then heated at121∘C for 60min (ZM-100 GBPI Packaging Test InstrumentsCo Ltd Guangzhou China) The heated fish bone wasrinsed with tap water 5 times and drained off before grinding(MKCA6-2 Masuko Co Tokyo Japan) The bone paste wasdried at 105∘C for 6 h and then coarsely milled (RT-08HKKaichuangtongheTechnologyDevelopmentCo Ltd BeijingChina) Average particle size of the obtained fish bone power

was determined to be about 22 120583m using a Mastersizer 2000analyzer (Malvern Instruments Ltd Worcestershire UK)The fish bone powder was mixed with deionized water (DI)and further diminished using a high-energy wet bead mill(MiniZeta 03 Netzsch Selb Germany) according to themethod as described by Yin et al [5] Average size of thefish bone particles in the emulsion after milling for 1 2 4and 6 h was about 048 030 018 and 012 120583m respectivelywhich were analyzed using a Nano ZS90 analyzer (MalvernInstruments Ltd Worcestershire UK) Morphologies of thefish bone particles observed using field emission scanningelectron microscope (ULTRA PLUS-43-13 Zeiss Germany)were shown in Figure 1

23 Preparation of Surimi Gel The vacuum-packaged frozensurimi was tempered at room temperature for 40min beforebeing cut into approximately 2 cm times 2 cm times 4 cm cubesSurimi cubes were comminuted using a silent cutter (Mul-tiquick 3 Braun Germany) at speed 3 for 30 sec Sodiumchloride (2) was added to extract myofibrillar protein Fishbone of different size (22 048 030 018 and 012 120583m) at1 g dried fish bone100 g surimi paste was added Moisturecontent was adjusted to 78 using ice water (0∘C) Themixture was blended and ground in a stainless steel mortarusing twin pestles (CA 1 Kinn Shang Hoo Iron WorksTaiwan) at an agitation speed of 45 rpm for 30min Finaltemperature of the paste was below 10∘C The paste wasstuffed into a polyethylene sausage casing (25 cm diameter)with one end presealed using a sausage stuffer (Tre-mss7khTrs Spade Italy) After stuffing the other end was sealedwith U-shape aluminum wire clips using a clipper (Hk12Hakanson Sweden) The samples were heated with twodifferent thermal treatments (1) 90∘C for 30min (directcooking) (2) 40∘C for 1 h setting followed by 90∘Ccooking for30min Cooked gels were submerged in ice water for 15minand stored overnight in a refrigerator (4∘C)

24 Determination of Gel Strength Gel strength of the silvercarp surimi gels incorporated with MFB of different particlesizes was determined by the method as described by Cao etal [20] The chilled surimi gels were equilibrated at roomtemperature (sim25∘C) for 2 h Sampleswere cut to 25 cmcylin-der and subjected to fracture by penetration using a TA-XTtexture analyzer (StableMicro Systems Surrey UK) equippedwith a spherical probe (diameter 50mm and crossheadspeed of 1mms) Breaking force (g) indicating gel strengthand penetration distance (mm) denoting deformability wererecorded

25 Determination of Calcium Ion Concentration Calciumion concentration in the surimi paste incorporated withMFBof different particle sizes was measured according to themethod as described by Yin et al [5] with somemodificationSurimi paste prepared as described above was added with 4times the volumes of DIwater and homogenized at 5000 rpmfor 1min (IKA T18 Cole-Parmer Shanghai China) Thehomogenate was then centrifuged at 10000timesg for 30min(J-26XP Beckman Coulter Inc Fullerton CA USA) Aftercentrifugation the supernatant was filtrated (Number 1


(a) (b) (c)

Figure 1 Morphologies of fish bone particles (a) Micro fish bone powder (b) micron fish bone particles in the emulsion with 1 h of high-energy wet media milling and (c) micron fish bone particles in emulsion with 6 h of milling

Waterman Xinhua Filter Paper Co Ltd Hangzhou China)and diluted with DI water The concentration of calciumion in the dilution was analyzed using an atomic absorptionspectrophotometer (AA-6300c Shimadzu Kyoto Japan)

26 Determination of TGase Activity TGase activity wasmeasured by the method of Yin and Park [18] with slightmodifications Silver carp surimi was added with 4 volumesof extraction buffer (10mM NaCl and 10mM Tris-HClpH 75) and homogenized (Ika T18 Cole-parmer Co LtdShanghai China) at 5000 rpm for 1min The homogenatewas centrifuged (J-26XP Beckman Coulter Inc FullertonCAUSA) at 16000timesg for 30min under 4∘CThe supernatantwas filtrated and used as crude extract Fish bone emulsionprepared as described above was centrifuged at 10000timesgfor 30min (Beckman Coulter Inc Fullerton CA USA)and filtrated Filtrate was added to an assay mixture (15120583MMDC 10mgmL DMC 3mM DDT and 50mM Tris-HClpH 75) at a volumetric ration of 1 to 4 For the controlsample calcium chloride was added to the assay mixtureand reached a concentration of 017mM The crude enzymewas added and vortexed After incubating the mixture at40∘C for 10min EDTA solution was added to terminatethe catalytic reaction Fluorescence intensity of the mixturewas immediately measured (RF-1501 Shimadzu Co KyotoJapan)

27 SDS-PAGE Protein patterns of all surimi gel sampleswere revealed using SDS-PAGE according to Laemmli [21]with some modification Surimi gel samples were homog-enized (Ika T18 Cole-Parmer Co Ltd Shanghai China)at 10000 rpm for 1min and solubilized using 5 sodiumdodecyl sulfate solution (90∘C) Stacking and separatinggels were made using 5 (wv) and 12 (wv) acrylamiderespectively Each lane was loaded with 10120583g protein Afterrunning gels were fixed and stained with 0125 Coomassiebrilliant blue R-250 and destained inDIwater containing 50methanol and 10 acetic acid

28 Scanning Electron Microscopy (SEM) Surimi gel was cutinto pieces (5mm times 5mm times 1mm) and fixed with 25glutaraldehyde in 02M phosphate (pH 72) for 2 h at room

temperature The sample was rinsed three times using 02Mphosphate (pH 72) The fixed sample was dehydrated ingraded ethanol solution with serial concentrations of 3050 70 80 95 and 100 Samples were submergedin acetic acid isopropyl ester (substituting ethanol) and thencritical-point-dried (HCP-2 Hitachi Koki Co Ltd TokyoJapan) using CO

2as the transition fluid Dried sample was

mounted on a bronze stub and sputter-coated with gold Thespecimenwas observed using a scanning electronmicroscope(Quanta 3D Dual Beam FEI Co Tokyo Japan) at anacceleration voltage of 15 kV

29 Determination ofWater Holding Capacity (WHC) WHCof gel sample was measured according to the method ofShi et al [22] Cylindrical gel samples were cut into athickness of about 5mm weighed accurately and placedbetween two layers of filter paper (Number 1WatermanpaperXinhua Filter Paper Co Ltd Hangzhou China) Samplewas then placed at the bottom of a centrifuge tube (50mL)and centrifuged at 3000timesg for 15min (TDL-5A FulgorInstruments Ltd Shanghai China) WHC was calculated aspercentage of water retained after centrifugation

210 Color Evaluation Color parameters 119871lowast (lightness) 119886lowast(redness to greenness) and 119887lowast (yellowness to blueness)were measured using a CR-400 colorimeter (KonicaMinoltaOsaka Japan) Whiteness was calculated according to anequation developed by Park [23] for surimi gel

211 Statistical Analysis All the data were obtained from atleast triplicatemeasurements Analysis of variance (ANOVA)was carried out using the SAS program (V8 SAS InstituteInc Carry NC USA) Differences among mean values wereevaluated by the Duncan multiple range test (DMRT) using a95 confidence interval


31 Gel Texture Effects of added MFB with different particlesize on breaking force and penetration distance of silvercarp surimi gels prepared with two thermal treatmentsare illustrated in Figure 2 Breaking force and penetration


f e e e e e

d dc

bc ba

g f f f f f

d e cdbc ab a

0

4

8

12

16

20

24

Pene

trat

ion

dist

ance

(mm

)

0

100

200

300

400

500

600

700Br

eaki

ng fo

rce (

g)

22 048 030 018 012ConParticle size (120583m)


Figure 2 Breaking force and penetration distance of surimi gels with different size of fish bone particles and thermal treatments Con controlsample without added fish bone Black bars gel cooked at 90∘C for 30min Grey bars gel incubated at 40∘C for 1 h followed by 90∘C cookingfor 30min Different lowercases above the error bar indicate significant differences among samples with fish bone of different particle size(119875 lt 005)

distance of directly cooked gel (90∘C30min) containingMFB were (119875 lt 005) higher than the control Whengels were prepared with setting before cooking (40∘C1 h +90∘C30min) MFB with an average particle size between048 and 012 120583m significantly (119875 lt 005) increased breakingforce and penetration distance However MFB with anaverage particle size of 22120583m had no effect (119875 gt 005) onbreaking force while significantly (119875 lt 005) decreasingpenetration distance

Breaking force and penetration distance of gels withsetting increased as MFB particle size decreased (119875 lt005) More calcium ions released from smaller fish boneparticles increased the activity of TGase in silver carp surimi(Figure 3) Increased calcium ion release contributed to theformation of MHC cross-links in the surimi gel (Figure 4)In addition to being an endogenous TGase activator calciumions in conjunction with setting (40∘C for 1 h) possiblyenhanced the unfolding of silver carp myofibrillar proteinsConsequently more exposure of the reactive residues imbed-ded inside the myofibrillar proteins might contribute to theformation of more 120576-(120574-glutamyl) lysine cross-links and ahigher degree of hydrophobic interactions [14] On the otherside reduction of fish bone particle size contributed positivelyto maintaining better surimi gel matrices (Figure 5) Theaddition of 1 MFB with an average particle size at 012 120583mresulted in increased breaking force and penetration distanceof gel with setting by approximately 19 and 8 respectivelyover the control (without added fish bone) The effectivenessof MFB (028120583m) addition on improvement of breakingforce and penetration distance was more pronounced withAlaska pollock surimi gel (25 and 14) [18] It mightbe related to different endogenous TGase activity myosinreactivity and endogenous calcium content from different

ed

c c

b

a

FE

DC

BA

0

05

1

15

2

Ca2+

(mm

olk

g)

0

5

10

15

20

25

30

35

TGas

e act

ivity

(Um

L)


Figure 3 Endogenous TGase activity and calcium ion concen-tration from surimi paste as affected by added fish bone particlesize Column TGase activity line calcium ion concentration Concontrol sample without added fish bone Different letters above theerror bar indicate significant differences among samples with fishbone of different particle size (119875 lt 005)

fish species Compared to silver carp myosin from Alaskapollock is reported to be more reactive and tends to formlarger polymers during cross-linking reaction [10]

Breaking force and penetration distance of surimi gelwithout setting increased gradually but not significantly (119875 gt005) as MFB particle size decreased (Figure 2) This slightincrease might have been attributed to the formation of aldquocalcium bridgerdquo between negatively charged residues on twoadjacentmyofibrillar proteins Generally the strength of ionicbonds in surimi gel is much weaker than that of hydrophobicinteractions and covalent bonds [10]


STD 22Con 048 030 018 01290∘C

150KD

10KD

20KD

25KD

37KD

50KD75KD

100KD

250KD

(a)

STD

MHC

AC

22 048 030 018 012Con40∘C90∘C

(b)

Figure 4 SDS-PAGE patterns of silver carp surimi gels with different size of fish bone particles and thermal treatments (a) Gel cooked at90∘C for 30min (b) gel incubated at 40∘C for 1 h followed by 90∘C cooking for 30min Numbers designate average fish bone particle size(120583m) STD protein standard Con control sample without added fish bone MHC myosin heavy chain AC actin

lowastlowast

lowast

Con 22120583m 048120583m 030120583m 018 120583m 012 120583m

(a)

lowastlowast

lowast

Con 22120583m 048120583m 030120583m 018 120583m 012 120583m

(b)

Figure 5 SEM images of silver carp surimi gels with different size of fish bone particles and thermal treatments (a) Gel cooked at 90∘Cfor 30min (b) gel incubated at 40∘C for 1 h followed by 90∘C cooking for 30min Con control sample without added fish bone Numbersdesignate average size of fish bone particles

Results suggested that effects ofMFBon surimi gel texturemight vary by fish bone particle size thermal treatments andfish species

32 TGase Activity As shown in Figure 3 addition ofmicronfish bone obviously activated TGase from silver carp surimiIt was consistent with the reports by Yin and Park [18]and Hemung [24] Activity of crude TGase extracted fromsilver carp surimi without added fish bone was 823UmLextract at 40∘C It significantly (119875 lt 005) increased as theadded fish bone particle size decreased Activities of crude

TGase extract incubated in assay with fish bone particle sizeat 22 048 030 018 and 012 120583m were 1266 1719 18232366 and 2966UmL respectively Increased activity ofendogenous TGase with decreased fish bone particle size wasdue to the release of more calcium ions in the surimi paste(Figure 3) Calcium compounds in the fish bone possess lowsolubility Furthermore they are imbedded in the collagenmatrix which makes them even harder to dissolve in waterDuring the wet milling process specific surface area of fishbone particle markedly increases and the collagen matrixis destroyed facilitating the release of calcium ion [6] The


calcium ion concentration in the surimi paste without fishbone was 017mmolkg Calcium ion concentration signif-icantly increased with decreasing of fish bone particle sizeand reached the maximal at 095mmolkg Optimal calciumion concentration for full activation of endogenous TGaseextracted from carp however depends on the purificationprocedure It has been reported that optimal calcium ionconcentrations for crude TGase extract and purified TGasefrom carp were at 5mM and 50mM respectively [25 26]

33 MHC Cross-Linking Influence of fish bone particle sizeon the cross-linking of MHC during gel formation wasanalyzed using SDS-PAGE Compared to gel without setting(Figure 4(a)) MHC of silver carp surimi gel markedlydisappeared after setting (Figure 4(b)) Reduction of MHCafter setting could be attributed to the formation of 120576-(120574-glutamyl) lysine cross-links andor proteolytic degradationOgata et al [27] reported that the degradation of MHC wasobserved in the carp surimi sample incubated at 37∘C for10min with addition of endogenous protease (cathepsin L)and accompanied by the appearance of the resultant productwhich had a molecular weight of sim27KD No obviouslyvisible band with a molecular weight of sim27KD was detectedon the SDS-PAGE after setting (Figure 4) implying that thereduction of MHC in this study was mainly related to thecross-linking reaction

Gradual reduction of MHC from the surimi gel withsettingwas observed asMFBparticle size decreased due to theactivation of endogenous TGase (Figure 3) resulting in theformation of more 120576-(120574-glutamyl) lysine cross-links of MHCMHC intensity of gel without setting remained constant asthe particle size of fish bone changed These results werein agreement with the report by Wang et al [28] that theintensities of MHC cross-links from silver carp surimi incu-bated at 35∘C for various time significantly increased withcalcium ion concentration up to 180mmolkg surimi pasteHowever MHC cross-links of gel without setting were notsignificantly affected by calcium ion concentration rangingfrom 0 to 540mmolkg surimi paste Changes of MHC onthe SDS-PAGE gel corresponded well with the changes of geltexture values (Figure 2)

The significant reduction of MHC bandrsquos intensity wasnoted when setting was employed neither MHC cross-linksnor protease-induced small molecular bands were present(Figure 4) As discussed above no visible band for protease-induced sim27 kDa [27] was observedMHC cross-links whichare not shown aboveMHC on the SDS-PAGE possibly couldnot enter the polyacrylamide gel system because the size ofcross-links was too large

34 Microstructure Figure 5 shows scanning electronmicroscopy (SEM) images of the internal structures ofsurimi gels added with different particle size of MFB andprepared with two thermal treatments Fibrous matrixwhich is a characteristic of heat-induced protein gel wasclearly observed in the surimi gels Control gels (withoutadded fish bone) and gels with MFB of particle size between048 and 012 120583m formed continuous structures (Figure 5)while surimi with MFB particles size of 22120583m formed

f ed cd cd bc

cd cd bc bc b a

0

20

40

60

80

100

Wat

er h

oldi

ng ca

paci

ty (

)


Figure 6 Water holding capacity of the surimi gels with differentsize of fish bone particles and thermal treatments Black bars gelcooked at 90∘C for 30min Grey bars gel incubated at 40∘C for 1 hfollowed by 90∘C cooking for 30min Different lowercases indicatesignificant differences among different treatments (119875 lt 005)

discontinuous structures with large pores (asterisks inFigure 5) In contrast to the porous and coarse networksof directly heated gel (Figure 5(a)) networks of surimigel prepared with two-step heating (setting and cooking)were denser and more compact along with a concomitantappearance of homogeneous surface (Figure 5(b)) Heat-denatured surimi proteins align in an ordered fashion todevelop a fine gel network when rapidly unfolded proteinsare associated in slow heating fashion (ie setting) [29] Inaddition the formation of larger amount of 120576-(120574-glutamyl)lysine cross-links after setting contributed to the stabilizationof the gel network

35 Water Holding Capacity (WHC) Higher WHC valuesindicate less expressible water in the surimi gel During theheat-induced gelling process the gel network formed whilebinding water and entrapping other ingredients WHC of thegels prepared with setting was significantly (119875 lt 005) higherthan that of gel without setting for all particle sizes (Figure 6)as setting induced more compact and denser gel networksby the function of endogenous TGase (Figure 3) WHCincreased from 4620 to 6496 and 6084 to 7393 forthe samples without and with setting respectively as MFBparticle size decreased from 22120583m to 012 120583m It might beattributed to the increased WHC contributed by fish boneaddition [3] As mud obviously holds more water than sandthe size of particle is a significant factor affecting WHC

36 Color Attributes Color parameters of gels from silvercarp under different thermal treatments with various fishbone particle size are shown in Table 1 119871lowast value of controlgel and gel with MFB of particle size below 048 120583m slightlybut significantly (119875 lt 005) increased after setting while thatof gel with MFB of particle size at 22 120583m slightly decreased(119875 lt 005) Yellowness value (+119887lowast) decreased (119875 lt 005)and whiteness value (119871lowast minus 3119887) increased (119875 lt 005) aftersetting for all fish bone particle sizes 119871lowast values of gels withMFB of particle size below 048 120583m regardless of setting


Table 1 Color parameters (lightness (119871) greenness (119886lowast) yellowness (119887lowast) and whiteness (119908)) of silver carp surimi gels with different size offish bone particles (120583m) and thermal treatments

Color parameter 119871 119886lowast 119887lowast 119908

Without setting

Con 7427 plusmn 049g minus205 plusmn 007a 244 plusmn 021g 6695 plusmn 060b

22 7479 plusmn 065f minus271 plusmn 009cd 906 plusmn 043a 4760 plusmn 128i

048 7652 plusmn 048a minus267 plusmn 006c 826 plusmn 021b 5174 plusmn 054g

030 7635 plusmn 023ab minus274 plusmn 045de 781 plusmn 020c 5293 plusmn 061f

018 7610 plusmn 035bc minus281 plusmn 003f 778 plusmn 037c 5274 plusmn 093f

012 7585 plusmn 043cd minus280 plusmn 003ef 736 plusmn 033d 5377 plusmn 090e

With setting

Con 7549 plusmn 047de minus236 plusmn 004b 191 plusmn 026h 6976 plusmn 058a

22 7511 plusmn 051ef minus294 plusmn 007h 827 plusmn 018b 5030 plusmn 032h

048 7627 plusmn 048ab minus288 plusmn 005g 753 plusmn 026d 5368 plusmn 059e

030 7581 plusmn 046cd minus303 plusmn 005i 731 plusmn 026d 5388 plusmn 067e

018 7570 plusmn 029cd minus310 plusmn 005j 685 plusmn 032e 5513 plusmn 093d

012 7559 plusmn 027d minus309 plusmn 005j 636 plusmn 022f 5650 plusmn 056c

The different lowercases in the same column indicate the significant differences (119875 lt 005) Data are expressed as means plusmn standard deviations

were significantly (119875 lt 005) higher than that of gel withMFB of particle size at 22120583m As particle size decreased119871lowast values of gels with MFB of particle size below 048120583mregardless of setting gradually decreased (119875 lt 005) Thismight be due to the decreased light scattering effect of watermolecular as a result of increased WHC of the gel (Figure 6)Yellowness value of gels with and without setting decreasedgradually (119875 lt 005) and whiteness value significantly (119875 lt005) increased with decreasingMFB particle sizeThus boththermal treatment and fish bone particle size affected colorattributes of silver carp surimi gels

4 Conclusions

Gel properties of silver carp surimi were significantly affectedby thermal treatments andMFB particle size Textural valuesWHC and whiteness of gels prepared with setting increased(119875 lt 005) as the MFB particle size decreased Improvementof gel texture and WHC resulted from reduced bone particlesize which was primarily due to the function of endogenousTGase and calcium ion from theMFB In addition reductionof MFB particle size contributed positively to maintainingintegrity of the three-dimensional myofibrillar gel networksMFB with an average particle size below 048 120583m possessesthe potential to be used to improve gel qualitywhile providingadditional dietary minerals for silver carp surimi products

Additional Points

Practical Applications Fish bone is a natural calcium sourceFish bone incorporated into silver carp surimi gels exhibiteddifferent properties which were affected by its particle sizeand heatingmethods subjected to surimi paste Improvementof the surimi gel texture was achieved by addition of the fishbone with particle size below 048 120583m when combined withsetting The practical application of this work is providinga theoretical foundation and basic data support for usingcalcium-enriched fish bone to improve gel texture while

providing additional dietary calcium for silver carp surimiproducts

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge financial support fromthe National Natural Science Foundation of China (no31601501) and the earmarked fund for China AgricultureResearch System (no CARS-46-23)

References

[1] T Larsen S HThilsted K Kongsbak and M Hansen ldquoWholesmall fish as a rich calcium sourcerdquo British Journal of Nutritionvol 83 no 2 pp 191ndash196 2000

[2] J Toppe S Albrektsen B Hope and A Aksnes ldquoChemicalcomposition mineral content and amino acid and lipid profilesin bones from various fish speciesrdquo Comparative Biochemistryand Physiology Part B Biochemistry and Molecular Biology vol146 no 3 pp 395ndash401 2007

[3] G-C Wu M Zhang Y-Q Wang K J Mothibe and W-XChen ldquoProduction of silver carp bone powder using superfinegrinding technology suitable production parameters and itspropertiesrdquo Journal of Food Engineering vol 109 no 4 pp 730ndash735 2012

[4] T Yin H Du J Zhang and S Xiong ldquoPreparation and charac-terization of ultrafine fish bone powderrdquo Journal of Aquatic FoodProduct Technology vol 25 no 7 pp 1045ndash1055 2016

[5] T Yin J W Park and S Xiong ldquoPhysicochemical propertiesof nano fish bone prepared by wet media millingrdquo LWT - FoodScience and Technology vol 64 no 1 pp 367ndash373 2015

[6] J Zhang T Yin S B Xiong Y J Li U Ikram and RLiu ldquoThermal treatments affect breakage kinetics and calcium


release of fish bone particles during high-energy wet ballmillingrdquo Journal of Food Engineering vol 183 pp 74ndash80 2016

[7] W W Xie T Yin J Zhang R Liu S M Zhao and S BXiong ldquoEffects of fish bone powder particle size on calciumbioavailability of fish bone powder-fish protein hydrolysatesmixedrdquo Food Science vol 35 pp 211ndash216 2015

[8] K I Jeyasanta V Aiyamperumal and J Patterson ldquoUtilizationof trash fishes as edible fish powder and its quality characteris-tics and consumer acceptancerdquo World Journal of Dairy amp FoodSciences vol 8 pp 1ndash10 2013

[9] A R Abdel-Moemin ldquoHealthy cookies from cooked fishbonesrdquo Food Bioscience vol 12 pp 114ndash151 2015


[11] S Benjakul W Visessanguan and Y Kwalumtharn ldquoThe effectof whitening agents on the gel-forming ability and whiteness ofsurimirdquo International Journal of Food Science and Technologyvol 39 no 7 pp 773ndash781 2004

[12] D Jia J You Y Hu R Liu and S Xiong ldquoEffect of CaCl2

on denaturation and aggregation of silver carp myosin duringsettingrdquo Food Chemistry vol 185 pp 212ndash218 2015

[13] N Lee and J W Park ldquoCalcium compounds to improve gelfunctionality of Pacific whiting and Alaska pollock surimirdquoJournal of Food Science vol 63 no 6 pp 969ndash974 1998

[14] J Yongsawatdigul and S Sinsuwan ldquoAggregation and confor-mational changes of tilapia actomyosin as affected by calciumion during settingrdquo Food Hydrocolloids vol 21 no 3 pp 359ndash367 2007

[15] China Fishery Ministry Fishery Year Book China AgriculturePress Beijing China 2015




[19] Y Q Ding Y M Liu H Yang et al ldquoEffects of CaCl2on

chemical interactions and gel properties of surimi gels from twospecies of carpsrdquo European Food Research and Technology vol233 no 4 pp 569ndash576 2011



[22] L Shi X Wang T Chang C Wang H Yang and M CuildquoEffects of vegetable oils on gel properties of surimi gelsrdquoLWTmdashFood Science and Technology vol 57 no 2 pp 586ndash593 2014

[23] JW Park ldquoSurimi gel colors as affected bymoisture content andphysical conditionsrdquo Journal of Food Science vol 60 no 1 pp15ndash18 1995

[24] B O Hemung ldquoProperties of tilapia bone powder and itscalcium bioavailability based on transglutaminase assayrdquo Inter-national Journal of Bioscience Biochemistry and Bioinformaticsvol 3 pp 306ndash309 2013

[25] H Kishi H Nozawa and N Seki ldquoReactivity of muscletransglutaminase on carp myofibrils and myosin Brdquo NipponSuisan Gakkaishi vol 57 no 6 pp 1203ndash1210 1991

[26] P K Binsi and B A Shamasundar ldquoPurification and charac-terisation of transglutaminase from four fish species Effect ofadded transglutaminase on the viscoelastic behaviour of fishmincerdquo Food Chemistry vol 132 no 4 pp 1922ndash1929 2012

[27] H Ogata F Aranishi K Hara K Osatomi and T IshiharaldquoProteolytic degradation of myofibrillar components by carpcathepsin Lrdquo Journal of the Science of Food and Agriculture vol76 no 4 pp 499ndash504 1998

[28] J Y Wang C C Liu S Z Zhao et al ldquoOptimal conditionsfor maximal cross-linkage of myosin heavy chain (MHC) andgelation of surimi product from silver carp (Hypophthalmichtysmolitrix)rdquo Food Science vol 29 no 11 pp 223ndash227 2008

[29] A M Hermansson ldquoAggregation and denaturation involved ingel formationrdquo in Functionality and Protein Structure E I PourEd pp 81ndash103 American Chemical Society Washington DCUSA 1979

Research ArticleEffects of Beeswax Coating on the Oxidative Stability ofLong-Ripened Italian Salami

Marcello Trevisani Matilde Cecchini Daniela SiconolfiRocco Mancusi and Roberto Rosmini

Department of Veterinary Public Health and Animal Pathology University of Bologna Bologna Italy

Correspondence should be addressed to Marcello Trevisani marcellotrevisaniuniboit



Copyright copy 2017 Marcello Trevisani et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Beeswax coating of foods put a barrier to oxygen light and vapour that can help to prevent oxidation of fat and pigments andwater loss The amounts of 2-thiobarbituric acid reactive substances (TBARS) and water activity (Aw) were assessed in Italiansalami coated with beeswax at 55 days of ripening and compared with controls at 5 6 and 7 months of shelf life The results werecorrelatedwith sensory quality TBARS levels were below 08mg kgminus1 in the beeswax-coated salami until 6months of aging (median0697max 0795) and significantly higher in the uncoated salami (median 1176max 1227) A slight correlation between the amountof TBARS and Aw was observed in beeswax-coated salamis whereas this effect was masked in controls by the large Aw variabilityobserved at 7 months Beeswax coating prevents case hardening and facilitated the peeling

1 Introduction

Beeswax is natural glazing agent that can be used in foodto prevent water loss and provide protection during storageIt is often used to prevent water loss and retard shrinkageand spoilage in fruit and cheese Refined beeswax coatingis a natural alternative to plastic envelopes that does notharm the environment and meets the criteria for GRASstatus defined by the FDA for food packaging materials[1] It is approved for food use in most countries andin the European Union under the E number ldquoE901rdquo [2]Colour changes occur due to oxidation phenomena involvingmyoglobin during ripening of salami Moreover shrinkagedue to dehydration results in aspect modifications mainlyascribable to fat aggregation [3] Even after salami has reachedwater activity (Aw) and pH values that make the productshelf stable without refrigeration environmental conditionsfor storage (relative humidity relative air speed tempera-ture and light) need to be controlled to prevent excessivewater loss product shrinkage and too hard consistencyoxidative changes and excessive growth of moulds Duringdistribution salami is often wrapped in plastic film withreduced oxygen and water vapour permeability to prevent

contamination by dirt and off-odour and protect the productsfromoxygen and loss or uptake ofmoistureOxygenmoisturebarrier properties of beeswax are intermediate between lowand high density polyethylene [4] It is also a barrier tophotooxidation Beeswax is also one of the most effectivematerials employed to decrease water vapour permeability ofedible films due to its high hydrophobicity and solid state atroom temperature [5 6] These properties have been used topreserve and improve the sensorial quality of salami duringaging Local producers in the area of Bologna (Italy) usedto coat salami with beeswax after the products are shelfstable (ie water activity has decreased to values below 092)They use the natural beeswax to limit an excessive water lossduring storageWax coating also prevents case hardening andmould development andmade the peelability easyThis studywas aimed at assessing the oxidative stability and sensorialcharacteristics of salami after beeswax coating


21 Reagents The reagents were as follows trichloroaceticacid (TCA) 99 1133-tetramethoxypropane (TMP) 99



2-thiobarbituric acid (TBA) 98 (Sigma-Aldrich Italy)ethylenediaminetetraacetic acid disodium salt (EDTA)(AnalR VWR) propyl gallate (Fluka) hydrochloridric acid37 (Merck) sodium hydroxide 20 water solution (CarloErba Italy) TCA 100 pv (100 g TCA 99 water up to100mL) TMP stock solution (1000mg kgminus1) extractingsolution (75mL TCA 100 208mL HCl 025M 1 g EDTA1 g propyl gallate and water up to 1000mL) TBA reagent(TBA 80mM in in NaOH 1M pH corrected at 40ndash42 withHCl 1M) TMP (5mg kgminus1) working solution (250120583L TMPstock solution 375mL TCA 100 and HCl 025M up to50mL)

22 Samples Twelve salamis ldquoFelino IGPrdquo were provided bya local producer at approximately 55 days of ripening Thesalamis had the typical characteristics (length 40ndash45 cmdiameter approximately 6 cm weight 10ndash12 Kg weight loss35ndash375 and water activity 090ndash092) The salamis derivedfrom a single lot were divided into two groups Six were usedas control The remaining were brushed washed and putback to dry in ventilated cells Then they were wrapped ina cotton gauze and tied and covered with beeswax (yelloworganic beeswax melted and held at 62ndash64∘C) for 3ndash5 sec-onds The resulting wax layer had a thickness of 2-3mmTherefore all salamis were hang up to dry in well-aired cellarsup to 5ndash7 months packed in cartoons and stored in a darkroom at 5∘C plusmn 1∘C (relative humidity 85ndash90) until analysesComparative assessments were made on two salamis for eachtreatment group (wax coated and uncoated) at 5 6 and 7months

23 Quantification of TBARS Three slices (5mm thick) weretaken from centre and intermediate parts from eachsalami These samples were minced for 10 seconds with aMoulinette and 25 g of the homogenate was analysed for2-Thiobarbituric Acid Reactive Substances (TBARS) usingthe method developed by Wang et al [7] for meat and meatproducts The entire protocol (sampling and analyses) wasrepeated two times (ie after 2-3 days) using other portionsof each salami A total of eight measurements (2 salamis 2replicates and 2 repetitions) for each treatment group andstorage time were obtained Sample homogenates were put invials in an ice bath mixed with 20mL of chilled extractingsolution and homogenised at 16000 rpm for 2 minutes withUltra-turrax (model T25 basic IKA Labortechnic Italy)Additional 5mL of extracting solution was used to washthe blades of the Ultra-turrax (final dilution 1 10) thenthe extracted samples were filtered (Whatman paper filtern4) Samples were continuously maintained in a chilled bathuntil the filtration Two mL of the filtrates was mixed with2mL of the TBA reagent and incubated at 40∘C in a waterbath for 90 minutes and then chilled in fridge at 6ndash8∘C for30 minutes Therefore the absorbance at 532 nm was read(5 replicates) on a spectrophotometer (Perkin-Elmer modelLambda 1) A calibration curve was designed using standardsat concentration in the range of 0025 to 07mgmLminus1 of TMP(5mg kgminus1) working solution By using the above-mentionedTMP solutions malondialdehyde (MDA) standards in the

range of 015 to 426 nmolmLminus1 were prepared Nine TMPstandards (from 20 to 560120583L of the TMP working solution)were mixed with 2mL of TBA reagent 300 120583L of TCA 100and HCl 025M up to 4mL A blank solution was madeas described before but without TMP Vials with the TMPstandards and blank were incubated as described for thesamples and the absorbance values at 532 nm (Abs532) wereread (average of 5 replicates) Ten-point standard calibrationcurves were designed The coefficient R2 must be between0995 and 1 in order to accept the curves for TBARS quan-tification The samplesrsquo TBARS concentration was calculatedby interpolation of their measured absorbance values (Abs)The resulting value was multiplied by the dilution factor ofthe sample (25 g in 25mL) and extract (1 2) to correct forthe final concentration Results are expressed as mg kgminus1 ofMDA equivalents TBARS

24Water Activity Thewater activity (Aw)was assessedwitha dew point water activity meter (Aqualab Series 3 DecagonUS) using the procedures recommended by the producer

25 Sensory Test A hedonic test was conducted with eightuntrained assessors who scored the acceptability of 3 attrib-utes (texture taste and flavour) using the following 1ndash10 pointscale texture (1 = hard 10 = soft) presence of acid taste (1 =sharp burning 10 =mild acidic) rancid off-flavour (1 = none3 = slightly perceived 10 = strong)With this aim the salamisanalysed for the TBARS at 6 and 7months of storage were cutin slices and the panel was asked to comparatively assess thequality of the salami (blind test between beeswax-coated ornoncoated salami)

26 Statistical Analysis TBARs values recorded for differentcategories (wax or not coating) and periods (0 1 and 2months of storage) were summarized graphically as box andwhiskers plots Statistical analyses were performed usingthe R package ldquostatsrdquo (version 2153) [8] Data relative tosamples from different categories and period were analysedwith Bartlettrsquos test to assess homoscedasticity (homogene-ity of variance) When departures from normality of datawere observed nonparametric alternatives to the analysis ofvariance (ANOVA) were used In particular the Wilcoxonsigned-rank test was used for comparing theMDA equivalentTBARS values observed in salami packaged with or withoutwax Differences among samples taken at 5 6 and 7 monthswere analysed with the Kruskal-Wallis rank sum test Whendifferences were considered to be significant at 119901 le 001 thesignificance of individual pair differences (aging periods) wastested for inequality using the multiple comparison test afterKruskal-Wallis using the R package ldquopgirmessrdquo


31 Effect of the Beeswax Coating on the TBARS Concen-tration Slower drying and ripening at low temperatures ofthe beeswax-coated salami resulted in less lipid oxidativechanges The concentration of TBARS (MDA equivalents)is reported in Table 1 Values were below 08mg kgminus1 in


Table 1 TBARS concentrations in salami according to aging period and beeswax coating

Ageing Coating TBARS (MDAmg kgminus1) Wilcoxon1 signed-rank test(months) Mean2 Median Range

5 Beeswax 0693a (plusmn0017) 0696 0653ndash0713119901 = 00039

Control 1016b (plusmn0045) 1029 0938ndash1059


Control 1166c (plusmn0045) 1176 1104ndash1227

7 Beeswax 1106b (plusmn0039) 1098 1048ndash1173119901 = 00039

Control 1869d (plusmn0023) 1872 1819ndash1897Note mean median and range calculated from 119899 = 8 measurements (ie 2 samples 2 replicates and 2 repetitions for each treatment group) Samples withsignificant differences in their malondialdehyde level are indicated by different letters 1significant differences detected between samples at the same agingperiod (Wilcoxon test)2Significant differences detected between samples at different aging period (Kruskal-Wallis test) chi-square = 153934 119901 value = 00004543

the beeswax-coated salami until 6 months of aging (median0697 max 0795) and significantly higher in the uncoatedsalami (median 1176 max 1227) At 7 months the MDAmedian values were equal to 1098 and 1872mg kgminus1 in waxcoated and uncoated salami respectively Limits for TBARS(MDA equivalents) have been suggested at 05mg kgminus1 ofmeat for threshold of consumer detection of rancidity [9ndash11]and 10mg kgminus1 for sausage products [12] However detectionlimits have not been set for salami The lipid peroxidationin raw ripened sausages (salami) involves transformationof primary products of lipid degradation (alkyl free radicalwith a group of conjugated bonds) into secondary productsincluding MDA [13ndash15] and this is correlated to the increaseof TBARS concentrationThe presence of high TBARS valuesin sausages after ripening can be explained by availability ofoxygen consequently to the mechanical process [16] How-ever the use of vacuum stuffing antioxidants the protectiveeffect of some starters [17] and also the storage of ripeningsausage in vacuum or modified atmosphere without oxygen[18] can significantly contribute to an increased oxidativestability The latter condition can occur with the use ofbeeswax coating In a study of Novelli et al [19] concerningldquoMilanordquo salami with similar fat content (approximately30) the TBARS values were equal to 139 plusmn 108mg MDAkgminus1 atgt3months of aging which is a value higher than thoseof the beeswax-coated salami at 7 months of aging

Even if the consumers donot perceive any flavour deterio-ration lipids oxidation involves loss of unsaturated fatty acids(nutrient loss) and the end products of lipid oxidationmay bemutagenic and carcinogenic Malondialdehyde (MDA) canreact with DNA and form MDA adducts [20 21] Storageof ripening sausage in vacuum or modified atmosphere hasresulted in increased oxidative stability of raw sausage after 2and 5months of storage [18] Beeswax coating appears to givesimilar protection

32 Correlation between TBARS Concentration Aw and Sen-sory Evaluations The beeswax-coated salami had signifi-cantly higher mean Aw values from 0903 to 0888 between5 and 7 months of aging The uncoated salami had Aw valuesin a range of 0812 to 0821 (Table 2) The sensory test did notreveal relevant differences in the taste and flavour of different

Table 2 Changes in the water activity values (Aw) in salamis withor without beeswax coating

Ageing period Control Beeswax coating5 months 0821 plusmn 0001a 0903 plusmn 0002c

6 months 0812 plusmn 0002a 0899 plusmn 0002d

7 months 0820 plusmn 0011a 0888 plusmn 0004e

Aging period at the beginning of the shelf life = 5 monthsDifferences related to coating F calculated = 119281 F critical = 475 119901 value= 221 lowast 10minus13Differences related to aging (in salami with beeswax coating) F calculated =2303 F critical = 514 119901 value = 00015Means with different letters are significantly different

treatment groups whereas texture was softer in the beeswax-coated salami (Figure 1) The softer texture was related tothe lower loss of water There is a statistically significantrelationship between TBARS and Aw values but only for thebeeswax-coated salami (Spearmanrsquos rank correlation rho =minus0623119901 value = 00011)Within this group the concentrationof TBARS slightly increased in consequence of the waterloss but this effect was masked in controls by the largeAw variability observed at 7 months (Aw = 0820 plusmn 0011range 0814ndash0832) (Table 2 and Figure 2) Any differencewas perceived during the sensory test with regard to sharpburning taste (Wilcoxon paired sign test 119901 value gt 005)Also the presence of rancid off-flavour was not perceivedand only two panel members (out of eight) reported valueabove the limit of perception in the uncoated salami at 7months of aging The panel test probably did not give riseto a negative score (rancid off-flavour) because the TBARSconcentration was relatively low (1819ndash1897mg MDA kgminus1)also in these samples An unpleasant fruity flavour wasperceived by some panel members in the salami coated withbeeswax at 7 months of aging This flavour deteriorationmight be related to spoilage by heterofermentative lactic acidbacteria in consequence of the higher Aw [22 23] Thereare no published studies available that define TBARS valuesthat are associated with the presence of rancid off-flavours inldquoFelinordquo salami

Sojic et al [24] reported that vacuumandMAPpackagingcan contribute to better oxidative and sensory stability of


00102030405060708090

100Texture (1 = hard 10 = soft)

00102030405060708090

100

00102030405060708090

100

Rancid off-flavour (1 = none 3 = sligthly perceived 10 = strong)(1 = sharp burning 10 = mildly acidic)

Taste

6m

wax

6m

cont

rol

7m

wax

7m

cont

rol

6m

wax

6m

cont

rol

7m

wax

7m

cont

rol

6m

wax

6m

cont

rol

7m

wax

7m

cont

rol

Figure 1 Box and whiskers plot for sensory quality of salami at 6 and 7 months of aging Notes box indicates quartiles and the central linethe median The lines (ldquowhiskersrdquo) show the largest or the smallest observation The symbol Q indicates average values

0810 0820 0830

10

12

14

16

18

Aw control

MD

A c

ontro

l

0885 0895 0905

06

07

08

09

10

11

Aw beeswax

MD

A b

eesw

ax

Figure 2 Relationship between TBARS and Aw values

dry fermented sausage and that TBARS values are negativelycorrelated with odour and taste of salami aged for 7 monthsBanon et al [25] observed that the main causes of loss ofsensory quality of salami limiting their shelf life under thetested retail (aerobic) conditions include flavour deteriora-tion associated with rancidity bitterness and mouldy off-flavour together with hardening and loss of juiciness

4 Conclusions

The results of this preliminary study indicate that beeswaxcoating can be a useful alternative to the plastic packagingThis natural coatingmaterial can be used to increase the agingperiodwithout compromising the texture which remains softand is appreciated for its better flavour Beeswax effectively

reduces the development of lipid peroxidation products inthese salamis

Additional Points

Practical Applications Beeswax coating can be used to pre-serve and improve the sensorial quality of salami duringaging It can be a natural packaging material that does notharm the environment and has good oxygenmoisture barrierproperties

Competing Interests



Acknowledgments

This study was supported by the School of Specialization inInspection of Food of Animal Origin of the University ofBologna Italy

References

[1] CFR (Code of Federal Regulations) Title 21 Chapter IPart 582mdashsubstances generally recognized as safe Section5821975mdashbleached beeswax April 2013 GPOrsquos Federal DigitalSystem httpswwwgpogov

[2] European Commission ldquoCommission Regulation (EU) No102011 of 14 January 2011 on plastic materials and articlesintended to come into contact with foodrdquoOfficial Journal of theEuropean Union L vol 12 pp 1ndash89 2011

[3] L Fongaro C Alamprese and E Casiraghi ldquoRipening ofsalami assessment of colour and aspect evolution using imageanalysis and multivariate image analysisrdquoMeat Science vol 101pp 73ndash77 2015

[4] B Cuq N Gontard and S Guilbert ldquoEdible films and coatingas active layersrdquo in ActiVe Food Packaging M L Rooney Edpp 111ndash142 Blackie Academic and Professional Glasgow UK1995

[5] V Morillon F Debeaufort G Blond M Capelle and AVoilley ldquoFactors affecting the moisture permeability of lipid-based edible films a reviewrdquo Critical Reviews in Food Scienceand Nutrition vol 42 no 1 pp 67ndash89 2002

[6] L Yang and A T Paulson ldquoEffects of lipids on mechanical andmoisture barrier properties of edible gellan filmrdquo Food ResearchInternational vol 33 no 7 pp 571ndash578 2000

[7] B Wang R D Pace A P Dessai A Bovell-Benjamin andB Phillips ldquoModified extraction method for determining 2-thiobarbituric acid values inmeat with increased specificity andsimplicityrdquo Journal of Food Science vol 67 no 8 pp 2833ndash28362002

[8] R Core Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2013 httpwwwR-projectorg

[9] J I Gray and A M Pearson ldquoRancidity and warmed-overflavorrdquo in Restructured Meat and Poultry Products A MPearson and T R Dutson Eds pp 221ndash269 Van NostrandReinhold Co New York NY USA 1987

[10] C Severini T De Pilli and A Baiano ldquoPartial substitution ofpork backfat with extra-virgin olive oil in lsquosalamirsquo productseffects on chemical physical and sensorial qualityrdquo Meat Sci-ence vol 64 no 3 pp 323ndash331 2003

[11] Y-S Choi J-H Choi D-J Han et al ldquoEffects of replacing porkback fat with vegetable oils and rice bran fiber on the quality ofreduced-fat frankfurtersrdquo Meat Science vol 84 no 3 pp 557ndash563 2010

[12] J G Bloukas E D Paneras and G C Fournitzis ldquoEffect ofreplacing pork backfat with olive oil on processing and qualitycharacteristics of fermented sausagesrdquoMeat Science vol 45 no2 pp 133ndash144 1997

[13] P A Morrissey P J A Sheehy K Galvin J P Kerry and DJ Buckley ldquoLipid stability in meat and meat productsrdquo MeatScience vol 49 no 1 pp S73ndashS86 1998

[14] M A Fellenberg and H Speisky ldquoAntioxidants their effects onbroiler oxidative stress and its meat oxidative stabilityrdquo WorldrsquosPoultry Science Journal vol 62 no 1 pp 53ndash70 2006

[15] K M Wojciak and Z J Dolatowski ldquoOxidative stabilityof fermented meat productsrdquo ACTA Scientiarum PolonorumTechnologia Alimentaria vol 11 no 2 pp 99ndash109 2012

[16] C Summo F Caponio and A Pasqualone ldquoEffect of vacuum-packaging storage on the quality level of ripened sausagesrdquoMeatScience vol 74 no 2 pp 249ndash254 2006

[17] H Bozkurt and O Erkmen ldquoEffects of starter cultures andadditives on the quality of Turkish style sausage (sucuk)rdquoMeatScience vol 61 no 2 pp 149ndash156 2002

[18] I Valencia D Ansorena and I Astiasaran ldquoStability of linseedoil and antioxidants containing dry fermented sausages a studyof the lipid fraction during different storage conditionsrdquo MeatScience vol 73 no 2 pp 269ndash277 2006

[19] E Novelli E Zanardi G P Ghiretti et al ldquoLipid and cholesteroloxidation in frozen stored pork salameMilano andmortadellardquoMeat Science vol 48 no 1-2 pp 29ndash40 1998

[20] L J Marnett ldquoLipid peroxidationmdashDNA damage by malon-dialdehyderdquo Mutation ResearchmdashFundamental and MolecularMechanisms of Mutagenesis vol 424 no 1-2 pp 83ndash95 1999

[21] L J Marnett ldquoOxy radicals lipid peroxidation and DNAdamagerdquo Toxicology vol 181-182 pp 219ndash222 2002

[22] B Ray ldquoSpoilage of specific food groupsrdquo in Fundamental FoodMicrobiology pp 213ndash232 CRC Press Boca Raton Fla USA3rd edition 2003

[23] M Trevisani and R Rosmini ldquoIgiene e tecnologie dei prodotticarneirdquo in Igiene e Tecnologie Degli Alimenti di Origine AnimaleG Colavita Ed pp 169ndash201 Le PointVeterinaire ItalieMilanoItaly 2012

[24] B Sojic N Hromis L Petrovic et al ldquoEffect of packagingmethod and storage period on fatty acid profile and TBARSvalue of traditional sausage (Petrovska Klobasa)rdquo Journal onProcessing and Energy in Agriculture vol 19 pp 105ndash107 2015

[25] S Banon R Serrano and M Bedia ldquoFactors limiting the shelf-life of salami pieces kept in retailing conditionsrdquo Italian Journalof Food Science vol 26 no 3 pp 289ndash299 2014







Editorial Board




Contents




























Acknowledgments




Miguel Elias









1 Introduction












119908de-


















































S saprophyticus


M lylae


[27 43]

KocuriaK varians

K kristinae


[42 44ndash46]






























































Curvacin L curvatus



















LAB

Acidification






YeastsAntioxidant






























































4 Conclusions






Acknowledgments


References
















































































































































































































1 Introduction




































Table2Color

ofthes

urface

andcrosssectio

nof

pork

andpo

ultry-po

rkkabano

sybefore

andaft

erthed

ryingprocess

(a)

Kabano

sy

Surfa

cecolor

Before

drying

process

Afte

rdryingprocess

K180

K270

K360

119871lowast

119886lowast


119886lowast


119886lowast


119886lowast

119887lowast

Pork


aplusmn07199


aplusmn20180

abplusmn1243a

bplusmn13382

aplusmn18164


Poultry-po


aplusmn19147


aplusmn22152


aplusmn22149


(b)

Kabano

sy

Crosssectio

ncolor

Before

drying

process

Afte

rdryingprocess

K180

K270

K360

119871lowast

119886lowast


119886lowast


119886lowast


119886lowast

119887lowast

Pork


aplusmn03156


bplusmn08154


bplusmn17161


Poultry-po


abplusmn19106

aplusmn0653a

bplusmn05555



abAv

eragev

aluesfor

thes

amed


arkedwith

different

lette


at119901le005

















(a)







(b)


















4 Conclusion








References




































1 Introduction





119908)































2





2[26] The












2level The


2










2O CO CO

2 alcohols carbonyl












119908 there











119908inhibit








119882= 086










119908and stored






119908ensures that





3and 150mgkg NaNO

2 They also


































2) and glucose















































2









Acknowledgments


References



































































































































































































































































































































1 Introduction



















2












A

AA A

A A

A

A A A

A A

A

A A A

A A

TcTo1To2


0

2

4

6

8

10

12Fr

ee su

lfhyd

ryl c

onte

nt (m

ol105g

pro)









AB

B B AB

B

AA A C

C C

B A A B AA

TcTo1To2


00

02

04

06

08

10

12

14

16Ca2+

-ATP

ase (

umol

Pim

g pr

om

in)





AA A A

A

A

A

AA A

A

A

AA A A

A

A

0

10

20

30

40

50

60

Surfa

ce h

ydro

phob

icity

TcTo1To2







MHCXLMHC

ACTMTN T

MLC 1


37KD

25 KD

20 KD

90 d60 d30 d15 d7 d0 d(a)

MHC

AC


TMTN T

MLC 1


37KD

25 KD

20 KD

90 d60 d30 d15 d7 d0 d(b)







AA A A

AB ABB B B B A

BB

A ABA A

0

50

100

150

200

250

300

350Br

eaki

ng fo

rce (

g)

TcTo1To2


(a)

AA A A

BA

AB AB A A

A

B AA A

B A

0

2

4

6

8

10

12

14

Pene

trat

ion

dist

ance

(mm

)

TcTo1To2


(b)






C C C B

B B

BB B

AA

BA A A AA

A

54

56

58

60

62

64

66

68

70

Whi

tene

ss

TcTo1To2






4 Conclusion


Additional Points




Acknowledgments


References










































1 Introduction

















(a) (b) (c)















f e e e e e

d dc

bc ba

g f f f f f

d e cdbc ab a

0

4

8

12

16

20

24

Pene

trat

ion

dist

ance

(mm

)

0

100

200

300

400

500

600

700Br

eaki

ng fo

rce (

g)






ed

c c

b

a

FE

DC

BA

0

05

1

15

2

Ca2+

(mm

olk

g)

0

5

10

15

20

25

30

35

TGas

e act

ivity

(Um

L)






STD 22Con 048 030 018 01290∘C

150KD

10KD

20KD

25KD

37KD

50KD75KD

100KD

250KD

(a)

STD

MHC

AC

22 048 030 018 012Con40∘C90∘C

(b)


lowastlowast

lowast

Con 22120583m 048120583m 030120583m 018 120583m 012 120583m

(a)

lowastlowast

lowast

Con 22120583m 048120583m 030120583m 018 120583m 012 120583m

(b)











f ed cd cd bc

cd cd bc bc b a

0

20

40

60

80

100

Wat

er h

oldi

ng ca

paci

ty (

)









Without setting







With setting









4 Conclusions


Additional Points



Competing Interests


Acknowledgments


References










































1 Introduction




































00102030405060708090


00102030405060708090

100

00102030405060708090

100


Taste

6m

wax

6m

cont

rol

7m

wax

7m

cont

rol

6m

wax

6m

cont

rol

7m

wax

7m

cont

rol

6m

wax

6m

cont

rol

7m

wax

7m

cont

rol


0810 0820 0830

10

12

14

16

18

Aw control

MD

A c

ontro

l

0885 0895 0905

06

07

08

09

10

11

Aw beeswax

MD

A b

eesw

ax



4 Conclusions



Additional Points


Competing Interests



Acknowledgments


References


























traditional meat products: improvement of quality and safety

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