Traditional Meat Products: Improvement of Quality and Safety

Journal of Food Quality

Lead Guest Editor: Marta Laranjo Guest Editors: Régine Talon, Andrea Lauková, Maria J. Fraqueza, and Miguel Elias Traditional Meat Products: Improvement of Quality and Safety Journal of Food Quality

Traditional Meat Products: Improvement of Quality and Safety

This is a special issue published in “Journal of Food Quality.” 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 is properly cited. Editorial Board

Encarna Aguayo, Spain Rossella Di Monaco, Italy María B. Pérez-Gago, Spain Riccarda Antiochia, Italy Hüseyin Erten, Turkey Witoon Prinyawiwatkul, USA Jorge Barros-Velázquez, Spain Susana Fiszman, Spain Eduardo Puértolas, Spain José A. Beltrán, Spain Andrea Galimberti, Italy Juan E. Rivera, Mexico Á. A. Carbonell-Barrachina, Spain Efstathios Giaouris, Greece Flora V. Romeo, Italy Marina Carcea, Italy Vicente M. Gómez-López, Spain Jordi Rovira, Spain Maria Rosaria Corbo, Italy Elena González-Fandos, Spain Amy Simonne, USA Egidio De Benedetto, Italy Alejandro Hernández, Spain Giuseppe Zeppa, Italy Alessandra Del Caro, Italy Jesús Lozano, Spain Antimo Di Maro, Italy Sara Panseri, Italy Contents

Traditional Meat Products: Improvement of Quality and Safety Marta Laranjo, Régine Talon, Andrea Lauková, Maria J. Fraqueza, and Miguel Elias Volume 2017, Article ID 2873793, 2 pages

The Use of Starter Cultures in Traditional Meat Products Marta Laranjo, Miguel Elias, and Maria João Fraqueza Volume 2017, Article ID 9546026, 18 pages

The Effect of Drying Parameters on the Quality of Pork and Poultry-Pork Kabanosy Produced according to the Traditional Specialties Guaranteed Recipe Marta Chmiel, Lech Adamczak, Katarzyna Wro'nska, Dorota Pietrzak, and Tomasz Florowski Volume 2017, Article ID 1597432, 7 pages

Health and Safety Considerations of Fermented Sausages Askild Holck, Lars Axelsson, Anette McLeod, Tone Mari Rode, and Even Heir Volume 2017, Article ID 9753894, 25 pages

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

Effects of Micron Fish Bone with Different Particle Size on the Properties of Silver Carp (Hypophthalmichthys molitrix) Surimi Gels TaoYin,JaeW.Park,andShanbaiXiong Volume 2017, Article ID 8078062, 8 pages

Effects of Beeswax Coating on the Oxidative Stability of Long-Ripened Italian Salami Marcello Trevisani, Matilde Cecchini, Daniela Siconolfi, Rocco Mancusi, and Roberto Rosmini Volume 2017, Article ID 8089135, 5 pages Hindawi Journal of Food Quality Volume 2017, Article ID 2873793, 2 pages https://doi.org/10.1155/2017/2873793

Editorial Traditional Meat Products: Improvement of Quality and Safety

Marta Laranjo,1,2 Régine Talon,3 Andrea Lauková,4 Maria J. Fraqueza,5 and Miguel Elias1,6

1 Instituto de Cienciasˆ Agrarias´ e Ambientais Mediterranicasˆ (ICAAM), Universidade de Evora,´ PolodaMitra,Ap.94,´ 7002-554 Evora,´ Portugal 2Instituto de Investigaçao˜ e Formaçao˜ Avançada (IIFA), Universidade de Evora,´ Evora,´ Portugal 3Universite´ Clermont Auvergne, INRA, MEDIS, Clermont-Ferrand, France 4Institute of Animal Physiology, Slovak Academy of Sciences, Koˇsice, Slovakia 5CIISA, Faculty of Veterinary Medicine, University of Lisbon, Avenida da Universidade Tecnica,´ Polo´ Universitario´ do Alto da Ajuda, 1300-477 Lisbon, Portugal 6Departamento de Fitotecnia, Escola de Cienciasˆ e Tecnologia, Universidade de Evora,´ PolodaMitra,Ap.94,7002-554´ Evora,´ Portugal

Correspondence should be addressed to Marta Laranjo; [email protected]

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

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

What are traditional meat products?Traditionalmeatprod- M.Trevisanietal.studiedtheeffectofbeeswaxcoating ucts are high sensory quality foods, usually with high nutri- of foods on the oxidative stability of Italian salami. Beeswax tional value, produced in a small scale, using ingredients coating constitutes a barrier to oxygen, light, and vapour, and procedures from ancient times. Producers must have the which may prevent oxidation of fat and pigments and ability to satisfy the expectations of consumers, regarding water loss. Furthermore, the authors concluded that beeswax sensory, nutritional aspects and safety, considering that they coating prevents case hardening and facilitated the peeling. are increasingly more demanding and more informed, turn- T.Yinetal.haveevaluatedthequalityofsilvercarp ing this into an important challenge. Usually, a meat product (Hypophthalmichthys molitrix) surimi (SCS) gels incorpo- is defined as foods that consist of or contain meat. The flesh of ratedwithfishboneandfoundoutthatsizereductionofthe an animal, typically a mammal or bird, is considered as meat. fish bone improved the quality of the SCS gel maintaining However, fish meat is also present in our diet and several better gel matrices. traditional processed products come from it. Thus, traditional M. Chmiel et al. have studied two types of kabanosy meat products sensu lato include food products derived from sausages, Traditional Specialties Guaranteed (TSG), from both meat and fish meat. Poland and concluded that their drying process could be All over the world, different processes are used to pre- shortened without compromising the authenticity of the serve meat and the particular know-how of people living products and with the concomitant advantages to producers. in different regions gave rise to a great diversity of meat R. Zhang et al. investigated the effects of different ozone products according to their traditions and historic use. These treatments on the physicochemical characteristics of myofib- traditional processes, their particularities, and their effect rillar proteins from silver carp (H. molitrix) surimi during on the quality and safety of meat products are important frozen storage. research topics. The scope of this edition was to disseminate A. Holck et al. reviewed the possible health effects of the high-quality research related with traditional meat products ingredients used in fermented sausages. Recent attempts to and review recent developments on the quality and safety improve sausages from the nutritional point of view include improvement of traditional meat products worldwide, such partial replacement of saturated by unsaturated fats, reducing astheuseofstarterculturesortheapplicationofnew the use of sodium chloride or replacement by potassium preservation methods. chloride, and the use of selected starter cultures. They also Among the 16 submitted manuscripts, six have been review the processing and postprocessing strategies to inhibit selected to be part of this special issue. the growth of food pathogenic microorganisms, such as 2 Journal of Food Quality

Escherichia coli, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, Clostridium botulinum,andToxo- plasma gondii, and reduce their presence in the products. The paper “The Use of Starter Cultures in Traditional MeatProducts”byM.Laranjoetal.reviewstheroleandmode of action of bacterial and fungal starter microbiota, focusing on the development of starter cultures better adapted to the meat matrix. Furthermore, omics approaches on starter cultures are revised, because the use of these techniques allows rapid screening of strains for desirable functional characteristics. Submitting authors come from five different countries, four European (Italy, Poland, Norway, and Portugal) and one non-European, China. We are pleased to introduce this special issue, which includes six papers that provide new insights on the manufacturing and processing of traditional meat products sensu latoandwewishthatthereadersofthisjournalfindthisissue of 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 their valuable participation in the evaluation process. M. Laranjo acknowledges a Post-Doc research grant from Fundac¸ao˜ para aCienciaˆ e a Tecnologia (FCT) (SFRH/BPD/108802/2015). Marta Laranjo Regine´ Talon Andrea Laukova´ Maria J. Fraqueza Miguel Elias Hindawi Journal of Food Quality Volume 2017, Article ID 9546026, 18 pages https://doi.org/10.1155/2017/9546026

Review Article The Use of Starter Cultures in Traditional Meat Products

Marta Laranjo,1,2 Miguel Elias,1,3 and Maria João Fraqueza4

1 Instituto de Cienciasˆ Agrarias´ e Ambientais Mediterranicasˆ (ICAAM), Universidade de Evora,´ Polo´ da Mitra, Ap. 94, 7002-554 Evora,´ Portugal 2Instituto de Investigaçao˜ e Formaçao˜ Avançada (IIFA), Universidade de Evora,´ Evora,´ Portugal 3Departamento de Fitotecnia, Escola de Cienciasˆ e Tecnologia, Universidade de Evora,´ Polo´ da Mitra, Ap. 94, 7002-554 Evora,´ Portugal 4CIISA, Faculty of Veterinary Medicine, University of Lisbon, Avenida da Universidade Tecnica,´ Polo´ Universitario´ do Alto da Ajuda, 1300-477 Lisbon, Portugal

Correspondence should be addressed to Marta Laranjo; [email protected]

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 objectives related to meat products’ quality and safety improvement, the selection of particular strains constituting a starter culture should be carried out in the context of its application, since its functionality will depend on the type of sausage and process conditions. Also, strain selection should comply with particular requirements to warrant safety. The aim of the current review is to update the knowledge on the use of starter cultures in traditional meat products, with focus on dry-fermented products. In this manuscript, we will try to give answers to some relevant questions: Which starter cultures are used and why? Why are LAB used? What are their role and their specific mode of action? Which other groups of microorganisms (bacteria and fungi) are used as starter cultures and how do they act? A particular revision of omics approach regarding starter cultures is made since the use of these techniques allows rapid screening of promising wild strains with desirable functional characteristics, enabling the development of starter cultures better adapted to the meat matrix.

1. Introduction Fermented meat products may be manufactured without the use of starter cultures, although their use can help to Starter cultures or starters are individual or mixed formula- ensure safety, standardising product properties (including tions of selected strains with a particular enzymatic activity flavour and colour), and shorten the ripening period. Nev- that when added in a defined concentration to a substrate ertheless, well-adapted and qualified presumption of safety transformitintoafoodproductwithspecificcharacteristics (QPS) strains must be used and the establishment of the [1]. This concept applied to meat products could be described starter culture must be verified in order to guarantee the as viable microorganisms that are able to multiply themselves expected performance. inside meat products, increasing their preservation, control- Probiotics are live microorganisms that confer a health ling their hygienic safety, and potentiating their acceptability benefittothehostwhenadministeredinadequateamounts by consumers, maintaining or improving their nutritional [3]. Probiotics have been used in food products, food sup- quality [1]. plements, and pharmaceutical products. Due to increasing The preliminary use of starters in meat products resulted concerns over health, probiotic foods (e.g., probiotic dairy from adding a portion of the final meat products to their raw products) are now accepted in the world market. Recently, the materials, meaning that part of the already fermented batch possibility of developing probiotic meat products has been of sausage was thrown back into the new mix. This already discussed [4]. By using probiotic starter microorganisms, fermented product contained the necessary microorganisms potentialhealthbenefitscanbeintroducedtomeatproducts to start the fermentation of the new batch. This is known as and it is already possible to produce probiotic meat products back-slopping or back-inoculation [2]. [5, 6]. Nevertheless, the potentially beneficial effects on 2 Journal of Food Quality human health from eating a probiotic sausage still need Additionally, microbial substances, namely, bacteriocins, confirmation [7, 8]. produced by Gram-positive species of the LAB group, such The starter groups used nowadays in meat industry are, as, for example, nisin and other lantibiotics or pediocin-like by order of importance, lactic acid bacteria (LAB), Gram- bacteriocins, have an antimicrobial role with an effect on positive catalase-positive cocci (GCC+) (mainly staphylo- preservation and safety. cocci), moulds, and yeasts. Starter cultures have a number of advantages: Lactic acid bacteria (LAB) are a group of Gram-positive (i) They are of known quantity and quality. bacteria belonging to the Firmicutes. They are catalase- negative, either rod-shaped (bacilli) or spherical (cocci), (ii) They reduce the ripening time. characterised by an increased tolerance to acidity (low pH (iii) They increase safety by outcompeting undesirable range), and have a low GC (guanine-cytosine) content. microorganisms. Although many genera of bacteria produce lactic acid as (iv) They enable the manufacture of a product of constant a primary or secondary end-product of fermentation, the quality all year round in any climatic zone, as long term lactic acid bacteria (LAB) is conventionally reserved as proper natural conditions or fermenting/drying for genera in the order Lactobacillales, which includes Aero- chambers are available. coccus, Carnobacterium, Enterococcus, Lactococcus, Lacto- bacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Theaimofthecurrentreviewistoupdatetheknowledgeon Tetragenococcus, Vagococcus,andWeissella [9]. As food fer- the use of starter cultures in traditional meat products, with mentation agents LAB are involved in making yogurt, cheese, focus on dry-fermented products. cultured butter, sour cream, sausage, cucumber pickles, In this manuscript, we will try to give answers to some olives, and sauerkraut, some species may spoil beer, wine, and relevant questions on this subject, through the analysis of processed meats [10]. published studies with some applied results. Which starter Gram-positive catalase-positive cocci (GCC+) are the cultures are used and why? Why are LAB used? What is second most important group of meat starters and are com- their role and their specific mode of action? Which other posed of nonpathogenic coagulase-negative staphylococci groups of microorganisms (bacteria and fungi) are also used (CNS). The most important starters from this group are as starter cultures and how do they act? What is their strains belonging to the genera Staphylococcus and Kocuria function? A revision related to omics methods applied to the [11]. screening of autochthonous strains with desirable functional At the beginning of the ripening process, the surface characteristics, allowing the development of well adapted mycobiota is mainly composed of yeasts; however, as 𝑎𝑤 de- starterculturestothemeatmatrix,willbedone. creases, moulds outcompete yeasts and predominate in the final product [12]. Moulds colonise the surface of fermented 2. Starter Cultures in meat products, in some cases conferring particular charac- Dry-Fermented Meat Products teristics, however, in other cases being considered signs of spoilage. The first generation of meat starter cultures was generally Yeasts are characteristic components of the mycobiota based on microorganisms isolated from vegetable fermenta- growing on fermented sausages. Their origin is mainly related tion, such as L. plantarum and members of the genus Pedio- to the environment and to the meat used as raw material, coccus. Then, a second generation of starter cultures com- since yeasts are naturally found on fresh meat. The most com- prising meat-borne strains, such as L. sakei and coagulase- mon genera are Candida, Rhodotorula, Debaryomyces,and negative staphylococci (CNS), was developed, harbouring Trichosporon. In fermented meats, the lactic acid produced by phenotypic traits of technological relevance [17]. More LAB changes the environment, favouring the development of recently,effortshavebeendedicatedtothestudyofthephys- yeasts, which use all of the nutrients and energy and grow fast iological and technological properties of LAB and CNS iso- [13]. lated from traditional fermented sausages, in order to develop Meat preservation by fermentation has been carried out functional starter cultures that enhance safety and nutritional for thousands of years, but the idea of starter cultures was advantages while maintaining industrial performance [5, 18]. first introduced for dry sausages in the 1940s with Patent US The manufacturing of dry-fermented sausages involves 2225783 A [14]. The first commercial starter culture wasa spontaneous fermentation commanded by bacteria (LAB) strain of Pediococcus acidilactici that was made available in and GCC+ and, less importantly, by fungi, namely, moulds the US in 1957 [15]. In Europe, the first starter culture to be and yeasts [19]. introduced was strain M53 from the genus Kocuria,isolated Most meat starter cultures commercially available are from a Finnish sausage, which was used to prevent colour and combined cultures of LAB (mainly Lactobacillus spp. and aroma defects [16]. Pediococcus spp.) and GCC+ (primarily Staphylococcus spp. Starter cultures play an essential role in the manufacture and Kocuria spp.). These bacteria are responsible for the of fermented food products. Starters composed of LAB microbial reactions that occur during meat fermentation, strains produce the lactic acid that acts on meat proteins such as acidification, catalase activity, and bacteriocin pro- modifying their water biding capacity, thus contributing duction [11]. to texture, moisture content, flavour, and aroma of the Several studies have addressed the importance of using products, and definitively acts on its microbiological safety. starter cultures in traditional dry-fermented meat products Journal of Food Quality 3 not only for safety or conformity reasons, but also for acid [9]. Homofermentative LAB include some lactobacilli uniformity purposes [20–22]. and most enterococci, lactococci, pediococci, streptococci, Although most studies about the use of starter cultures tetragenococci, and vagococci that ferment hexoses through are on dry-fermented sausages [23–25], a few works on other glycolysis by the Embden-Meyerhof-Parnas pathway. On meat products, such as hams or fresh sausages, have also been the other hand, heterofermentative LAB ferment pentoses reported [26]. mainly through the phosphoketolase pathway and include Inoculation of starter cultures in dry-fermented meat leuconostocs, some lactobacilli, oenococci, and Weissella products may occur either by incorporation as an ingredient species. in the meat batters or by surface inoculation. Relevant technological features for LAB starters include Bacteria are usually incorporated in the meat batters at fast production of lactic acid; growth at different temper- concentrations between 5 and 8 log colony forming units atures, salt concentrations, and pH values; gas production (cfu)/g [23]. Yeasts may be inoculated either on the surface from carbohydrates; catalase activity and hydrolysis of hydro- of the sausage or in the meat batter at a concentration typ- gen peroxide; nitrate and nitrite reduction; moderate prote- ically between 4 and 6 log cfu/g. Moulds are always surface- olytic and lipolytic enzymatic activities; good performance inoculated, due to their strictly aerobic character, frequently in combined starters with other microbial components by dipping in an aqueous solution of spores at concentrations [29]. 2 ranging from 3 to 4 log spores/cm . However, fermentation conditions must be controlled to avoid excessive pinholes, gas pockets, and off-flavours, resulting from gas production from carbohydrates [30]. 2.1. Their Role in Quality Improvement of Sausages. The selec- Additionally, the production of hydrogen peroxide may result tion of starter cultures for quality improvement of sausages is in undesirable oxidation, known as greening [30]. Further- basedontechnologicallyrelevanttraits.Theautochthonous more,itmustbetakenintoaccountthatproteolyticand microbiota of sausages and other meat products, as well as the lipolytic activities should be moderate, to avoid undesirable microbiota of the processing environment of the production sensory changes. units, may be a good starting point for the isolation of As for the role of LAB in the quality of dry-fermented potential starters, because those strains are well adapted to meat products, LAB participate in the coagulation of muscle the meat environment [19]. proteins by acidifying the batters, which results in increased Bourdichon and coworkers [27] presented a list of micro- slice stability, firmness, and cohesiveness of the final product organisms used in food fermentation in a wide range of food [31, 32]. Besides, they contribute to the flavour of the final matrices (dairy products, meat, fish, vegetables, legumes, product through the formation of noticeable acidic and cereals, beverages, and vinegar). vinegary (acetic acid) tastes. Moreover, the existing acidic conditions may increase the activity of cathepsin D, which is 2.1.1. Bacteria: LAB and GCC+. When selecting starter cul- againresponsibleformuscleproteolysis[33]. tures for dry- and semidry-fermented sausages, LAB and Several authors have reported the use of LAB starter CNS strains with useful metabolic activities and benefits cultures for the production of fermented sausages [34–38]. during fermentation should be used. For example, Wang and coworkers reported the inoculation with L. sakei as beneficial for microbiological quality against (1) Lactic Acid Bacteria (LAB).Lacticacidbacteria(LAB) the growth of foodborne pathogens, also improving sensory are Gram-positive, non-spore-forming cocci or bacilli with characteristics [34]. a low GC content [28]. They generally are nonrespiratory and lack catalase. They produce lactic acid as one of the main (2) Gram-Positive Catalase-Positive Cocci (GCC+).Gram- fermentation products of carbohydrates. They lack genuine positive catalase-positive cocci GCC+, mainly nonpathogen- catalase and do not possess cytochromes. All LAB grow ic coagulase-negative staphylococci (CNS), are also impor- anaerobically,butunlikemostanaerobes,theygrowinthe tant in the fermentation process of sausages, since they presence of O2 as “aerotolerant anaerobes” [9]. improve the quality of the final product, while standardis- According to the current taxonomic classification, they ing the production process. They enhance colour stability, belong to the phylum Firmicutes, class Bacilli, order Lacto- contribute to flavour development, and reduce spoilage. The bacillales. Six different families include all genera, as shown ones most frequently isolated from fermented sausages are in Table 1 (http://www.uniprot.org/taxonomy/186826). summarised in Table 2. Lactic acid bacteria are among the most important The use of coagulase-negative staphylococci (CNS) as groups of microorganisms used in food fermentation. They meat starter cultures contributes to an adequate colour contribute to the taste and texture of fermented products development based on their nitrate reductase activity. On the and inhibit food spoilage bacteria by producing growth- otherhand,theircatalaseactivityreducesoxidativedamage inhibiting substances and large amounts of lactic acid. and their metabolism contributes to flavour. The flavour- Basedonsugarfermentationpatterns,therearetwobroad generating potential of CNS is even more important when metabolic categories of LAB: homofermentative and hetero- producing low-salt [47, 48] or low-fat [49, 50] sausages [51]. fermentative. The homofermentative pathway produces basi- However,thefullmetabolicpotentialofCNSshouldbe callyonlylacticacid,whereastheheterofermentativepathway further explored, so that we may take advantage of more produces CO2 and ethanol or acetate in addition to lactic technological features of CNS [52]. 4 Journal of Food Quality

Table 1: Families and genera of LAB.

Family Genus Cellular morphology Sugar fermentation Aerococcaceae Aerococcus Cocci-tetrads Homofermentative Carnobacteriaceae Carnobacterium Bacilli Homofermentative Enterococcus Cocci Homofermentative Enterococcaceae Tetragenococcus Cocci-tetrads Homofermentative Vagococcus Cocci Homofermentative Lactobacillus Bacilli Strain-dependent Lactobacillaceae Pediococcus Cocci-tetrads Homofermentative Leuconostoc Cocci Heterofermentative Leuconostocaceae Oenococcus Cocci Heterofermentative Weissella Cocci/bacilli Heterofermentative Lactococcus Cocci Homofermentative Streptococcaceae Streptococcus Cocci Homofermentative

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

Family Genus Species Metabolic activities References S. xylosus (i) Nitrate reductase S. carnosus Staphylococcaceae Staphylococcus (CNS) (ii) Proteolytic [39–42] S. equorum (iii) Lipolytic S. succinus (iv) Catalase S. saprophyticus (i) Nitrate reductase M. luteus (ii) Antioxidative Micrococcus [27, 43] Micrococcaceae (iii) Catalase M. lylae (iv) Lipolytic (v) Proteolytic K. varians (i) Nitrate reductase Kocuria (ii) Proteolytic [42, 44–46] K. kristinae (iii) Lipolytic

Besides contributing to flavour, Staphylococcus and Kocu- frankfurter-type meat emulsion in degrading polychlori- ria also provide nitrate-reductase and antioxidant activities nated biphenyls (PCBs) [59]. Furthermore, quite a few works [53, 54]. have been published reporting the results obtained by the Numerous studies addressing the use of starter cultures in utilisation of mixed starter cultures (LAB and CNS) [25, 60– meat products have been published, with both single (either 66]. LAB or GCC+) and mixed cultures. Bacteriocinogenic LAB and selected strains of S. xylo- Several authors have reported the use of CNS starter cul- sus and S. carnosus are commercially available for use in tures for the production of fermented sausages. According to improving the safety, colour, and flavour of final products. It is Ravyts et al. [51], the success of CNS in flavour development also important to assess positive interactions, such as growth seems to be determined by acidification. and proteolytic activity, among the different starter cultures Hugas and Monfort [31] highlighted the need to use strains [67–70]. selected strains of GCC+ to ensure sensory quality. Besides, The effect of different starter culture combinations other authors have described the capability of S. xylosus and S. (Staphylococcus carnosus, Pediococcus pentosaceus,andLac- carnosus strains to modulate aroma through the degradation tobacillus sakei)onthequalityofTurkishtypefermented of amino acids and free fatty acids (FFAs) [55–57]. sausage (Sucuk) has been evaluated during ripening and it Autochthonous strains of S. xylosus have been recom- was concluded that the use of lipolytic starter cultures (S. mended for the production of very aromatic sausages in carnosus/L. sakei) would have a positive effect in acceler- Southern Europe, instead of the less adapted commercial ating ripening and enhancing the quality of dry-fermented starter cultures [58]. sausages [71]. Lusnicandcolleagueshavestudiedtheeffectofan Tremonte and coworkers demonstrated that S. xylosus added starter culture (S. xylosus and S. carnosus)toa and Kocuria varians are able to stimulate the growth of L. Journal of Food Quality 5 sakei strains, positively influencing the proteolytic activity of (ii) It metabolizes peroxides, protecting fat from oxida- strains in a combined use [66]. tion, thus preventing rancidity [12].

Casqueteandcolleagueshaveemphasisedtheimportance (iii) It reduces O2 levels on the product surface, thus of autochthonous starter cultures in improving homogeneity avoiding oxidative processes and improving meat and safety of fermented meat products, without depreciating colour [80]. their sensory characteristics [60–62]. Furthermore, they have highlighted the importance of choosing a starter formulation (iv) It contributes to the flavour of the final product, consisting of a combination of strains that is appropriate for by breaking up fats, proteins, and lactic acid, thus each ripening procedure [60]. favouring pH increase [12]. We may conclude that flavour and aroma of fermented The use of moulds as a seasoning for sausage can have sausages result from the combined action of different bacte- both desirable and undesirable consequences. The desirable ria: LAB produce lactic acid and small amounts of acetic acid, consequences are the creation of a successful product that ethanol, and acetoin; however, the proteolytic and lipolytic appeals to consumers. The undesirable consequences are activities of both LAB and GCC+ are essential to the sensory health risks associated with the growth of undesirable moulds quality of fermented sausages. that produce highly toxic secondary metabolites, mycotoxins, such as ochratoxin A (OTA), or penicillin produced by 2.1.2. Fungi: Yeasts and Moulds. Fungi generally contribute species of Penicillium [81]. to a characteristic flavour of some fermented meat products. Furthermore, surface moulding of fermented meat prod- Yeasts may be either inoculated in the meat batters or surface- ucts was observed during storage and can be a quality prob- inoculated, whereas moulds are always inoculated at the lem, because of the undesirable effects, mainly connected to surface of sausages. Surface inoculation has a further physical the production of off-flavours [81]. protective role. Surface mould inoculations were traditionally done with the autochthonous mycobiota, which was mainly composed (1) Yeasts. The first studies with yeasts in fermented sausages of Penicillium spp., Aspergillus spp., or Scopulariopsis spp. were conducted in the first decades of the 20th century, when The first toxicologically and technologically suitable mould the importance of the “fleur du saucisson” was recognized starter culture for meat products, P. nalg i ov e n s e strain, was and the use of pure yeast cultures for flavouring in fermented selected by Mintzlaff and Leistner in 1972 [82]. However, sausages began to be recommended. Later on, it was estab- nowadays, a wide assortment of industrialised starter cultures lished that yeasts are part of the microbiota of fermented is commercially available as an alternative to the inoculating sausages and their use as starter cultures was suggested, mixtures composed of autochthonous strains. because the addition of selected Debaryomyces strains could Some studies on the use of mould starter cultures have improve the curing, colour, and flavour of sausages [72]. already been performed [80, 83]. For example, quality traits of Several studies have tried to understand the role of yeasts wild boar mould-ripened salami manufactured with different as secondary microbiota in fermented meat products. Yeast selections of meat and fat tissue and with and without strains belonging to the genera Debaryomyces, Yarrowia, commercial bacterial starter cultures have been investigated Pichia, Rhodotorula, Cryptococcus,andTrichosporon have [84]. The use of a bacterial starter culture in the manufac- been isolated from meat products [73], with clear predomi- tureofmould-ripenedwildboarsalamiresultedinsignifi- nance of the Debaryomyces genus [13]. cantly lower peroxide values, lower TBARS concentrations, Someyeastshavebeenshowntocontributetoflavour and lower amounts of biogenic amines, namely, histamine, andtexturedevelopmentthroughoutthecuringofvarious cadaverine, and putrescine, associated with better sensory products [74–76]. Moreover, some studies have shown that evaluation scores. thecharacteristicflavourofdry-curedmeatproductsmaybe Application of commercial moulds to sausage surfaces developed through the influence of yeasts [77–79]. improves primarily the safety towards regarding mycotoxin Furthermore, the manufacture of dry-fermented sausages production. Moreover, the production of antibiotics, namely, with optimised concentrations of Debaryomyces spp. in the penicillin, also needs to be controlled [82]. Additionally, presence of LAB and CNS has been demonstrated to have sausageproducersachievemoreconsistentflavour,taste,and a positive effect on the final flavour and sensory quality by drying rate and a more uniform appearance. inhibiting the development of rancidity and generating ethyl Table 3 shows a list of moulds found in fermented meat esters that contribute to the proper sausage aroma [78]. products. Among the species mentioned in Table 3, P. nalg i ov e n s e (2) Moulds. Surface moulding of fermented meat products and P. g l adi oli are currently considered safe and are commer- is considered a desirable event in most European countries, ciallyavailabletobeusedasstarterculturesinmeatproducts which include Italy, Romania, Bulgaria, France, Hungary, [12]. Switzerland, Southern Germany, Spain, Austria, and Belgium [12].Infact,thepresenceofmyceliumatthesurfaceof 2.2. Antimicrobial Activity of Starter Cultures. Bacteriocins, sausages has several main advantages: natural antimicrobial peptides, and the acid lactic produced from glucose could be used to improve the quality and safety (i) It prevents excessive drying, allowing homogeneous of meat products by avoiding the presence of pathogens, such dehydration of the product [12]. as Listeria monocytogenes and spoilage microorganisms, and 6 Journal of Food Quality

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

Common species Uncommon species Penicillium nalgiovense P. waksmanii Mucor spp. P. gladioli Aspergillus ochraceus Scopulariopsis spp. P. camemberti E. herbariorum Cladosporium spp. P. chrysogenum E. repens Eupenicillium spp. P. aurantiogriseum A. niveus Eurotium spp. P. bre v i comp ac tum P. c itr inum Talaromyces spp. P. nordicum A. candidus Geotrichum candidum P. phoeniceum P. crustosum Talaromyces wortmannii Eurotium rubrum P. commune P. griseofulvum A. sclerotiorum P. ol s onii A. versicolor P. impli catum P. alii Scopulariopsis candida P. fellutanum P. s olitum improving the competitiveness of their producers for survival concentrations, and, to a lesser extent, oxygen availability are [85]. among the most important preservative conditions during A list of the main bacteriocins produced by LAB along meat fermentation [17]. with a list of bacteria they are effective against is summarised The main metabolic activities and their corresponding in Table 4. technologicalrolesforthemainmicrobialstartergroupsare Several L. sakei and L. curvatus have been reported shown in Table 5. asbacteriocinproducersandhavebeenusedasprotective In general, CNS are poorly competitive in the presence cultures, and their activity against L. monocytogenes has been of acidifying LAB strains [99]. On the other hand, strains of proved in meat products [87–90]. L. sakei have shown superior competitiveness, which could Lactococcus lactis and Enterococcus spp. strains isolated probably be explained by their specialised metabolic reper- from different food matrices have been shown to produce toire well adapted to the sausage environment, including the bacteriocins [91–93]. arginine deiminase (ADI) pathway [100] and the utilisation Pediococcus acidilactici MCH14 pediocin-producing of nucleosides [101]. strain and the pediocin PA-1 itself have been demonstrated Genus-specific and species-specific PCR and real-time to inhibit the growth of the foodborne pathogens L. monocy- RT-PCRmethodshavebeenusedtomonitorandquantifythe togenes and Clostridium perfringens in Spanish dry-fer- populations of the inoculated starter cultures [24]. Moreover, mented sausages and frankfurters [94]. RT-PCR-DGGE and RNA-based pyrosequencing of the 16S Bacteriocins produced by strains of L. plantarum isolated rRNA gene have also been used to monitor the microbiota of from Portuguese traditional pork products have been shown fermented sausages [102]. to have a broad spectrum of activity [95]. LAB starter cultures have been used in the production 2.4. Safety of Selected Meat Starter Cultures. Meat starter of Nham, which is a Thai-style fermented pork sausage, cultures or food cultures (FC) are safe live bacteria, yeasts, for their antilisterial activity in order to reduce the severity or moulds used in food production, and they are in them- of postacidification and increase the shelf life of Nham at selves a characteristic food ingredient (http://www.effca.org/ ambient temperature [96, 97]. content/food-culture). Food starter cultures (microorgan- Additionally, also S. xylosus strain SX S03/1 M/1/2 has isms) used directly in food production are regarded as food been shown to produce a thermostable bacteriocin which ingredients in the European Union (EU). Starters enter in a could be used as starter culture or meat additive to prevent category of food ingredients with a very long history of use possible handling or meat processing contamination [98]. in a great variety of food products. If a starter is added to a food product, the requirements established in the General 2.3. Competitiveness of Starter Cultures. One of the most Food Law should be accomplished by the food operator. The important properties of meat starter cultures is the ability food cultures used as starters in the fermentation of foods to colonize the meat environment, in competition with the are not subject to EU premarketing regulation, unless they autochthonous microbiota and dominating the microbial are regarded as being novel to the EU market and their con- community of fermented products. The starter culture must sumers. Many starters were selected from fermented foods compete with the natural microbiota of the raw material, and several microorganisms are present in spontaneously which carries out the expected metabolic activities through fermented foods. However, regarding safety concerns, any its growth rate and survival under the prevailing conditions food cultures to be introduced in a food should be evaluated. during sausage production. Low temperatures, high salt The approaches for assessing the safety of microorganisms Journal of Food Quality 7

Table 4: LAB bacteriocins, bacteriocin producers, and susceptible pathogenic bacteria.

Bacteriocin Bacteriocin producer Susceptible pathogenic bacteria Listeria monocytogenes Staphylococcus aureus Enterococcus spp. Brochothrix thermosphacta Sakacin Lactobacillus sakei Pseudomonas spp. Campylobacter spp. Escherichia coli Klebsiella spp. Other LAB Listeria monocytogenes Staphylococcus aureus Clostridium perfringens Clostridium tyrobutyricum Bacillus cereus Plantaricin L. plantarum Enterococcus spp. Brochothrix thermosphacta Pseudomonas spp. Salmonella spp. Escherichia coli Other LAB Listeria monocytogenes Staphylococcus aureus Brochothrix thermosphacta Curvacin L. curvatus Pseudomonas spp. Escherichia coli Other LAB Listeria monocytogenes Staphylococcus aureus Nisin Lactococcus lactis Clostridium tyrobutyricum Other LAB Listeria monocytogenes Pediocins Pediococcus spp. Enterococcus spp. Other LAB Adapted from Fraqueza et al. [86]. entering the human food chain differ considerably depending external experts and is based on the history of use, body of on the applicable legislation, if any. knowledge, and the absence of adverse effects at the strain Several approaches have been delineated in order to level. consider the starter cultures safe. The Qualified Presumption Food cultures with a long history of safe use in food of Safety (QPS) list is the EFSA fast track risk assessment are considered as traditional food ingredients and are legally tool that is used by EFSA panels when evaluating prod- permittedforuseinfoodsintheEUwithoutpremarket ucts with microorganisms that require a premarket autho- authorisation, as described earlier. As a consequence, EFSA risation (e.g., feed additive cultures, cell factories produc- panels do not evaluate microbial strains of food cultures. ing enzymes/additives/vitamins, novel microorganisms, and Nevertheless, the QPS list can be consulted when safety plant protection). This approach is restricted only to the evaluations of food culture are made. microorganisms related to regulated food and feed products Microorganisms, which are not on the QPS list, are not and is based on history of use, body of knowledge, and necessarily considered to be unsafe and their assessment the absence of adverse effects at the taxonomic unit level regarding antibioresistance, virulence, and biogenic amine [103, 104]. characterization should be done. The GenerallyRecognizedasSafe(GRAS) status is open to The International Dairy Federation (IDF) and the Euro- all types of food additives, which include food cultures. The pean Food and Feed Cultures Association (EFFCA) have determination of GRAS status is made by the FDA and/or proposed additional tools and methods to evaluate the safety 8 Journal of Food Quality

Table 5: Requirements for starter LAB, GCC+, yeasts, and moulds.

Microbial group Metabolic activity Technological role Modulate flavour (acid/tangy) Inhibit pathogens Acidification Develop texture Accelerate drying LAB Proteolysis Develop flavour Inhibit pathogens Antimicrobial Extend shelf life Antioxidant Protect colour Probiotic Compete in the gastrointestinal tract Nitrate reductase Develop typical red (cured) colour GCC+ Degradation of amino acids and FFAs Develop flavour Antioxidant Yeasts Proteolytic Prevent rancidification Lipolytic Moulds Antioxidant Prevent rancidification Adapted from [17].

of food cultures with the unique target of keeping a high level Safety hazards associated with CNS were mostly limited offoodsafetyandtoprotecthumanlifeandhealth.According to the presence of antibiotic resistance [108]. CNS strains to Laulund et al. [105], whatever the strategy applied, it is resistant to multiple antibiotics have been reported [109]. imperative to have an evaluation of the food cultures’ safety Kastner et al. [110] detected the tetracycline resistance genes at three levels: (a) at the strain level, (b) during production, tetKinStaphylococcus spp. starter cultures. and (c) in the process it is applied to and throughout the shelf The detection of antibiotic resistant (AR) strains among life of the food. LAB has resulted in their recognition as a reservoir of AR genes horizontally transmissible to pathogens through the 2.4.1. Assessment of Antibioresistance. The One Health con- food chain, which constitutes a problem [111, 112]. Antibi- cept recognises that the health of people is connected to otic multiresistant strains of lactobacilli and other LAB thehealthofanimalsandtheenvironment.Thefoodchain have been isolated from dry-fermented meat products [113– has been recognized as one of the main routes for the 120]. LAB possesses a broad spectrum of natural (intrinsic) transmission of antibiotic-resistant bacteria between animal and acquired antibiotic resistance. However, only resistance and human populations [106]. Antibiotic resistant bacterial acquired by mutation or horizontal gene transfer poses a risk strains may be a potential direct link between the indigenous for public health [121]. microbiota of animals and the human gastrointestinal tract. The most common resistance genes detected in LAB Bacterial strains selected as starters with technological isolated from dry-fermented sausages are the tetracycline or food protective characteristics to be introduced in food resistance genes tetM, tetW, a n d tetS and the genes coding always need to be phenotypically assessed for antibiotic for erythromycin resistance, ermBandermC [117, 120]. These resistance to clinically relevant antibiotics. The phenotypic are genes linked to mobile elements, and if the phenotypic testing based on determination of a minimum inhibitory expression of antibiotic resistance is expressed, their presence concentration (MIC)foraselectedgroupofantimicrobials is considered a hazard. should be performed. The absence of phenotypic antibiotic resistance is preferred, but if a resistance profile is observed, 2.4.2. Detection of Strains Producers of Biogenic Amines. aproperanalysisofthewholegenomepotentiallycombined Any strains to be incorporated as starters in fermented with information that the observed resistance is not transfer- meat products should be assessed for their (in)ability to able is needed; only then can the strain(s) be considered safe mediate the production of biogenic amines. Strategically, for use in food culture [107]. the use of Lactobacillus spp. or Pediococcus spp. non-BA The possibility of antimicrobial resistance transfer from producer strains could dominate and avoid the presence of viable microorganisms to other microorganisms is related high contents of BA in meat products. Several authors have to the genetic basis of the resistance being considered most reported the important role of starter cultures in decreasing plausible, when the resistance is mediated by added/acquired the content in biogenic amines [47, 48, 122–126]. genes. Regarding this possibility, several safety assessments have been done by several authors on the species usually 2.4.3. Toxigenic Potential. Among LAB, enterococci play an selected for starters, such as CNS or LAB. important role in food fermentation and may contribute to Journal of Food Quality 9 the organoleptic uniqueness of some products, but they are inability of S. carnosus to form biofilms may explain why it is also responsible for community-acquired and nosocomial rarely recovered from meat processing environments [142]. infections [118]. Some of the most important virulence LAB biofilms may be used to control the formation of factors include the production of hydrolytic enzymes, namely, biofilms by the foodborne pathogens Listeria monocytogenes, gelatinase,lipase,andDNase,haemolyticactivityandthe Salmonella Typhimurium, and Escherichia coli O157:H7 [143]. production of cytolysin, the presence of adhesins, and the Genes potentially responsible for biofilm formation and ability to form biofilms [127]. cellular aggregation that may assist the organism to colonize Two studies with enterococci strains isolated from several meat surfaces have been identified in L. sakei strain 23K [144]. Portuguese dry-fermented sausages revealed that although Moreover, the analysis of microenvironments through the meat enterococci harbour antibiotic resistance and produce scanning electron microscopy (SEM) evidenced the presence biofilms, a reduced number of virulence factors were detected of microchannels that favour microbial flow, while the ability [118, 128]. However, a third study with Portuguese dry- of L. sakei to form biofilm guarantees the correct colonisation fermented products from northern Portugal has detected of the different meat niches, throughout the fermentation phenotypic and genotypic evidence of potential virulence process (2017). factors among Enterococcus spp. isolates, which is a reason of Biofilm formation in LAB species has been reported concern [129]. to be a stress response and survival strategy in stressful Some members of the CNS group, primarily S. epider- environments [145, 146]. Some reports have also described midis, are common nosocomial pathogens, and the presence the genes responsible for quorum sensing, adhesion, and of regulatory elements involved in the control of virulence- biofilm formation [147–150]. factor synthesis has recently been identified. Remarkably, Another possible biocontrol strategy to avoid the pres- strains of S. xylosus were isolated from patients who had an ence of pathogens in meat industry could be the use of underlying disease, while the same species has been reported bacteriocins and enzymes; this is considered important for to be involved in infections of poultry [130]. the maintenance of biofilm-free systems and thus for the Although CNS of food origin have not been found to quality and safety of foods. produce nosocomial infections, some strains that produce enterotoxins have been described. Vernozy-Rozand et al. [131] 2.5. Functional Starter Cultures. Functional starter cultures reported enterotoxin E to be the most common enterotoxin arestartersthathaveatleastonefunctionalproperty,which in S. equorum and S. xylosus, although it is reported that the may contribute to food safety and/or offer one or more occurrence of staphylococcal enterotoxin genes in CNS from organoleptic, technological, nutritional, or health advantages slightly fermented sausages was rare, detecting only entC in [151]. They offer additional functionalities compared to plain S. epidermidis [132]. starter cultures and are a way of improving the fermentation Absence of genes coding for staphylococcal enterotoxins process of meat products and achieving tastier, safer, and or enterotoxin-like superantigens is a requirement for strains healthier products. selected as starter cultures, and the S. xylosus and S. carnosus strains currently used as starter cultures or isolated from 2.5.1. Bioprotective Cultures. Biological preservation has fermented meat products generally lack toxin genes [11]. gained increasing attention as a means of naturally control- The analysis of virulence factors in strains of S. epi- lingtheshelflifeandsafetyoffoods.Theuseofprotec- dermidis, S. simulans, S. xylosus, S. kloosii,andS. caprae tive starter cultures in the manufacture of fermented meat revealed sometimes high percentage of incidence of the products is a well-established technology [86]. Bioprotective following virulence traits: production of slime, 𝛼-haemolysin, starters may contribute to the safety and increase in shelf life 𝛽-haemolysin, DNase, TNase, hyaluronidase, and TSST-1 and of fermented meat products through the release of organic production of enterotoxins SEA, SEB, SEC, and SED [133]. acids [152], the production of bacteriocins against important food pathogens, mainly L. monocytogenes [153], and the 2.4.4. Strains with Ability of Biofilm Formation. In food control of biological hazards [86]. industry, biofilm formation is undesirable for hygienic and Potential protective starter cultures to use in fermented safety reasons, as it can allow the attachment of food-spoilage meat products have been identified [154] and tested [4, or pathogenic microorganisms to food or food surfaces [134]. 155–157]. The use of bioprotective starter cultures ensures Nevertheless, several authors believe that colonization of food safety, while increasing shelf life, without compromising the surfaces by starters could be desirable, as it would inhibit nutritional value of fermented meat products or depreciating colonization by pathogenic or spoilage bacteria [135]. their sensory quality. Among CNS, biofilm formation has been studied in S. aureus [136], S. epidermidis [136], S. hominis [137], S. sciuri 2.5.2. Probiotics. According to the currently adopted defini- [135], and S. equorum [138]. S. capitis, S. cohnii, S. epidermidis, tion by the Food and Agriculture Organization/World Health S. lentus,andS. saprophyticus have all also been reported to Organization (FAO/WHO) [158], probiotics are defined as form biofilms [139], though due to different genetic determi- “live microorganisms which when administered in adequate nants [140]. These studies concluded that, in general, biofilm amounts confer a health benefit on the host.” formation is a strain-dependent characteristic. Furthermore, Probiotics are nonpathogenic health-promoting microor- the capacity of S. xylosus to form biofilms may contribute to ganisms that when ingested in defined amounts may have a its survival of food processing [141]. On the other hand, the positive effect on human physiology and health [29]. In 1965, 10 Journal of Food Quality

Lilly and Stillwell proposed probiotics to be “microorganisms characteristics and a lack of negative features, enabling promoting the growth of other microorganisms.” To act as safe the development of starter cultures based on indigenous probiotic microorganisms, strains should be of species and technological bacteria from traditional sausages, which are genera normally present in the human gastrointestinal tract thus better adapted to the meat matrix [22, 172]. [159]. The first genome sequence of a starter to be published was Probiotics are LAB (or bifidobacteria), mainly Gram- the one of the LAB L. sakei 23K[144].Despitethesmallsized positive Lactobacillus species. genome (1,883 protein-coding genes), L. sakei contains seven In general, health benefits of probiotic foods are based on rRNA gene clusters [144]. This redundancy may contribute the presence of selected strains of LAB that, having passed to its ability to grow in complex microbial ecosystems [173]. through the stomach and the small intestine, survive in the With regard to gene products, the L. sakei genome shares the large intestine and confer a health benefit on the host [160]. highest level of conservation with Lactobacillus plantarum, LAB with probiotic properties may have a positive influ- which can be used as a starter in fermented meat, dairy, and ence on product taste, flavour, and aroma, as well as on vegetable products [144, 174, 175]. Genome analysis revealed functional and physiological properties [8]. a specialized metabolic repertoire to adapt and grow on Some LAB strains are able to produce nutraceutical meat products. Important cellular functions are encoded compounds [161]. Studies on Lactococcus lactis highlight the by a redundancy of genes likely to enhance the organism’s possibility of developing LAB meat starter cultures for in situ robustnessandmostprobablyhelpittooutgrowothercom- production of vitamins, by overexpression and/or disruption peting bacteria. As a unique ability among lactic acid bacteria, of relevant metabolic genes [162–164]. L. sakei is able to use meat components, such as purine Although dairy products are the most commonly used nucleosides, abundant in meat, upon glucose depletion, to food vehicles for the delivery of probiotics, several studies grow and produce energy. Genes possibly responsible for dealing with the use of probiotics in fermented meat products biofilm formation and cellular aggregation, which may assist to improve their nutritional value as functional foods have in colonising meat surfaces, were also identified [144]. been reported [5, 154, 165–167]. The draft genome sequence of L. sakei subsp. sakei strain The commercial application of probiotics in meat prod- LS25, a commercial starter culture for fermented sausages, uctsisnotacurrentprocedure,mostlybecauseoftechnolog- has been released [176]. Slightly larger than the one of L. ical issues. Although fermented meat products are processed sakei 23K, this genome has 1,972 predicted protein-coding without heating, probiotics may still be inactivated due to low genes and 7 rRNA operons [176]. Compared to the L. sakei pHorwateractivityvalue,aswellasbythepresenceofnative 23K genome [144], 1,618 genes are orthologous, but 250 microorganisms or curing salts. The most important problem seem to be unique to LS25, including a set of genes for is to find a compromise between technology, safety, quality, carbohydrate metabolism, various transporters, and dehy- and health-beneficial value of food [160]. For recent reviews, drogenases/oxidoreductases [176]. please refer to Neffe-Skocinska´ et al. [168] and Vuyst et al. [8]. Complete or draft genome sequences of Pediococcus Some species involved in sausage fermentation, such as pentosaceus and Pediococcus acidilactici strains, from diverse L. plantarum, have been engineered to produce an excess Korean fermented food products, have been released, but of folate (vitamin B11) [162]. This gives the possibility of none isolated from meat products [177–179]. fortifying meat products with vitamins and other essential Genomes of several strains of starter CNS have also compounds, thus producing healthier meat products [29]. been published, namely, S. xylosus SMQ-121 [180], S. xylosus Today, the use of probiotic starters in any fermented S04002 [181], and S. carnosus TM300 [182]. food claiming health benefits should be scientifically demon- The draft genome sequence of S. xylosus SMQ-121 strated according to the legal requirements of EU for labelling revealed the absence of genes coding for toxins or viru- [169, 170]. lence factors. Furthermore, only four antibiotic resistance genes were found: two genes encode proteins that belong 3. Omics of Meat Starter Cultures to the major facilitator superfamilies involved in phenicol and fluoroquinolone resistance; another gene encodes a 󸀠 The main bacterial species used in meat fermentation are putative aminoglycoside 3 -phosphotransferase for resistance LAB and CNS. Lactobacillus sakei, Lactobacillus curvatus, to aminoglycosides; and the last one encodes trimethoprim Lactobacillus plantarum (mainly in Europe), and Pediococcus resistance. Nevertheless, this strain was found to be sensitive pentosaceus and Pediococcus acidilactici (mainly in the US) to amikacin, chloramphenicol, ciprofloxacin, and trimetho- are the starters commonly used for their fermentative role prim [180]. in dry-sausage production, while Staphylococcus xylosus and A genome comparison of several S. xylosus meat starter Staphylococcus carnosus are known for their involvement in cultures, including strain S04002, with other S. xylosus strains thedevelopmentandstabilityofcolourandaromaproduc- causing cow and goat mastitis, among others, has shown the tion [171]. presence of aroma compounds in S. xylosus S04002 [181]. Using comparative genomics, transcriptomics, proteom- S. carnosus TM300 genome has the highest GC content ics, and metabolomics, the diversity of strains naturally of all sequenced staphylococcal genomes [182]. It contains present in traditional fermented sausages is being explored. only one prophage and one genomic island characterised These approaches allow rapid high-throughput screen- by a mosaic structure composed of species-specific genes. ing of promising wild strains with desirable functional All starter cultures features, such as nitrate/nitrite reduction, Journal of Food Quality 11 several sugar degradation pathways, two catalases, and nine Next generation sequencing methods will improve osmoprotection systems, are present. It lacks most virulence knowledge related to microbiota and strain characterization factors, namely, the typical S. aureus toxins, as well as biofilm involved in dry-fermented meat products. Future work must formation genes, highlighting its nonpathogenic status [182]. be done regarding these novel approaches and certainly Following the publication of the genome sequences of novelvisionofstarterbehaviouronparticularproductswill several strains, global approaches based on transcriptomics be given. and proteomics have been developed in order to better understand the adaptation of starters to the meat environment and 4. Conclusions their interactions with the ecosystem and the meat substrate. Genes involved in safety and technologically relevant The increasing knowledge and exigence level of consumers properties of food associated CNS, such as antibiotic resis- have forced the search for high value traditional meat prod- tance, haemolysins, toxins, amino acid decarboxylases, bind- ucts. Consequently, the number of production units (meat ing proteins to extracellular matrix (ECM), lipases, proteases, transforming) has increased, sometimes in low developed stress response factors, and nitrate dissimilation, have been regions in a bewildered way. detected using DNA microarrays [183]. The production of traditional meat products, namely, S. xylosus C2astrainresponsetonitrosative[184]or dry-fermented, dry-cured sausages, is still a very tradi- nutrients and osmotic stress [185] has been investigated tional and laborious process subjected in several cases to through DNA microarrays. S. xylosus has been shown to uncontrolled natural environmental conditions. This poses a counteract nitrosative stress by developing several oxidative problemtotheproducerssincetheirmeatproductswillnot stress resistance mechanisms, such as modulation of the be uniform throughout time. Thus, it is necessary to find solu- expression of genes involved in iron homeostasis, detoxifying tions contributing to the reproducibility of products charac- enzymes, and DNA and protein repairs [184]. S. xylosus teristics. The use of starter cultures based on autochthonous adapted its metabolism to the meat nutrients and anaerobic microbiota selection may play here an important role. In conditions by simultaneously using glucose and lactate as fact, the use of these starters in sausages production may carbon sources and by using meat peptides and amino acids. improve their sensorial characteristics and contribute to their S. xylosus respondedtotheosmoticstresscausedbythe biopreservation and safety, extending their shelf life, and to addition of salt (NaCl) by overexpressing genes involved increased meat products uniformity. in transport and synthesis of osmoprotectants, particularly Selected starter cultures provide a powerful tool for + + glycine betaine, and Na and H extrusion [185]. To over- driving the fermentation of meat products, allowing desired come the damaging effects of oxidative and nitrosative stress, quality and safety targets to be reached. Their use in meat staphylococci have developed protection, detoxification, and fermentation results in acceleration of fermentation time, an repair mechanisms controlled by a network of regulators improvement of safety (by reducing undesirable microorgan- [186]. isms), and a better quality of the final product. The selection Among the overexpressed proteins in S. xylosus biofilm, of a starter culture should be carried out in the context of several related to exopolysaccharide biosynthesis were its application, since functionality will depend on the type of reported [187]. Furthermore, with overexpression of some sausage, the technology applied, the ripening time, and the proteins involved in amino acids metabolism, translation, ingredients and raw materials used. Future knowledge will be and secretion, nitrogen metabolism appeared as quite active gained with omics methods approach. in sessile cells of S. xylosus. Additionally, protein secretion systems were also upregulated in biofilms, suggesting more active protein trafficking in sessile S. xylosus cells [187]. Conflicts of Interest L. sakei 23Kstrainglobaltranscriptomeresponseduring The authors declare that they have no conflicts of interest. growth on ribose [188] and L. sakei La22 strain transcriptomic response to meat protein environment [189] have been studied using DNA microarrays. Acknowledgments The ribose uptake and catabolism in L. sakei 23K is highly regulated at the transcriptional level, and it is closely This work was funded by National Funds through FCT- related to the catabolism of nucleosides. A global regulation Fundac¸ao˜ para a Cienciaˆ e a Tecnologia under the Project mechanism seems to allow fine tuning of the expression of UID/AGR/00115/2013. M. Laranjo acknowledges a postdoc enzymes, which control the efficient use of available carbon research grant from FCT (SFRH/BPD/108802/2015). sources [188]. 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Research Article The Effect of Drying Parameters on the Quality of Pork and Poultry-Pork Kabanosy Produced according to the Traditional Specialties Guaranteed Recipe

Marta Chmiel, Lech Adamczak, Katarzyna WroNska, Dorota Pietrzak, and Tomasz Florowski Division of Meat Technology, Department of Food Technology, Faculty of Food Sciences, Warsaw University of Life Sciences-SGGW, 166 Nowoursynowska Street, 02-787 Warsaw, Poland

Correspondence should be addressed to Marta Chmiel; [email protected]

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

Academic Editor: Maria J. Fraqueza

Copyright © 2017 Marta Chmiel et al. This is an open access article distributed under the Creative Commons Attribution License, which 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 quality features of TSG (traditional specialties guaranteed) pork and poultry-pork kabanosy. After heat treatment and 24-hour cooling at ∘ ∘ 4–6 C, the products were placed in three chambers at 15 C with differentiated air relative humidity: 60, 70, and 80%, respectively. The drying process was carried out until all variants of kabanosy achieved the required final yield of the product< ( 68%). Color components, water activity and shear force, water, protein, fat, and salt content, and the TBARS indicator values were determined. The drying process might be shortened (∼50%) by a reduction of humidity in the drying chamber from 80 to 60%. The changes in the content of chemical components in pork kabanosy compared to poultry-pork ones demonstrated the different dynamics of the drying of the two types of kabanosy and the need for the selection of optimum drying conditions relative to raw material composition.

1. Introduction whose production history dates to the 1920s/30s [10]. The word “kabanos” probably comes from the name of the pork, Sausages are one of the oldest meat products and have been “kabanina,” which was obtained from a characteristic species manufactured for nearly two thousand years. They are con- of pig, known as a “kaban.” In the nineteenth century, a sumedallovertheworldduetotheirattractiveflavorprofile. kaban in Poland and Lithuania was a young male, extensively Dry and semidry sausages are considered by consumers to be fattened with potatoes to obtain a delicate meat with a high two of the most delicious and highly sought after products degree of intramuscular fat (marbled meat with intramuscu- [1–3]. The growing interest in this type of products results lar fat content above 3%), which in turn positively affected from their diversity, the use of different raw materials, and its juiciness and tenderness. In 2011, pork kabanosy were their degree of fragmentation, not to mention the spices or registered in the European Union as a product of traditional smoking methods used. Moreover, their high desirability is specialties guaranteed (TSG). Kabanosy are long, thin, and also affected by their unique taste and aroma [4, 5]. In recent evenly wrinkled pork sausages in natural casings, sheep years, there has been an increase in consumer demand in intestines. The product is subjected to drying and smoking terms of food quality and its safety and effects on health [6]. processes, which affects the formation of their specific color. Growing consumer interest has been noted in food with a The color of the surface of pork kabanosy should be dark red smaller level of additives [7], as well as traditional and natural withahintofcherry,whileslightlycreamyfatparticlesand products, especially in the countries of Eastern Europe [8, dark red meat pieces should be visible in the cross section [10]. 9]. This group of products includes kabanosy, which are In the case of poultry kabanosy, the color is lighter, which popular in Poland. Kabanosy are a Polish traditional product istheresultoftheuseofpoultrymeat.Kabanosyshould 2 Journal of Food Quality be characterized by the aroma of cured meat with a delicate (v) Heat treatment: kabanosy were dried in a Jugema hint of cumin and black pepper. The characteristic feature smoking-cooking chamber (Jugema, Sroda´ Wielko- ∘ of kabanosy is their unique smell, which is mainly due to polska, Poland) for 15 minutes at 40 C, smoked with ∘ smoking. warm smoke for 30 minutes at 50 C, and then baked ∘ ∘ The drying process also plays an important role in the at 75 Cuntil70 C was reached in the geometric center distinctive, unique taste of kabanosy, emphasizing the advan- of the bar; after the treatment, kabanosy were cooled tages of their aroma and taste. Drying is one of the oldest for 1 hour in an off chamber. methods of food preservation [11–16]. According to European (vi) Cooling: kabanosy were cooled in a refrigerator for 24 ∘ Parliament and Council Regulation (EU) number 1044/2011 hours at 4–6 C. [10], pork kabanosy (TSG) should be dried for 3 to 5 days ∘ at a temperature of 14–18 C, with an air relative humidity of (vii) Drying: kabanosy were divided into three variants, 80%,toachievethedesiredyield(≤68%).Thedryingprocess andtheseweresubjectedtoadryingprocessinalabo- significantly affects the quality of the produced kabanosy ratory drying chamber (PHU Chłodnictwo, Warsaw, and is a highly energy-consuming step that determines the Poland) at three different drying air relative humidi- ± ± ± economy of production [11, 14]. Therefore, one aim of this ties (K1–80% 2%, K2–70% 2%, and K3–60% ∘ ± ∘ study was to determine the effect of differentiated air relative 2%) at the same temperature of 15 C 2 C. The humidity in the drying chamber (80, 70, and 60%) on the humidity and temperature in the chambers were qualityofporkkabanosy.Theconsumptionofpoultrymeat monitored using data loggers (EL-USB-2 models, has increased rapidly all over the world in the last decade, Lascar Electronics Ltd., Erie, USA). The drying pro- and poultry production has become the fastest growing meat cess was carried out until all variants of kabanosy sector [17]. Due to the nutritional value and low price of achieved the TSG required [10] final yield of the prod- poultry meat resulting from its high supply, we also decided to uct, that is, below 68%. The final yield was controlled produce poultry-pork kabanosy with a chemical composition each 12 h of the drying process. For kabanosy dried in similar to pork kabanosy TSG in this study. a chamber at 60% humidity, the required final yield below 68% was achieved after 24 h of the process; at a 2. Materials and Methods humidity of 70% after 36 h; and at a humidity of80% after 48 h of drying. 2.1. Kabanosy Production. The basic raw materials for the Before and after the drying process, a range of measure- production of pork kabanosy were class I pork, 30%; class ments were performed for each of the three production series, IIA, 40%; class IIB, 30%. In the case of poultry-pork kabanosy, foreachofthethreevariantsofkabanosy,andforbothpork ∗ ∗ ∗ the composition was chicken thigh meat, 80% and pork jowl, and poultry-pork ones. The color components 𝐿 , 𝑎 , 𝑏 of 20%. For both types of kabanosy, the same spice composition thesurfaceandcross-sectionalareaofthebar,wateractivity was used (with respect to raw materials), that is, black pepper (𝑎𝑤), and the texture, that is the shear force (only after the (0.15%), sugar (0.20%), nutmeg (0.05%), and cumin (0.07%). drying process), were measured on the unground kabanosy. Porkandpoultry-porkkabanosywereproducedinthree Ground kabanosy (laboratory grinder Zelmer Diana 886.8, series with precut (pieces measuring 5 cm), cured (2% based Zelmer, Rzeszow, Poland, grid hole diameter 3 mm) were on the weight of raw materials for 24 hours before production, ∘ subjected to the measurements of basic chemical component under refrigeration 4–6 C) pork or poultry meat and pork content, that is, water, protein, fat and salt, and the TBARS jowl. The production process was conducted according to the indicator was also determined. following scheme: 2.2. Kabanosy Yield at Different Stages of the Production Pro- (i) Grinding: class I pork meat or part of chicken thigh cess. The yield of heat treatment after 24 hours and the final meat (30%) was ground on a mesh of Ø 10 mm; class yield after the drying process at different air humidities in IIA, IIB pork meat, part of chicken thigh meat (50%), thechamberatalevelof80,70,and60%,respectively,were andjowlweregroundonameshofØ8mm;the determined during the process of pork and poultry-pork raw material grinding was conducted in a Mesko kabanosy production. The yields were determined relative to WN60 laboratory grinder (Mesko-AGD, Skarzysko-˙ the initial weight before the heat treatment. Kamienna, Poland). ∗ ∗ ∗ (ii) Mixing: ground meat was mixed using a Kenwood 2.3. Measurement of Color Components on the 𝐿 𝑎 𝑏 Scale. Major mixer (Kenwood, Havant, UK) for 5 minutes to The measurements of color components were performed using a Minolta CR-200 camera (Konica Minolta, Wroclaw, thoroughlymixtheingredients;thespiceswereadded ∘ Poland, light source D65, 2 observer, measuring head hole during the mixing. ∗ 8 mm) calibrated according to white standard (𝐿 97.81, (iii) Bar stuffing and molding: sheep intestines (Ø 22mm) ∗ ∗ 𝑎 −0.45, 𝑏 1.88).Themeasurementsweretakenineachof were filled using a Dick manual stuffer (Friedr, Dick the 5 repetitions on the surface and cross section of kabanosy, GmbH & Co. KG, Deizisau, Germany) and then taking the average as a result of the measurement. formed into 25 cm bars. (iv) Settling: the bars were deposited on a smoking stick 2.4. Content of the Basic Chemical Components. The moisture for 1 hour at room temperature. content was determined according to PN-ISO 1442:2000 [18] Journal of Food Quality 3

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

Final yield (%) Kabanosy Thermal treatment yield (%) Yield after cooling (%) K1 - 80% K2 - 70% K3 - 60% a a a Pork 78.5 ± 5.3 74.9 ± 4.4 59.2 ± 6.8 55.3 ± 3.5 50.8 ± 2.8 a a a Poultry-pork 84.2 ± 2.1 82.0 ± 3.5 65.0 ± 3.6 61.2 ± 2.9 60.7 ± 3.2 a Average values in rows marked with different letters differ significantly at 𝑝 ≤ 0.05.

∘ by drying samples at 105 C (SUP-65 dryer, Wamed, Warsaw, to PN-ISO 4121:1998 [23]. Kabanosy prior to the assess- Poland). The protein content was determined by the Kjeldahl ment were conditioned for half an hour at room tempera- method according to PN-75/A-04018:2002 (Velp Scientifica ture. UDK 129 Distillation Unit, Poland) [19]. The fat content was determined by Soxhlet extraction (Buchi¨ Extraction System 2.9. Statistical Analysis of the Results. The results were sub- B-811, Donserv, Poland) according to PN-ISO 1444:2000 jected to statistical analysis using ANOVA analysis and a [20]. The salt content was determined using potentiometric detailed Tukey’s HSD test, at a significance level of 𝛼 = 0.05. methods according to PN-ISO 1841-2:2002 [21], using a 702 Statistica ver. 10 PL (StatSoft, Inc., Tulsa, USA) was used. The SM Titrino (Metrohm AG, Herisau, Switzerland) device. effect of air relative humidity in the drying chamber on yield andonselectedqualityfeaturesofporkandpoultry-pork

2.5. Measurement of Water Activity (𝑎𝑤). Measurement of kabanosy was determined. water activity was carried out using an Aqua Lab CX-2 apparatus (Decagon Devices, Inc., Pullman, USA). The sample was 3. Results and Discussion prepared by cutting a flat rectangle from kabanosy, and the ∘ analysis was performed at a temperature of 25.0±1.5 C, three 3.1. Yield of Kabanosy. The yields of kabanosy at different times for each sample, and the average was taken as a result stages of the production process are shown in Table 1. In of the measurement. the case of both pork and poultry-pork kabanosy, there was no significant (𝑝 > 0.05) effect derived from air relative humidity on the final yield of these products (Table 1). Dried 2.6. TBARS Indicator Determination. Thiobarbituric acid sausages can lose up to 30% of their weight throughout the reactive substances (TBARS) values were determined accord- production process and such sausages in the US market are ing to the extraction method of Shahidi [22]. Absorbance was considered to be “moderately” dry products. In contrast, dry measured at 532 nm using a spectrophotometer (Hitachi U- sausages are those that are characterized by a 40% weight 1100;Geminibv.,Apeldoorn,Netherlands)againstablank loss [24]. The weight loss of the product is faster in the case containing 5 mL of 2-thiobarbituric acid (TBA) and 5 mL of of a product dried at lower relative air humidity [25]. The 10% trichloroacetic acid (TCA). A constant coefficient of 2.34 tendency observed in this study was, therefore, consistent was employed for converting the absorbance units to TBARS with the literature data. The period of drying of kabanosy values, which were expressed as mg malondialdehyde per kg in the chamber with 80% humidity (yield below 68%, as sample (mg MAD/kg). required by TGS) was considerably longer in comparison with the drying time of the kabanosy under a humidity of 2.7.Measurement of Texture-Shear Force. Measurement of the 70and60%.Theuseoflowerrelativehumidityinthedrying shear force was performed using a ZWICKI 1120 endurance process reduced the duration of the process by 50%; however, apparatus (Zwick, Ulm, Germany). Warner-Bratzler’s device this may result in lower product yield. The observed average equipped with a flat knife was used for this purpose. The 10-percentage point difference in the yield of pork kabanosy maximum shear force 𝐹max was read out at a head shift of dried at 80 and 60% relative air humidity was caused not only 50 mm/min. The samples were prepared directly before tests by the varying relative air humidity in the chamber but also by cutting the kabanosy into 10 cm sections and subjecting by the varied duration of the process. The different drying them to 30 minutes of conditioning at room temperature. dynamics of both types of kabanosy suggest the need for The measurements were taken at three points on each ofthe the selection of optimum drying conditions in terms of raw prepared sections, for three different bars from each variant material composition. of kabanosy. The results were then averaged. ∗ ∗ ∗ 3.2. 𝐿 𝑎 𝑏 Color Components of Kabanosy. The results of ∗ ∗ ∗ 2.8. Sensory Evaluation. The sensory quality of kabanosy 𝐿 , 𝑎 and 𝑏 color component measurement on the surface after the drying process in chambers with different levels of and cross sections of kabanosy are summarized in Table 2. ∗ humidity was assessed using a 10-point intensity scale for A reduction of color component 𝐿 , and thus the lightness such sensory characteristics as surface color, hardness, aroma, ofthesurfaceafterthedryingprocess,wasnotedinpork and taste. However, overall acceptability was assessed on a kabanosy. The lowest value for this component was observed hedonic scale (where 0 points corresponded to unacceptable in the case of kabanosy subjected to drying in a chamber and a 10-point evaluation as very desirable). The evalua- with a relative air humidity of 60%. The effect of different tion was conducted by a trained 10-person team according relative air humidities in terms of the color lightness on the 4 Journal of Food Quality ∗ ∗ ± 0.7 ± 0.4 ± 0.4 ±0.2 𝑏 𝑏 a b b b ± 0.4 2.0 ± 0.9 7.1 ± 0.8 1.3 ∗ ∗ ± 0.6 4.8 a a b 𝑎 𝑎 a K3, 60% K3, 60% ± 1.8± 2.2 16.4 14.9 ± 1.7± 0.6 16.1 9.8 ∗ ∗ a a 𝐿 b b 𝐿 ± 1.3 38.2 ∗ ∗ ± 0.6 45.6 ± 0.4 52.4 ± 0.3 42.8 𝑏 𝑏 a b a ab ± 0.7 1.6 ∗ ± 1.2 4.3 ± 1.5 6.2 ± 1.1 8.5 ∗ a 𝑎 a a 𝑎 ab K2, 70% K2, 70% After drying process After drying process ± 0.8 15.4 ± 0.6 9.4 ∗ ± 2.0± 2.2 18.0 15.2 ∗ a b 𝐿 a a 𝐿 Surface color ± 0.5 55.5 ∗ ± 0.3 48.0 ∗ ± 1.8± 1.5 39.2 43.1 𝑏 Cross section color 𝑏 a (a) ab (b) a a 𝑝 ≤ 0.05 . ± 1.2± 0.6 2.7 5.3 ± 0.9± 0.6 6.8 9.1 ∗ ∗ a a a a 𝑎 𝑎 K1, 80% K1, 80% ± 0.7± 1.9 19.9 14.7 ± 1.9 10.6 ∗ ± 0.3 15.6 ∗ a a 𝐿 a 𝐿 ab ∗ ∗ 𝑏 𝑏 ∗ ∗ 𝑎 𝑎 Table 2: Color of the surface and cross section of pork and poultry-pork kabanosy before and after the drying process. Before drying process Before drying process ∗ ∗ 𝐿 𝐿 55.3 ± 2.1 15.8 ± 1.1 3.7 ± 0.8 51.9 58.8 ± 0.9 9.7 ± 0.6 6.9 ± 1.3 53.9 45.4 ± 0.2 21.1 ± 0.4 8.9 ± 1.8 40.4 50.5 ± 2.9 13.8 ± 0.9 12.9 ± 1.7 44.2 Average values for the same differentiator rowsin marked withdifferent letters differ significantly at b Kabanosy Pork Kabanosy Pork Poultry-pork Poultry-pork a , Journal of Food Quality 5 surfaceofporkkabanosywasnotsignificant(𝑝 > 0.05). humidity. This could be due to significant differences inthe Similar relationships were found for poultry-pork kabanosy process of drying in the various research series, as evidenced (Table 2). According to De Maere et al. [26], reduction of the by, among others, high standard deviations. ∗ 𝐿 component values during the drying process may result Inthecaseofpoultry-porkkabanosy,adecreaseinwater from the reduction of water content in the product. content was observed from 53.1% before the drying process For both the pork and poultry-pork kabanosy, a decrease to 45.8% after drying in a relative air humidity of 60%. ∗ ∗ in the values of the 𝑎 and 𝑏 color components was observed Protein, fat, and salt content changed from 21.7, 21.9, and during the drying process in chambers with different relative 2.7%, respectively, before the drying process to 25.4, 25.6, air humidity compared to the kabanosy before the drying and 3.1% after drying in the chamber with 60% relative air process. This applies to both the color of the bar and the cross humidity. Also in this case, there were no significant (𝑝> ∗ ∗ section of the products. There was a tendency for 𝑎 and 𝑏 0.05) differences of chemical composition of poultry-pork values to be lower with a decrease in relative air humidity kabanosy dried in chambers of different relative air humidity in the drying chamber. Pork kabanosy dried with an relative (data not shown). air humidity of 60% were characterized by significantly (𝑝< According to European Parliament and Council Reg- ∗ ∗ 0.05) lower values for components 𝑎 and 𝑏 than kabanosy ulation (EU) number 1044/2011 [10], kabanosy should be dried at 80% humidity (Table 2). In contrast, for the poultry- characterized by the following chemical composition: protein pork kabanosy, drying relative air humidity had no significant content of at least 15%, water up to 60%, fat up to 35%, and (𝑝 > 0.05) effect on the values of those components (Table 2). salt maximum 3.5%. The poultry-pork kabanosy produced ∗ ∗ ∗ Measurements of color components 𝐿 , 𝑎 ,and𝑏 were in this study, irrespective of the drying conditions, met all also taken on the cross sections of kabanosy. The color on the the above requirements. In the case of pork kabanosy, only cross sections of kabanosy dried with a relative air humidity aslightexcessofortheaveragesaltcontentwasfoundin of 70% and 60% was characterized by significantly lower products dried at 60% relative air humidity. ∗ values for component 𝐿 compared to the values obtained on the cross sections of kabanosy dried at 80% humidity 3.4. Water Activity (𝑎𝑤), TBARS Indicator Value, and Texture (Table 2). In the case of poultry-pork kabanosy, a significant of Kabanosy. The water activity of both pork and poultry- (𝑝 < 0.05) effect of relative air humidity was noted on the pork kabanosy decreased with a decrease in relative air lightnessoftheircrosssectioncolor;however,thiseffect humidity in the drying chamber (Table 3). There was no was ambiguous. Poultry-pork kabanosy dried in a chamber significant𝑝 ( > 0.05) effect of drying air humidity on water with a relative air humidity of 60% were characterized by activity in pork kabanosy. Poultry-pork products dried at ∗ significantly lower values for component 𝐿 ,andsotheywere 60% humidity were characterized by a significantly𝑝 ( < 0.05) thedarkest,comparedtokabanosydriedatahumidityof70% lower water activity than those dried at 70 and 80% humidity. (Table 2). Also in the studies presented by Collell et al. [1], a relationship Pork kabanosy dried in chambers with a relative air was demonstrated between the fall in 𝑎𝑤 and water content humidity of 70 and 60% were characterized by significantly during the drying process. ∗ (𝑝 < 0.05) lower levels of yellow color (𝑏 )inthecross In the present study, there was no significant𝑝 ( > 0.05) sections of the bar compared to kabanosy dried at a relative effect of air relative humidity on the value of the TBARS air humidity of 80% (Table 2). indicator in the case of pork and poultry-pork kabanosy In the case of poultry-pork kabanosy, significant differ- (Table 3). entiation of this color component was observed between the There was no significant (𝑝 > 0.05) effect of air relative values determined for the cross section of the product dried humidity on the shear force of either pork or poultry-pork at 70 and 60% of relative air humidity (Table 2). kabanosy. There was only a tendency pointing to an increase in the shear force of kabanosy with a decrease in relative 3.3. Content of the Basic Chemical Components in Kabanosy. humidity in the drying chamber. According to Arnau et The average content of basic chemical components in pork al. [14], the first method to obtain an acceptable texture kabanosy before the drying process was 52.1% water content, with a short drying period in dry-cured meat products is 24.8% protein content, 19.6% fat content, and 3.0% salt to accelerate the drying process by decreasing the relative content. During the drying process, as expected, there was a humidity and increasing the temperature of the drying air. decrease in the water content and increase in the components This was not confirmed in our research. constituting the dry matter of the product. Olivares et al. [27] indicate that the concentration of protein and fat in 3.5. Sensory Quality of Kabanosy. A reduced relative humid- the product during the drying process is mainly due to the ity of the air in the drying chamber did not affect such sensory watercontentdecreaseresultingfromweightloss.Thehighest qualities of pork and poultry-pork kabanosy as hardness, average values for protein (29.5%), fat (23.5%), and salt (3.6%) aroma, taste, and overall acceptability (Table 4). A significant content were found in the case of drying of the pork kabanosy (𝑝 < 0.05) impact on the color of pork kabanosy was noted in a chamber with 60% relative air humidity. Despite the 10- as a result of the relative air humidity. Kabanosy dried at the percentage point differences in the yield of products dried at relative humidity of 60% received lower scores for color than 80% and 60% of relative air humidity, no significant differ- kabanosy dried at a humidity of 80%. An opposite trend was ences (𝑝 > 0.05) were found in the chemical composition observedinthecaseofthecolorofpoultry-porkkabanosy, of kabanosy dried in chambers with different relative air but this was not significant (Table 4). Also, some trends in 6 Journal of Food Quality

Table 3: Water activity (𝑎𝑤) and TBARS indicator values in pork and poultry-pork kabanosy before and after the drying process; shear force value after drying process.

(a)

Water activity TBARS (mg MAD/kg of the product) Kabanosy Before drying After drying process Before drying After drying process process K1, 80% K2, 70% K3, 60% process K1, 80% K2, 70% K3, 60% a a a a a a Pork 0.943 ± 0.007 0.940 ± 0.004 0.927 ± 0.011 0.919 ± 0.011 0.54 ± 0.17 0.50 ± 0.11 0.60 ± 0.23 0.56 ± 0.18 Poultry- 0.956 ± 0.005 0.940a ± 0.002 0.935a ± 0.004 0.926b ± 0.003 0.55 ± 0.08 0.53a ± 0.08 0.55a ± 0.12 0.55a ± 0.11 pork (b) Shear force (𝑁) Kabanosy After drying process K1, 80% K2, 70% K3, 60% a a a Pork 91.3 ± 14.9 99.3 ± 6.9 103.4 ± 8.0 a a a Poultry-pork 83.6 ± 17.9 85.5 ± 22.9 94.4 ± 14.9 a,b Average values for the same differentiator in rows marked with different letters differ significantly at 𝑝 ≤ 0.05.

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

Characteristic (points) Kabanosy K1, 80% K2, 70% K3, 60% a ab b Pork 9.4 ± 0.6 8.4 ± 0.3 6.8 ± 0.3 Surface color a a a Poultry-pork 7.4 ± 0.5 8.2 ± 0.3 9.26 ± 0.3 a a a Pork 9.4 ± 1.2 8.4 ± 0.9 7.2 ± 0.6 Hardness a a a Poultry-pork 8.8 ± 0.2 8.6 ± 0.2 9.6 ± 1.0 a a a Pork 6.8 ± 0.8 6.8 ± 0.7 6.6 ± 0.3 Aroma a a a Poultry-pork 8.2 ± 0.6 8.2 ± 0.8 8.0 ± 0.9 a a a Pork 7.4 ± 0.3 7.2 ± 0.8 6.2 ± 0.8 Taste a a a Poultry-pork 7.4 ± 0.6 8.6 ± 0.5 8.0 ± 0.5 a a a Pork 6.2 ± 0.5 6.0 ± 0.5 7.2 ± 0.9 Overall acceptability a a a Poultry-pork 6.4 ± 0.8 6.6 ± 1.4 6.8 ± 1.4 a,b Average values for the same differentiator in rows marked with different letters differ significantly at 𝑝 ≤ 0.05. changesinthescoresgivenintheevaluationofhardness not significantly differentiated. Kabanosy dried in air of 60% and taste of kabanosy were observed. In the case of pork humidity were thus characterized by the lowest final yield; kabanosy, scores given for these discriminants were lower and, moreover, the duration of drying in this case was the with the decrease in relative humidity; and in the case of shortest. No significant effect of drying relative air humidity poultry-pork kabanosy, marks were higher (Table 4). This was found on the value of the TBARS indicator in the case confirms the need for a separate examination of the effect of of pork or poultry-pork kabanosy; however, reduced water humidity in the drying chamber, depending on the type of activity and water content were observed with a decrease rawmaterialusedinkabanosyproduction. in air relative humidity and analogically an increase was observed in protein and fat content in the products. After the drying process, the products were darker both on the surface 4. Conclusion ∗ and in cross section (a lower value for component 𝐿 )than Pork kabanosy produced at differentiated drying relative air before the process. Also, relative air humidity in the drying humidities met the requirements for the recipe for tradi- chamber affected the color components of kabanosy. The tional specialty guaranteed (TSG) regarding the content of changes in the contents of particular chemical component basic chemical components, water, protein and fat, and the in pork kabanosy compared to poultry-pork ones point to assumed final yield; in the case of the salt content, these the different dynamics of the drying process of both types of assumptions were only met in the case of the product dried kabanosy and the need for the selection of optimum drying in air at 70 and 80% relative humidity. TSG assumptions conditions in terms of raw material composition. regarding the chemical composition were also fulfilled by Differentiation in relative air humidity in the drying poultry-pork kabanosy, regardless of relative air humidity in chambers affected the dynamics of changes, mainly in terms thechamber.Thefinalyieldoftheproductswasproportional of the color parameters and water activity of produced to the humidity prevailing in the drying chamber, but this was kabanosy. Moreover, pork kabanosy dried at the relative Journal of Food Quality 7

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Review Article Health and Safety Considerations of Fermented Sausages

Askild Holck,1 Lars Axelsson,1 Anette McLeod,1 Tone Mari Rode,2 and Even Heir1

1 NofimaAS,NorwegianInstituteofFood,FisheriesandAquacultureResearch,P.O.Box210,1431As,˚ Norway 2NofimaAS,NorwegianInstituteofFood,FisheriesandAquacultureResearch,P.O.Box8034,4068Stavanger,Norway

Correspondence should be addressed to Askild Holck; [email protected]

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

Academic Editor: Marta Laranjo

Copyright © 2017 Askild Holck et al. This is an open access article distributed under the Creative Commons Attribution License, which 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 are produced using widely different recipes and manufacturing processes. Over the last years, eating fermented sausages has been associated with potential health hazards due to their high contents of saturated fats, high NaCl content, presence of nitrite and its degradation products such as nitrosamines, and use of smoking which can lead to formation of toxic compounds such as polycyclic aromatic hydrocarbons. Here we review the recent literature regarding possible health effects of the ingredients used in fermented sausages. We also go through attempts to improve the sausages by lowering the content of saturated fats by replacing them with unsaturated fats, reducing the NaCl concentration by partly replacing it with KCl, and the use of selected starter cultures with desirable properties. In addition, we review the food pathogenic microorganisms relevant for fermented sausages (Escherichia coli, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, Clostridium botulinum,andToxoplasma gondii) and processing and postprocessing strategies to inhibit their growth and reduce their presence in the products.

1. Introduction polycyclic aromatic hydrocarbons in the products. Hazards can also be both of direct microbiological nature, the sausages Meat is especially rich in proteins, vitamins, and minerals potentially being contaminated with food pathogens, and and is an important element in human diet [1]. Due to its of indirect microbiological nature by metabolic activity of perishable nature, meat historically had to undergo different microorganisms causing presence of biogenic amines and methods of conservation. One strategy was mincing the mycotoxins. meat with salt and spices and lowering the water content Raw meat is an ideal medium for growth of many by drying. Fermented sausages were thus created and are microorganisms due to its high moisture content (70–80%), treasured traditional foods. Nowadays, a large number of and its abundance of proteins, peptides, and amino acids, different sausages are produced using widely different recipes growth factors, and minerals. In addition, it usually contains and manufacturing processes. In 1995, the production of fermentable glycogen and has a pH favorable for many fermentedsausagesintheEUwasestimatedtobeabout microorganisms. This is why raw meat is a highly perishable 750,000 tons [2]. Spain produces around 200,000 tons per product and should be preserved. For fermented sausages, year, while France produces another 110,000 tons [3]. The this preservation consists of a number of strategies (hurdles) production figures for 2014 for Norway and Finland were working together. These include lowering of pH by ferment- 7300 tons and 7000 tons, respectively [4]. ingsugarstomainlylacticacid,loweringofwateractivity(𝑎𝑤) Traditionally, fermented sausages were considered by salting, drying by evaporating water, inhibiting growth healthy and safe foods. More recently, eating fermented of aerobic bacteria by creating an anaerobic environment, sausages has been associated with health hazards caused by inhibiting microbial growth by addition of nitrate or nitrite, the high contents of saturated fats and NaCl, presence of and inhibiting surface growth by smoking or by addition of nitrite and degradation products such as nitrosamines, and specific molds. Together these hurdles generally lead to a use of smoking which can lead to toxic compounds such as shelf-stable product. However, traditional fermented sausage 2 Journal of Food Quality manufacturing processes do not ensure microbiologically final pH < 4.7. They have a moisture content between 45 and safe products. Several foodborne outbreaks attributed to dry 50%, are heavily smoked, are lightly spiced, and are usually or semidry fermented sausages (DFSs) (see references below) heated to an internal endpoint temperature between 43 and ∘ have demonstrated that actions must be taken to ensure 65 C. that these products are safe to consume. In most cases, the pathogen in question does not grow in the finished products 3. Sausage Ingredients Related to Health but survive long enough in high enough numbers to cause disease. 3.1. Fat. Consuming a healthy diet throughout the life course Here we give an overview of the literature pertaining helpspreventmalnutritioninallitsformsaswellasa to health issues and microbiological issues for fermented range of noncommunicable diseases and conditions [10]. The sausages and strategies to produce healthier and microbio- increased production of processed food, rapid urbanization, logically safer sausages. and changing lifestyles have led to a shift in dietary patterns. People are consuming more foods high in energy (fats and 2. Production of Fermented Sausages sugars). Energy intake (calories) should be in balance with energy expenditure. Evidence indicates that total fat should The large variety of fermented sausages and fermentation notexceed30%oftotalenergyintaketoavoidunhealthy processes that exist have been thoroughly described else- weight gain, with a shift in fat consumption away from satu- where [2, 5, 6]. Most often fermented sausages are produced rated fats to unsaturated fats so that saturated fats contribute from two-thirds of lean meat from animals such as pork and no more than 10% of the total energy intake [10]. Regarding beef and one-third of fat, nearly always pork backfat. In short, polyunsaturated fatty acids, controlled feeding and cohort meat is cut and mixed with fat, spices, salt, sugar, sodium studies of eicosapentaenoic acid (EPA) and docosahexaenoic nitrite (sometimes nitrate), and starter culture. Generally, the acid (DHA) intakes have demonstrated physiological benefits starter culture is a single species of lactic acid bacteria (LAB) on blood pressure, heart rate, triglycerides, and likely inflam- or a LAB mixed with other bacteria such as Staphylococcus mation, endothelial function, and cardiac diastolic function. xylosus or S. carnosus. The mix is stuffed into natural or Consistent evidence for a reduced risk of fatal coronary heart artificial casings of varying diameters and subjected to a disease and sudden cardiac death at consumption of approx. fermentation procedure where the LAB grow and convert 250 mg/day of EPA plus DHA was demonstrated [11]. In the sugar to lactic acid which leads to a pH decrease from industrialized countries, approx. 36–40% of the total calories around 5.8 down to 5.3–4.6, depending on the amount of in the food supply come from fat, nearly half of which is from available fermentable sugars and process conditions. The meat intake [12, 13]. staphylococci, when present, will contribute to flavor devel- A way to reduce the amount of fat in fermented sausages opment and reduction of nitrite and nitrate. Subsequently, the is to simply add less backfat to the batter. There are however sausages are dried until the desired 𝑎𝑤 is reached. Fermenta- limitations as to how large such a reduction can be before tion and drying steps are performed in smoke chambers and sensory and technological quality of the sausages are reduced drying rooms with controlled temperature and humidity. since fats contribute profoundly to taste, texture, and mouth Fermented sausages can be either dry or semidry [7]. feeling. In Norway, one of the large producers of fermented Generally, DFSs have 𝑎𝑤 ≤ 0.90, while, for semidry sausages, sausages has a commercial product called “Extra Salami,” 𝑎𝑤 ranges between 0.90 and 0.95 [8]. American type dry which is produced with 20% less fat than in the standard sausages such as Genoa salami, dry salami, and pepperoni salami recipe. An alternative strategy is to replace some of contain 25–40% moisture, are heavily spiced, are not heated the pork backfat with more healthy unsaturated fats or oils. ∘ above 26.7 C, have a firm texture, and are usually shelf-stable. Again, several challenges are associated with substitution of InEurope,thesefermentedsausagescanbefurtherdivided animal fat for oils in comminuted meat products. Incorporat- into Northern and Mediterranean types [9]. Northern type ing hydrophobic oils can be difficult as meat contains approx. products such as cervelatwurst, Westphalian salami, plock- 75% water and is hydrophilic. Also, increasing the content wurst, boerenmetworst, and Belgian salami often contain of unsaturated fatty acids increases the susceptibility to lipid beef and pork and are characterized by relatively short oxidation, which reduces shelf-life [14]. By emulsifying or ripening periods of up to 3 weeks and involve clearly sep- encapsulating the oil and by addition of antioxidants, this arated fermentation and drying periods. Rapid acidulation problem can in many cases be mitigated. to final pH values below 5 and smoking ensure microbi- In a set of experiments with DFSs, 25% of the pork ological safety and shelf-life. Mediterranean type sausages backfat was substituted for an emulsion with linseed oil [15]. such as Spanish salchichon´ and chorizo and Italian salami No oxidation problems were detected during the ripening are predominately pork products and involve longer ripening processinsuchsausageswithbutylhydroxytolueneand periods, up to several months, often without clear separation butylhydroxyanisole added as antioxidants. No substantial between fermentation and drying. Smoke is not applied and changes in odor, appearance, flavor, and oxidation status were acidulation to final pH values above 5 is slower. Instead observed. In Dutch-style fermented sausages, 15 or 30% of of smoking, the sausages are often covered with specific the backfat was replaced with pure commercial encapsulated molds. Semidry sausages, such as summer sausage, cervelat, fish oil, added either as such or as a preemulsified mixture Lebanon Bologna, and Mettwurst are usually fermented at with soy protein isolate [16]. Sausages with encapsulated fish ∘ higher temperatures, 32.5–38.1 C, for more than 18 h to a oil appeared to retain the overall quality, and no clear effects Journal of Food Quality 3 were found in the different sensory attributes when using 15 Potassium ions can give a bitter taste, which poses restrictions untrained assessors. on to how much can be introduced in a product. No changes In most experiments where oil partly replaced backfat, the in organoleptic characteristics of fermented sausages were oil was added together with stabilizers. In low fat fermented detected when KCl substitutions for NaCl were lower than sausages (total fat content 10%), 20% of the fat was substituted 40% [23]. Corral et al. observed the same for slow fermenting ∘ for preemulsified olive oil and added 𝜄-carrageenan [17]. The sausages fermented and dried at 10–12 Cfor57dayswhere application of vacuum packaging over the last two weeks of 16% of the NaCl was replaced with KCl [24]. Although a slight ripening improved the physicochemical characteristics of the reduction in aroma development was detected, the sausages sausages and resulted in sensory attributes equal to or better were judged to have the same overall quality as the controls than the high fat control sausages with 30% backfat. Likewise, with 2.7% NaCl. 32.8% of the fat could be replaced by a linseed oil carrageenan DosSantosetal.producedfermentedsausageswith50% gelled emulsion without loss of sensory qualities [18]. In reduction of NaCl (12.5 g/kg), sausages where 50% of the Pamplona-style chorizo, both low sodium ion and low fat NaCl was substituted for KCl, CaCl2,ora1:1mixtureof (20% less than standard recipe) sausages were produced [19]. KCl and CaCl2 [25]. A 50% NaCl reduction and a 50% Here 58% of the NaCl was substituted for 20% KCl and 38% substitution of the NaCl for KCl did not influence the CaCl2,and50%ofthebackfatwasreplacedwithanalginate fermentation and maturation process. Sausages with CaCl2 emulsion consisting of 64% water and 30% olive oil. 5% inulin showed a decrease in pH, an increase in 𝑎𝑤, and lower lactic was also added to sausages. These sausages retained sensory acid production. Overall, sensory acceptance decreased in notes similar to those of the traditional control chorizo and sausages with reduced sodium content. However, preference achieved a good acceptability rating. Fat can also be partly mapping identified a group of consumers that existed for replaced with other compounds. When 50% of the pork fermented sausages with 50% reduced NaCl substituted for backfat was replaced with konjac gel, a low-calorie ingredient KCl or a blend of KCl and CaCl2. De Almeida et al. produced with a high content of nondigestible fiber, the sausages had salami sausages with 60% reduction in NaCl and adding an overall acceptability similar to the control sausages [12]. different amounts of a 1 : 1 blend of KCl and CaCl2 [26]. The A “Super Salami” with 45% less fat and with 10% canola oil salt replacement mixtures did not affect the technological encapsulated in alginate and guar gum is available on the process, but the sausages had lower acceptability. The authors Norwegian market. The finished sausages contain 20% fat, suggested to enhance the sensory perception by addition of which 25% is saturated fat, 60% is monounsaturated, and of spices and other flavor enhancers. This strategy was 15% is polyunsaturated. A review of approaches to healthier successfully used when sausages were produced with 25% or formulations of comminuted meat products in conjunction 50% of their NaCl replaced with KCl and supplemented with with fat and salt has been published by Bolger et al. [14]. 2%yeastextract[27].Theincreasedvolatilecompoundsfrom catabolism of the yeast extract suppressed the sensory quality 3.2. Salt. Salt serves many important functions in fermented defects caused by KCl introduction. KCl is considered safe sausages, where it contributes to taste, texture, microbio- and exhibits an antimicrobial activity similar to that of NaCl logical safety, and overall acceptability. High sodium ion [28]. Replacing some of the NaCl with KCl should therefore + consumption (>2gNa /day, equivalent to 5 g salt (NaCl)/day) not influence antimicrobial safety of the sausages. General contributestohighbloodpressureandincreaseoftherisk implications of salt and sodium reduction on microbial food of heart disease and stroke [20]. Most people consume too safety have been reviewed earlier [29]. much salt, on average 9–12 grams per day, or around twice the recommended maximum level of intake. The principal 3.3. Nitrite. In addition to its important preservative effect, benefit of lowering salt intake is a corresponding reduction nitriteisinvolvedindevelopmentoftheredcuringcolor in high blood pressure. WHO Member States have agreed to formation and flavor development and acts as an antioxidant reduce the global population’s intake of salt by a relative 30% [30, 31]. by 2025. Reducing salt intake has been identified as one of the According to the Commission Regulation (EU) number most cost-effective measures countries can take to improve 1129/2011, nitrates (sodium nitrate, E251; potassium nitrate, population health outcomes. An estimated 2.5 million deaths E252) and nitrites (sodium nitrite, E250, and potassium couldbepreventedeachyearifglobalsaltconsumptionwere nitrite, E249) are listed as permitted food additives. Max- reduced to the recommended level. Meat and meat products imum dose authorized for use in cured meat products by contribute 21% to the sodium intake [21]. the EU is 300 mg/kg nitrate (for some products 250 mg/kg Fermented sausages contain high amounts of salt, which nitrate) and 150 mg/kg K-nitrite (or 150 mg/kg Na-nitrite) contributes to the microbiological safety and shelf-life by measured as ingoing amounts [32]. Nitrate may be reduced by binding water and making it unavailable for microorganisms. Gram+ catalase+ cocci (GCC+) to nitrite in the meat. Nitrate Salt also has a profound impact on the technological prop- is less used nowadays and primarily employed in dry cured erties of the meat and thus on the sausage texture. It facili- hams and dry sausages where long, slow curing processes tates solubilisation of myofibrillar proteins, increases binding necessitate a long-term reservoir for nitrite that is reduced properties of proteins to improve texture, and increases to nitric oxide in several reactions, which can then react with + viscosityofmeatbatters[22].AstheNa ions cause health myoglobininthemeattogivetheredcuredcolor[33–35].For issues, reducing the NaCl content and/or replacing some of nitrite, the residual amounts will vary with the formulation it with other salts like KCl or CaCl2 has been investigated. of the product, especially if ascorbate (vitamin C) is added to 4 Journal of Food Quality prevent oxidation and to improve the color of the product. products tested were in the range 0–9 mg/kg. In Germany, According to EFSA, the ingoing amount of nitrite rather 116 samples of cured meat products were tested, of which than the residual amount contributes to the inhibitory effect 85% were below 20 mg/kg. Some reduction of the total N- against microorganisms. nitrosamine content in DFSs appeared to be possible through Flavor is a complex stimulus involving taste, odor, texture, the addition of ascorbic acid [42]. A large number of agricul- and temperature. The meat, salt, lactic acid, and spices are tural food products, seafoods, meat products, vegetable oils, major contributors to flavor. Nitrite contributes to the cured sauces, and seasonings contain N-nitrosamines in the range meat flavor. Several experiments with bacon, frankfurters, 0.2 to a few 𝜇g/kg [43]. A benchmark dose methodology for and hams produced with and without nitrite have been developing tolerable daily intakes (TDIs) has been developed reviewed [36]. The results usually showed higher flavor scores based on a large lifetime cancer dose-response study of for products produced with nitrite. NDMA in drinking water given to rats [44]. Taking into The antioxidant properties of nitrite will inhibit develop- account inter- and intraspecies differences, a TDI range of 4.0 ment of rancid off-flavors [37]. The antioxidant properties are to 9.3 ng/kg/day was calculated. From these considerations, caused by nitrite being oxidized to nitrate by sequestering intake of NDMA from DFSs will generally be well below the oxygen, which is then not available for oxidizing fatty acids. TDI. Similarly, nitrogen oxide can easily sequester oxygen and Partly due to the health concerns in conjunction with be oxidized to NO2 [34]. In addition, the stable complexes nitrite, there has been a growing popularity of cured meats between nitrite-derived compounds and heme-bond iron produced as “natural” and “organic” without addition of 2+ inhibit the release of free Fe , which is therefore not available nitrate or nitrite [33, 45, 46]. These “natural curing” processes for initiation of lipid peroxidation [38]. The antioxidant consisted of adding a natural source of nitrate along with properties of nitrites have also been partly explained by nitrite a nitrate-reducing starter culture. Most often, the natural and dinitrogentrioxides reacting with unsaturated lipids to source was a concentrated vegetable extract of celery (Apium form nitro-nitroso derivatives and thus stabilizing the lipids graveolens var. dulce) with about 3% nitrate. Sometimes the against peroxidation changes [39]. extracts are pretreated to convert the nitrate to nitrite before From a health perspective, nitrates are relatively non- use. Others have been employing Swiss chard (Beta vulgaris toxic, but nitrites and nitrite metabolic compounds such as var. cicla) powders. This product contains 3.0 to 3.5% nitrate. nitric oxide and N-nitroso compounds have raised concerns A benefit of this product compared with celery extracts is that over potential adverse health effects [40]. The International it contains no allergens. Agency for Research on Cancer (IARC) has concluded that The World Health Organization estimates that the daily nitrates and nitrites are probably carcinogenic to humans dietary intake of nitrate is usually between 40 and 172 mg under conditions favoring nitrosation where an NO group [47]. A substantial amount of dietary nitrate comes via is covalently bound to carbon, sulphur, oxygen, or nitrogen fruits and vegetables. For example, approximately 98% of atoms in an organic molecule. During curing in acidic the dietary intake of Swedish children originates from fruits environment, undissociated nitrous acid picks up a hydrogen and vegetables and only 2% from cured meat products [48]. ion and splits off a water molecule. The resulting positively In contrast, dietary nitrite amounts to less than 20% of charged nitrosonium ion may then react with amino groups the daily nitrite exposure. The remaining 80% results from to form N-nitrosamines. Some of these N-nitrosamines endogenous bioconversion of dietary nitrate to nitrite in are carcinogenic. In meat, the most relevant nitrosamines saliva. Humans generally consume 0.3 to 2.6 mg nitrite each are N-nitrosodimethylamine (NDMA), N-nitrosopiperidine day [47]. Some reports estimate that cured meat contributes (NPIP), and N-nitrosopyrrolidine (NPYR). Formation of 4.8% of the daily nitrite intake [49]. these compounds is only possible when secondary amines are Nitric oxide is involved in regulation of blood pressure ∘ present, pH must be <5.5, and temperature must be >130 C and in regulations of gastrointestinal, respiratory, and gen- (NPYR) or the product must be stored for a long time at room itourinary tract functions and immunologic reactions [50]. temperature (NDMA, NPYR) [38]. N-nitrosamines can also The basal level of nitrate in blood is around 2 mg/kg and be formed from biogenic amines. In a survey of DFSs of both that of nitrite approx. 100-fold lower [50]. Lack of nitric North and South European types in Belgium, N-nitrosamines oxide production can lead to a number of conditions like were detected in 54 of 101 samples [41]. The total amount hypertension, atherosclerosis, and thrombosis and can be remained below 5.5 𝜇g/kg except in one sample with 14 𝜇g/kg. ameliorated by dietary nitrite interventions [51]. A number NPIP was the most prevalent N-nitrosamine present above of case control studies have been conducted worldwide to limit of detection in 28% of the sausages. There was only a determine if there is a link between gastric cancer and nitrate limited relation between N-nitrosamine content and residual intake [49]. No such link has been found. Other studies level of NaNO3 andnorelationshipwithNaNO2 level. The trying to link nitrates and nitrites consumption to brain, authorsassumedthattheamountsofN-nitrosamineswere esophageal, and nasopharyngeal cancers have been inconclu- low because the median concentrations of residual NaNO2 sive. and NaNO3 levels were lower than 20 mg/kg in the screened In conclusion, one might argue that the positive effects of products. EFSA refers to several surveys on residual levels curing are overwhelming against the small possibility of the of nitrite in cured meat products [32]. The range varied formationoflowdosesofnitrosamines.Theintakeofcuring considerably, but generally the average residue levels were agents from meat products is small in comparison with other low. For example, in France 74% of raw dried cured meat foods [34]. Journal of Food Quality 5

3.4. Smoke. Smoking is a traditional treatment of Northern seafood products. A number of products contain PAHs with type fermented sausages and is part of the conservation to undetectablelevelsofBaP.TheEFSAthereforeconcludedthat inhibitgrowthofmoldsandbacteriaontheproductsurface. benzo(a)pyrene is not a suitable indicator for the occurrence In addition, smoking adds a desirable smoky flavor, delays of PAHs in food, and one should rather use a specific group lipid oxidation, and adds color from light lemon to dark of four (PAH4) or eight PAHs (PAH8) based on the available brown depending on the kind of smoldering wood and the data relating to occurrence and toxicity. The EU Commission time/temperature regime of the process. Smoke develops has in the Commission Regulation (EU) 835/2011 established from the charring of wood, usually beech, oak, alder, hickory, an upper limit of BaP and PAH4 for smoked meat and or maple as well as fruit trees. The wood is normally cut smoked meat products. As of Sept. 1, 2014, the limit for BaP is into shavings or saw dust. The thermal composition of the 2 𝜇g/kg, and the total amount of PAH4 is 12 𝜇g/kg [58]. The wood followed by oxidation generates hundreds of differ- accumulation of PAHs in different smoked meat products is ent compounds, mainly H2O, CO, CO2, alcohols, carbonyl related very significantly to the parameters of smoking and compounds, carboxylic acids, esters, hydrocarbons, nitrogen thekindofwoodusedforsmokegenerationandevenonthe oxides, and phenols [52, 53]. Most smoke compounds would location of the product in the kiln which affects the tempera- notbeallowedbylawtobeaddedtofoodsinpureform; ture and the flow rate of the smoke [52]. Codex Alimentarius however, since the toxicity and concentration in the products Commission code of practice CAC/RCP 68/2009 specifies ten are very low, smoking is generally regarded as safe. Many of variables that need to be controlled to minimize and prevent the phenols such as guaiacol and its derivatives, cresol, pyro- PAH contamination of meat products during smoking [59]. catechols, and pyrogallol, show high antimicrobial activity. These variables are fuel type, smoking or drying method The content and distribution of these compounds in smoked (direct or indirect), smoke generation process (temperature, meats are related to their solubility in lipid and water phases airflow, friction versus smoldering, liquid smoke), distance of the products. It is not yet possible to predict exactly the between the food and the heat source, position of the food in concentration of smoke phenols that is necessary to inhibit relation to the heat source, fat content of the food, duration bacteria. The inhibitory concentration of smoke phenols for of smoking, and direct drying, temperature during smoking Listeria monocytogenes is in the range of 10–100 𝜇g/g, which and direct drying, cleanliness and maintenance of equipment, is in the same range as that found when mini-salamis (20 mm and finally design of the smoking chamber and the equipment diameter) were smoked with beech (35–75 𝜇g/g) [54]. The used for smoke/air mixture (which influences the smoke desirable smoky flavor is predominately from phenols such density in the smoking chamber). The importance of these as syringol, 4-methylsyringol, 4-allylsyringol, guaiacol, 4- factors has been reviewed by Ledesma et al. [53]. methylguaiacol, and trans-isoeugenol [52]. The content of PAHs in smoked meat products is usually Some hydrocarbons formed in smoke are hazardous to well below the maximum level set by the EU Commission human health, namely, the polycyclic aromatic hydrocarbons [52]. The greatest amount of BaP is deposited on the meat (PAHs). These are highly hydrophobic compounds consisting product casing, and only a minor fraction then migrates of two or more fused aromatic rings, mainly of hydrogen intotheproduct[53].ThecontentofPAHinsausageswill and carbon atoms. Compounds with four or more rings depend on the type of casing used. Both for dry fermented are less volatile and adsorb on soot and other combustion Petrovskakolb´ asa´ sausages from Serbia and traditional DFSs particles. There are 15-16 PAHs that are considered by the from Portugal, the PAH contamination level was lower when IARC and the European Union due to their carcinogenic and collagen casings were used [60, 61]. mutagenic properties [55, 56]. They are classified as carcino- One option to reduce PAH in meat products is by using genic, probably carcinogenic, possibly carcinogenic, and not liquid smoke. This is an easier, more rapid, and repro- classifiable. Benzo(a)pyrene (BaP) is the only compound in ducible process [53]. Liquid smoke is produced by chilling the carcinogenic group. Special attention has been given to and thereby condensing wood smoke. The liquid smoke is a group of eight of the PAHs (PAH8), which were used in then refined and filtered to remove toxic and carcinogenic previous cancer studies and in EFSAs risk evaluation [55]. impurities containing PAH. Use of liquid smoke is therefore The PAH compounds convert to diol epoxides and bind generally considered to be of less health concern than covalentlytoDNAandcauseerrorsinreplication,mutation, traditional smoking. and tumor genesis. BaP,when administered by the oral route, has been reported to produce tumors of the gastrointestinal 3.5. Starter Cultures. In a traditional process for producing tract, liver, lungs, and mammary glands of mice and rats and fermented sausages, bacteria, yeast, and fungi contribute to has also been associated with several other cancers [57]. various degrees to the final product. However, it is generally For nonsmokers the main source of PAH is foods. The accepted that LAB play the most prominent role, since the median dietary exposure across European countries was initial acidification is essential, both technologically and from calculated both for mean and for high dietary consumers a safety perspective [62]. Low pH and organic acids will and varied between 235 ng/day (3.9 ng/kg body weight (b.w.) inhibit contaminant spoilage flora and potential pathogens per day) and 389 ng/day (6.5 ng/kg b.w. per day), respectively, and ensure preservation. Acid conditions also aid in texture for benzo(a)pyrene alone, and 1168 ng/day (19.5 ng/kg b.w. per formation due to meat protein coagulation and in color for- day) and 3078 ng/day (51.3 ng/kg b.w. per day), respectively, mation through the reactions of nitrite and nitrogen monox- for PAH8. The two highest contributors to the dietary ide with myoglobin [62]. Although LAB also contribute to exposure were cereals and cereal products and seafood and aroma formation, mainly through organic acid production, 6 Journal of Food Quality other bacterial groups appear to be more important. These Mold growth on the external surface of DFS is desirable are the Gram-positive catalase-positive cocci (GCC+), in on some types of fermented sausages in many European particular the coagulase-negative staphylococci (CNS). CNS countries, especially around the Mediterranean, but also in, convert amino acids and free fatty acids to potent aroma for example, Hungary and Belgium. The distinct grey-whitish compounds essential for taste notes of fermented sausages. In appearance of these products is an attractive feature. In addition, CNS also possess highly active nitrate reductase and the traditional manufacture of these products, the process catalase, which contribute to color formation by producing relies on the fortuitous inoculation of the maturing sausages nitrite from nitrate [35] and the limitation of lipid oxidation by spores resident in the air. The different factories have that may cause rancidity, respectively [63, 64]. Traditional their own distinct “house flora,” which are adapted to the production of fermented sausages is based on spontaneous process and will eventually dominate the surface growth fermentation; that is, endogenous microorganisms present in andensuresomereproducibilityoftheproductquality.The the raw material will perform the microbial transformation surface molds contribute to the taste and aroma of the of the material. However, it has long been known that better sausages by lipolytic, proteolytic, and lactic acid oxidizing reproducibility could be obtained by adding a small portion activities, enhance general quality parameters through oxy- of a previous successful batch when starting a new, the so- gen consumption, which counteract rancidity development, called “back-slopping” technique [65]. This is the forerunner and improve color. The mold surface layer also modifies the to the use of starter cultures, that is, the intentional addition drying rate and thus prevents excessive drying of the sausages of premade microbial cultures to a fermentation process, [76]. The specific conditions prevailing on the sausage ∘ either single or mixed, in order to control and standardize the surfaces, for example, temperatures from 10 to 20 Cand process. The first-generation starter cultures for fermented relative humidity starting at 90–95% and decreasing during sausages were developed in the 1940s in the USA. However, the ripening period, select for certain genera of molds, in thesewerenotbasedonthedominatingmicroorganisms particular Penicillium and occasionally Aspergillus.Common found in spontaneous fermentation, or even isolated from species are P. nalg i ov e n s e , P. chr y s o g e num ,andP. nordi c um meat, but rather on their technological feasibility, for exam- [76–78]. Mold starter cultures have been developed, most ple, surviving freeze-drying, and their fast acid production often consisting of spores of P. nalg i ov e n s e [78, 79]. The main rate. These cultures, primarily strains of the genera Pediococ- selection criteria for these cultures are their low potential cus, were useful for the particular products produced in the for mycotoxin production (see below) and their ability to USA, that is, “summer sausages” with very short production outcompetethe“houseflora”whileretainingtheabilityto and maturation times [62]. However, they were less suitable produce sausages of acceptable taste, aroma, and appearance forproductsoftheEuropeantraditionwithlongerfermen- [76, 78–80]. tation and maturation times. Research in the 1960s, 1970s, Fungal surface colonization of maturing sausages starts and 1980s, also confirmed in many later studies, revealed that with salt and acid tolerant yeast species, such as Debaryomyces these types of sausages were dominated by L. sakei or the hansenii. However, along with the decrease in 𝑎𝑤,there related species L. curvatus and, to some degree, L. plantarum is generally a shift in the mycobiota towards molds [81]. [62, 66–68]. The second-generation LAB starter cultures, Although the role of yeasts in sausage fermentation is not nowwidelyused,areoftenbasedonthese[69].Molecular equallywellknownasforbacteriaormolds,itcanbe characterization by, for example, genome sequencing and significant in some products [82, 83]. Lipolytic, proteolytic, comparative genomics has shown that strains of L. sakei and lactate oxidation activities account for this effect [81–83]. isolated from meat and meat fermentation have evolved to be Starter cultures containing D. hansenii have been developed, perfectly adapted to this particular environment [70–72]. L. sometimes in combination with mold spores [81]. plantarum lacks this specific adaptation but is a fast-growing, All starter cultures are, by definition, “functional,” since highly flexible bacterium with the largest genome size of their activities contribute to the transformation of the raw the lactobacilli. Some specific nonstarter LAB (“house flora”) material and to the appearance and quality of the final strains of L. plantarum have been shown to outcompete com- product. However, the description of a starter culture as mercial starters based on L. sakei or L. curvatus in industrial “functional” often pertains to one (or several) additional sausage production [73]. GCC+ strains were isolated from function(s), beyond the normal properties of a starter culture. fermentedmeatproductsintheearly1900sandtheirrolein Several such additional functions have been described, for aroma formation and color stability was established in the example, properties that enhance food safety (see also below) 1950s [2, 69]. They were subsequently suggested for use as or have a technological advantage [64]. In recent years, in starter cultures for sausage production, first as single cultures, accordance with trends in consumer demands, functionality but later mixed cultures were shown to be superior to both a for enhanced health properties has been studied. Probiotic single GCC+ culture or a single LAB culture [2, 63, 64]. The startercultureshavebeenoneofthemainthemesinthis success of these mixed cultures is likely because they reflect research [84]. The term “probiotics” was coined in the the course and dynamics of a spontaneous fermentation 1950s as an antonym to “antibiotics.” The term subsequently better than a single culture and thereby retain the aroma and developed into a scientific concept and was defined as taste of the traditional products [63, 74, 75]. The GCC+ strains “live microorganisms that, when administered in adequate most often found in spontaneous fermentation and also used amounts, confer a health benefit on the host” by FAO/WHO as starters are CNS and belong to the species Staphylococcus in 2001. This definition was later reinforced as adequate carnosus, S. xylosus,andS. saprophyticus [64, 74]. and sufficient [85]. LAB, especially bacteria belonging to Journal of Food Quality 7 the genus Lactobacillus, are recognized as common inhabi- 4. Microbial Hazards Associated with tants of the human gastrointestinal tract and have received Fermented Sausages considerable attention in the last decades for their health- promoting properties and use as probiotics. The use of Although historically considered as safe, the characteristics probiotic strains in fermented products was first employed ofDFSscanprovidesurvivalandevengrowthofcertain in the dairy industry, and milk-based products are still the pathogens in these products. Surveys have shown the pre- most common vehicles for delivery of probiotics [86]. How- sence of pathogenic Escherichia coli, Salmonella Typhimu- ever, being products where LAB proliferate and dominate, rium, Staphylococcus aureus,andL. monocytogenes in dry fermented sausages are also potential carriers for delivery of fermented sausages. Clostridium botulinum and Toxoplasma probiotic LAB strains [64, 87, 88]. There are some significant gondii have also been reported as potential microbial risks for consumers of DFSs. challenges in using fermented sausages as probiotic products Pathogenic microorganisms can be introduced through in comparison with dairy products. The most important are contaminated raw materials or through cross-contamination as follows: (i) the meat raw material is not sterilized or from equipment or personnel during processing or at retail. pasteurized before the fermentation process and a probiotic Conditions during sausage processing and pathogen char- bacterium must therefore be as competitive as any starter acteristics determine the ability for pathogen growth and culture normally used for the fermentation to outcompete survival and also determine possible strategies for pathogen the endogenous flora; (ii) the mature sausage constitutes elimination to ensure product safety. a harsh environment with low 𝑎𝑤 and containing salt and nitrate; thus, survival of the probiotic after fermentation 4.1. E. coli. Pathogenic E. coli belong to various pathotypes, should be validated; (iii) the numbers of the probiotic after with verocytotoxigenic E. coli (VTEC), (synonymous to maturation and storage must be very high since the serving Shigatoxigenic E. coli (STEC)) predominantly associated with size and daily consumption of fermented sausage product are meat. VTEC strains produce Shiga-toxins 1 and/or 2. They generally less than a comparable dairy product; and (iv) the may carry different virulence factors responsible for varia- probiotic should produce an acceptable product with regard tions in clinical manifestations. A subgroup of VTEC causing totasteandquality[89,90].Therearetwomainalternatives severe infections of enterohemorrhagic colitis and possibly in the research and development of probiotic fermented hemolyticuremicsyndrome(HUS)characterizedbyacute sausages.Thefirstistoselectstrainsbasedontheirprobiotic renal failure and anemia is designated enterohemorrhagic E. properties and subsequently investigate the suitability of the coli (EHEC). More than 150 different serotypes of VTEC have strain(s) in the production of fermented sausages. Using been associated with human diarrheal infections. Serotype this strategy, already commercial probiotic strains have been O157:H7 strains have been the most known disease causing studied. The perhaps most well-documented probiotic strain, VTEC.Non-O157haveemergedwiththeserotypesO26,O45, Lactobacillus rhamnosus GG, has been used in several studies O103, O111, O121, and O145, also known as the “big six,” for this purpose, with varying success [91–94]. Although the being most frequently associated with human disease [99]. GG strain can perform the fermentation, there seems to be Raw meat ingredients contaminated through the slaughtering a balance between inoculum size, off-taste (due to excessive acid), and enough survival in the finished product, which is process are regarded a primary source of VTEC in DFSs. difficult to achieve [94]. Similar problems were encountered Cattle are regarded a primary O157:H7 VTEC reservoir using another well-documented strain, L. plantarum 299v although other animals such as sheep, swine, goat, and [95]. A better outcome was obtained with a new L. rhamnosus deer can also be carriers of VTEC. In outbreaks caused by strain, isolated from human intestine and with potential contaminated DFSs, VTEC serogroups of O157, O26, O111, probiotic properties [95]. The disadvantage of using such a andO103havebeencausativeagents[6].Lowcellnumbers strain is that it is not possible to use the wealth of previous (10–1000) are sufficient to cause disease [100, 101] and levels documentation, which a well-known strain might have, in lower than 1 cell (EHEC O111:NM) per 10 g were reported promoting the product. The second strategy that has been in a salami outbreak from Australia. Although growth of used for developing probiotic meat products is to use strains pathogenic E. coli during initial phases of fermented sausage isolated from successful meat fermentation, or even meat production can occur, combinations of low pH and 𝑎𝑤 inhibit startercultures[73,96].Suchstrainshavetobeassessedfor growth of E. coli in finished products [88]. However, extensive potential probiotic properties but are usually well adapted pathogen survival in finished products has been reported to the meat fermentation environment. These strains will [102–105]. Strategies for effective VTEC elimination in DFSs also suffer from the fact that their probiotic properties areachallengeforproducers.Ithasbeensuggestedthat will be poorly documented in comparison to well-known serotype O157:H7 strains have enhanced tolerance to acids documented strains. There have been attempts to launch compared to other serotypes and that this may have a role probioticmeatproductscommerciallyinGermanyandJapan in their capacity to cause outbreaks via low pH foods like [97], but the outcome in commercial terms is unclear. An DFSs,forexample[106,107].However,withinthisandother obstacle in the development of probiotic products in general serotypes, strains variations in acid resistance exist. The low is also that EFSA has so far rejected all health claims of infectious dose, the serious outcome of EHEC infections, probiotics using a very strict assessment in their approval and several reported outbreaks linked to VTEC contaminated process [85, 98]. DFSs highlight VTEC as the most serious safety risk in DFSs. 8 Journal of Food Quality

Effective strategies for VTEC reduction/elimination during antigen presenting cells and with T-cell receptors on specific the whole farm to fork chain are therefore required. T-cells[121].SEsarealsoabletopenetratethegutlining andactivateimmuneresponses,therebyleadingtovomiting 4.2. Salmonella. Salmonella are important zoonotic patho- [122]. The level of S. aureus present in the foods causing disease in an English survey ranged from no viable S. aureus gens with high economic significance in animals and humans. 10 7 As foodborne pathogens, the two S. enterica serovars Epi- detected to 1.5 × 10 cfu/g with a median of 3.0 × 10 cfu/g dermidis and Typhimurium are dominating among human [123]. cases. Serovar Epidermidis is associated with eggs and S. aureus does not compete well with the indigenous poultry while Typhimurium is linked to meat of pork and microorganisms in foods and will grow better in processed bovine origin [108]. Most salmonellosis infections are self- foodswherethecompetingflorahasbeendestroyed,for limiting, yet severe and life-threatening complications (e.g., example, in products contaminated after a heat treatment or sepsis) can follow. Infected animals are the primary source whenthefoodprocessgivesS. aureus aselectiveadvantage. of Salmonella where transmission to environments and Thiscanbethecaseforcuredmeats,sinceS. aureus can tolerate high amounts of salt and grow down to 𝑎𝑊 =0.86. foodslikelyoccursthroughfecalcontaminationandcross- ∘ S. aureus is able to grow in a wide range of temperatures (7 contamination. According to EFSA, 2.8% of the samples ∘ ∘ taken from minced meat and meat preparations from other to 48 C) with an optimum 37 C and pH (4 to 10), with an species than poultry intended to be eaten cooked tested optimum of 6 to 7 [124]. These characteristics enable S. aureus positive for Salmonella intheEUin2010[109].Infoods, to grow in a wide variety of foods. such as minced meat and meat preparations intended to be Although S. aureus can tolerate high salt and low pH eaten raw, 1.8% of samples were Salmonella positive. A coordi- and is often implicated in meat outbreaks (ham, pork, and nated approach has led to a significant reduction of human sausages), few incidences on food poisoning from fermented cases of salmonellosis in the EU in the last decade. Still, sausages are reported [123, 125–129]. Outbreaks caused by S. Salmonella werethemostcommoncausativeagentoffood- aureus are usually old, of which some have been registered by borne outbreaks reported in EU in 2013 [108]. Salmonella Center for Disease Control [130–134]. S. aureus is frequently have been implicated in several outbreaks linked to con- found in fermented sausages, but generally at levels too low sumption of DFSs where contaminated meat ingredients are to produce enterotoxin amounts sufficient to cause illness. a common source. Reported outbreaks seem to be dominated Although S. aureus can tolerate salt and nitrite, it is a poor by fermented sausages produced from pork meat contam- competitor under anaerobic conditions, at low pH and low 𝑆 temperatures. If sausages are fermented at no higher than inated with . Typhimurium although other serovars (e.g., ∘ Montevideo, Goldcoast) have also been causative agents [110– 25 C for 2 to 3 days and the initial count of S. aureus is below 4 113]. The infectious dose can be low where 10–1000 cells 10 cfu/g, the risk of enterotoxin formation is low [2]. For ∘ are sufficient to cause disease [114]. Studies have shown semidry sausages, fermentation up to 43 Ciscommoninthe Salmonella to be more sensitive than E. coli O157:H7 and L. US, and a rapid pH drop during manufacture will ensure monocytogenes to at least certain DFS manufacturing process inhibition of S. aureus. Consequently, the American Meat parameters [103, 115, 116]. As for reduction of other pathogens, Institute in 1982 specified the maximum time allowed to reach use of starter cultures has a positive effect on Salmonella pH 5.3 [2]. Apparently, the use of appropriate process controls reductions, for example [117, 118]. Reported differences in and starter cultures has significantly reduced the incidence Salmonella reductions are influenced by variations in recipes, of “summer sausages” outbreaks of S. aureus food poisonings processes, and strains and direct comparisons between stud- in the US [2]. North Carolina State University Meat lab has ies are difficult. At higher contamination levels, complete proposed in their HACCP program that to ensure safety elimination through traditional processing is difficult. products should be fermented to pH 5.3 or below within 1200 degree hours [135]. 4.3. S. aureus. S. aureus is common on skin and mucosal When chorizo was inoculated with S. aureus and without ∘ membranes of humans with estimates of 20–30% persistent a starter culture and fermented at 30 C, the pathogen grew and 60% for intermittent colonization [119]. The bacterium well. S. aureus growth was, however, reduced by using starter ∘ isalsofoundonfoodanimals.S. aureus produces a range culture, lower fermentation temperature (20 C), and higher of staphylococcal enterotoxins (SEs) of which some show concentrations of spices, nitrites, nitrates, and ascorbate emetic activity [120]. SEs are a major cause of food poisoning, [136]. In addition, no enterotoxin A was detected in the latter which typically occurs after ingestion of foods, particularly sausages after drying. Both strategies using specific starter meat and dairy products, that have been contaminated and cultures and starter cultures in combination with bacteriocins stored at elevated temperatures where S. aureus have grown have been shown to reduce the presence of S. aureus [137– andproducedtoxins.Symptomsareofrapidonset,duetothe 139]. S. aureus growth in Italian dry salami was affected by the preformed toxins in the food, and include nausea and violent initial pH, initial levels of S. aureus, lactic acid bacteria, day vomiting, with or without diarrhea. The disease is usually of fermentation, and interactions between these parameters resolved within 24–48 hours. Staphylococcal toxin SEA is [140, 141]. the most common cause of staphylococcal food poisoning Other species of staphylococci (CNS) are frequently worldwide. The SEs belong to a group of superantigen toxins, foundinfoods.SomearealsousedasstarterculturesinDFS. which bypass conventional antigen recognition by interaction Of a set of 129 such different strains, only one strain carried with major histocompatibility complex class II molecules on an enterotoxin gene, and 78% of the strains did not carry Journal of Food Quality 9 decarboxylases for biogenic amine formation. Although 78% problems, and death. The toxins bind irreversibly to periph- of the strains possessed at least one gene encoding antibiotic eral nerve endings and block the release of nevrotransmitters. resistance, these CNS were considered to pose a low safety An overview over reported outbreaks associated with meat hazard [142]. and fish has been given previously [157]. The rapid alert system for food and feed (RASFF) for the years 2010–2015 does 4.4. L. monocytogenes. Foods contaminated by L. monocyto- notreportanyoutbreaksofC. botulinum from fermented genes can cause listeriosis, infections varying from mild flu- sausages. C. botulinum that can affect man are often grouped like symptoms to life-threatening disease with a high fatality into proteolytic and nonproteolytic strains. The proteolytic rate in vulnerable populations. Ready-to-eat (RTE) products strains are the most hardy ones and can grow down to a pH consumed without prior heat treatments and containing of 4.6 or at 10% NaCl and down to 𝑎𝑤 of 0.94. They also have higher than 100 cells/g are considered to pose a direct risk to spores that can withstand boiling for extended periods. The human health. L. monocytogenes is ubiquitous in nature [143] combination of low pH, high NaCl, and low 𝑎𝑤 ensures that and contamination of DFSs can occur through contaminated C. botulinum will not grow in matured fermented sausage. In ingredients, preferably raw meat. The important role of addition, nitrate or nitrite is added to the sausage batter to contaminated processing equipment and environments as a inhibit growth of C. botulinum and other pathogens. Nitrate source of Listeria in DFSs has been indicated in several studies is reduced by GCC+ in the batter to nitrite. The mechanism [144–147]. Thus, L. monocytogenes are commonly found in by which nitrite inhibits C. botulinum is uncertain. Nitrite has DFSs with reported prevalence up to 40% [148]. Prevalence been reported to inhibit the phosphoroclastic system of C. in beef is usually in the range 0–10%, but with generally botulinum [158]. This could be of importance for inhibiting higher prevalence reported on pork meat [149, 150]. Never- C. botulinum the 2-3 initial days of sausage production where theless, only one outbreak in Philadelphia, USA, in 1986/1987 the water activity is high and before the fermenting lactic acid with possible epidemiological association to fermented meat bacteria have lowered the pH. is known. Fermented sausages have been evaluated to be Hospital et al. produced two types of Mediterranean products of low to moderate risk associated with listeriosis. fermented sausages, salchichon´ and fuet with final pH of 5.0 This is due to usually low levels of L. monocytogenes in and 5.2, respectively [159]. 𝑎𝑤 was between 0.88 and 0.90. these products and that a high minimum infectious dose One batch contained the maximum ingoing dose allowed by 4 (>10 cells) is normally required for illness. Some growth the EU 150 mg/kg NaNO3 and 150 mg/kg NaNO2.Theyalso of L. monocytogenes can occur in the initial phase of DFS made sausages with 25 and 50% nitrate and nitrite reductions processing, but the combinations of low pH (5.3–4.6) and and control sausages without nitrate/nitrite. In no cases was 𝑎𝑤 (≤0.90) generally restrict growth of the bacterium in the toxin production detected from spores added to the sausages, fermented sausage products. The extent to which DFSs can be even though the conditions for growth of C. botulinum considered safe is primarily dependent on the fermentation remained acceptable for 8–12 days during manufacture. Cell and drying process. With the wide specter of fermented free extracts from a meat isolate of Staphylococcus sciuri sausages produced, not all sausage recipes and processing have been shown to inhibit C. botulinum in vitro and may conditions may ensure products where the levels of L. mono- show some potential in inhibiting C. botulinum in fermented cytogenes are compliant with the microbial criterion of ≤100 sausages [160]. colony-forming units per gram [151]. It is therefore important The C. botulinum concern in conjunction with cured for the DFS producers to gather information on the safety of product is more relevant for nonfermented products which their products in terms of L. monocytogenes contamination could support growth than for fermented sausages. The use of and growth and implement processing parameters to assure nitrite in fermented sausages, the conditions in the sausages food safety. notbeingabletosupportgrowthofthebacterium,the The effects of using starter cultures for increased number of C. botulinum spores generally being very low if pathogen reductions have been shown in several studies, present, and the lack of registered outbreaks from fermented for example [152–154]. In general, enhanced reductions were sausages together point to a low risk of food poisoning from obtained in products with low pH and low 𝑎𝑤 and stored these products. under ambient conditions [103, 116, 155]. Reductions of L. monocytogenes during fermentation and drying in fermented 4.6. Toxoplasma gondii. T. gondii is an obligate, intracellular sausages are dependent on many factors, including strain parasite, which is widely distributed in the world. Conven- differences in their ability to tolerate and adapt to DFS tionally it is associated with handling cats and cat litter; conditions that are also dependent on recipe and processing however, Center for Disease Control and Prevention, USA, conditions [147, 156]. now estimates that 50% of toxoplasmosis is foodborne and that foodborne toxoplasmosis causes 327 deaths annually 4.5.C.botulinum.C.botulinumis a strictly anaerobic spore and is the leading cause of death from foodborne pathogens forming bacterium. Spores of C. botulinum occur in the after Salmonella in USA [161, 162]. Consuming undercooked soil and may enter the meat from contaminated hides. The meat products has been considered the major risk factor. botulinum neurotoxins are produced in growing vegeta- Healthy adults generally have no symptoms, whereas severe tive cells after the spores have germinated. The toxins can illness can occur in infected fetuses, newborns, immunocom- cause nausea, vomiting, fatigue, dizziness, dryness in mouth promised individuals, and transplant patients. Nitrite and and throat, paralysis of muscles, double vision, respiration nitrate, spices, low pH, and cold storage have no effects on 10 Journal of Food Quality the viability of T. gondii cysts [163]. The cysts do not survive histamine, with tyramine usually being the most abundant freezing for longer than 4 hours. Using frozen meat for the [168]. Contaminant Gram-negative enterobacteria and/or sausage batter will thus reduce the risk of infection. Regarding pseudomonads, present in the raw material, are the most DFS production, duration of the fermentation is critical to important BA producers before the onset of the fermentation T. gondii survival. Tissue cysts remain viable in fermented by LAB. High BA content of food products is often considered sausages after 12 h of treatment even in presence of 2% curing an indication of spoilage or hygiene failure in the handling salt. When fermented sausages were produced containing of the raw material [165, 168]. Good hygienic quality of the experimentally contaminated goat meat, no viable cysts were meatandarapidpHreductionintheinitialstageofthe detected in the final sausages after 12 days [164]. These and sausage production process are essential for inhibition and other risk evaluations conclude that fermentation over long control of BA production by these contaminants [169]. Salt periods reduces the risk of infection [163]. and nitrite tolerant Gram-positive bacteria, such as LAB and CNS, will initiate the fermentation and eventually dominate 5. Other Microbiology Related Health and the microflora. Prominent tyramine producers among LAB Safety Concerns relevant for sausage fermentation are L. curvatus and many enterococcal strains found in artisanal sausage manufacture 5.1. Biogenic Amines. Biogenic amines (BAs) are basic, non- in southern Europe [170]. Histamine producers are very volatile low-molecular weight, nitrogenous compounds, rare among sausage LAB and histamine, when present in common in living organisms where they perform various sausage, is considered to be produced by mainly contami- functions on, for example, the nervous, gastric, and intestinal nant enterobacteria [169]. However, specific strains of, for systems and on regulation of blood pressure [165]. They are example, L. buchneri and L. parabuchneri harbor the histidine formed as a result of normal metabolic activities in humans, decarboxylaseenzymeandareconsideredspoilageorganisms animals, plants, and microorganisms, generally through in cheese [171, 172]. Although never dominating a sausage fer- decarboxylation of the corresponding amino acids. BAs are mentation, such lactobacilli may be present as contaminants of considerable food safety concern as they may be present [169]. Other LAB relevant for sausage fermentation, such in various foods and when ingested in excessive amounts as L. sakei and L. plantarum, are generally nonaminogenic may cause certain diseases, or disease-like conditions, due [168,170,173].Aminoaciddecarboxylasesareuncommon to a disturbance of the normal physiological concentrations. in the most common CNS relevant for sausage fermentation, Symptoms of intoxication include headaches, flushes, nau- for example, Staphylococcus xylosus, S. saprophyticus,andS. sea, cardiac palpitations, and increased or decreased blood equorum [173]. However, occasional strains of S. carnosus and pressure. The most important BAs in foods are histamine, S. equorum may show BA production [142, 173]. putrescine, cadaverine, tyramine, tryptamine, phenylethy- Different strategies have been investigated to control lamine, spermine, and spermidine [166]. Of these, histamine and minimize BA formation in fermented sausages. The andtyraminearethemosttoxic.Presenceofsomeoftheother addition of specific inhibitory agents to the meat batter, such BAs may enhance the effects of histamine or tyramine [166]. as wine [174] or plant essential oils [175], is an example. Normal physiological concentrations of BAs are carefully Such additions reduce the initial contaminating flora, thereby regulated in the human body. For instance, the amines can reducing BA formation, but may also change the product be oxidized by monoamine oxidases (MAO) or diamine taste and appearance. Methods have been suggested for the oxidases (DAO). Hypersensitivity for BAs in some humans removal of BAs after their formation, such as the use of maybecausedbydecreasedactivityoftheseenzymesdue fermentative bacteria with amine oxidase activity [176] or to deliberate inhibition (MAO inhibitor drugs) or genetic the use of gamma radiation [177]. However, such procedures disposition [167]. Definitive toxicity levels or limits are there- are considered inappropriate since it may disguise incidents fore difficult to determine [165]. Amino acid decarboxylases of hygienic malpractice and/or spoilage [169]. The generally are the enzymes responsible for the formation of BAs. These recommended and most efficient way of reducing and/or enzymes are widely present in spoilage microorganisms, but controlling BA formation in fermented sausages seems to be also ubiquitous in desirable microorganisms, such as bacteria the use of nonaminogenic starter cultures [165, 168, 175, 178– important in fermented sausages, that is, LAB and CNS [168]. 182]. The use of a LAB starter culture results in a more High levels of biogenic amines may occur in foods such rapid pH decrease than a spontaneous fermentation, thereby as fish, fish products, and fermented foods (meat, dairy, inhibiting contaminant Gram-negative bacteria and thus some vegetables, beers, and wines). Generally, the potential the potential for BA formation at the initial stages of the of BA formation increases with the protein content of the process. The dominance of nonaminogenic LAB during the raw material as the breakdown of proteins provides the fermentation ensures minimal BA production. Nonamino- aminoacidprecursorsforBAs.Fishandcheesearethemost genic CNS will contribute to the effect. Mixed cultures of both implicated products in foodborne BA intoxication. No cases nonaminogenic LAB and CNS have been shown to perform of BA poisoning have implicated fermented sausages as the better than single starters, probably because each starter cause, although measured amounts of BAs have in some controls and dominates different parts of the microflora [169, instances reached similar levels as in fish related outbreaks 178]. To ensure dominance of the selected starters, the use [165]. of so-called autochthonous starter cultures is recommended The most important BAs present in fermented sausages [168,173,183].Thesearebacterialstrainsisolatedfromthe of food safety concern are tyramine, phenylethylamine, and particular products they subsequently should be used in Journal of Food Quality 11 as starters. Such starters are potentially better adapted to foods [191, 192]. A more practical approach is to carefully each specific process than commercial cultures and will also choose the environmental parameters during ripening, espe- preservethequalityandtasteoftheoriginalproduct.Ifcom- cially with regard to 𝑎𝑤 and temperature, in order to favor mercial cultures remain the only option, they should be tested colonization of starter cultures against OTA producing fungi for performance since highly competitive nonstarter LAB [193]. may dominate the fermentation, despite the use of starter cultures [73]. 5.3. Antibiotic Resistance. The growing level of resistance to In conclusion, the selection of starter cultures, especially antibiotics in bacteria presents a serious concern to human LAB, for use in fermented sausage production, should use and animal health and presents significant financial and the absence of amino acid decarboxylase activity as a basic societal costs. Antibiotic resistance (AR) in food bacteria is criterion. of concern because they may act as reservoirs for AR genes. Even if the relative amount of antibiotic resistant bacteria 5.2. Mycotoxins. The surface colonization of dry fermented in a particular fermented food product may be low, the sausages by fungi is nearly inevitable. The conditions are ideal absolute number can nevertheless be significant because large for, for example, Penicillium species, unless specific measures amounts of living bacteria are ingested when the food is are taken to minimize fungal growth, such as mechanical consumed. Food bacteria may carry transferable AR, which removal or the use of dipping regimes with antifungal could be transferred to commensal or pathogenic bacteria compounds, for example, sorbate solutions. Smoking may inthegastrointestinaltract.Thepresenceoftransmissible also inhibit the growth of fungi to some extent. One or AR genes should therefore be an important safety criterion more of these measures are often used in the Northern in the selection of starter cultures [180]. Enterococci are European, especially Scandinavian, tradition of fermented generally not used as starter cultures for fermented sausages sausage production where mold growth is undesirable. but may be involved in spontaneous fermentation. Entero- However, as mentioned, mold growth on the surface is a cocci have been thoroughly investigated with regard to AR desirable and characteristic feature of many products in some because of their clinical significance. AR is also frequently countries. A safety concern with regard to surface growth of detected among food enterococci [194]. Because enterococci molds on fermented sausages is mycotoxin production. Most harbor different gene transfer mechanisms (e.g., pheromone- Penicillium species are capable of producing one or more responsive plasmids, conjugative and nonconjugative plas- mycotoxins [184, 185], the most important being ochratoxin mids, and transposons), they may acquire these determinants A (OTA), patulin, citrinin, cyclopiazonic acid, and roquefor- fromotherenterococcalstrainsandtransferthemtopotential tine. In surveys of molds isolated from fermented sausages, pathogens [195]. This represents a possible risk related to the potentially toxigenic Penicillium strains are commonly found use of enterococci as probiotics or starter cultures [194, 195]. [79, 186]. Actual production of mycotoxins in the products Thus, no enterococcal strains are currently included in the has also been shown, though to a lesser degree [79, 80, 187]. QPS (qualified presumption of safety) list of EFSA (European P. nalg i ov e n s e strains were early selected as starter cultures Food Safety Authority) [196]. due to their apparent low toxigenic potential and useful Lactobacilli have a long history of safe use in fermented technological properties [78, 188]. This seems still to be the food, which supports their GRAS (generally recognized as best choice as more recent studies confirm low toxigenic safe) and QPS status granted by FDA (US Food and Drug potential [76, 79]. Administration) and EFSA, respectively. Many Lactobacillus Fungal starter cultures alone may not always be able to species are intrinsically resistant to a number of antibiotics, outcompete resident house flora, which has adapted over for example, streptomycin and vancomycin [180, 197]. How- long time. Other measures may be necessary to control ever, transmissible AR has frequently been detected, also mycotoxin production. OTA represents the most important in strains isolated from fermented sausages [180, 198–200]. mycotoxin produced by different molds relevant for sausage Tetracycline resistance, mediated by the tetM gene, and the production, that is, Penicillium strains [80]. P.verrucosum and ermB erythromycin resistance gene seem to be the most P. nordi c um are capable of producing OTA when they grow common [180, 199]. In vitro experiments have shown that on the sausages surface during both ripening and storage AR determinants can be transferred from meat associated [187]. OTA is undesirable because it is classified by IARC into LAB to other LAB and to pathogens [201, 202]. A similar “Group B” as a molecule with possible carcinogenic activity in pattern exists in CNS [180, 203, 204], showing that most AR humans [189]. Ozonated air has been suggested as a method genes are shared in nearly all meat associated Gram-positive for preventing the growth of OTA producing molds [187]. bacteria [180]. This may reflect the (mis)use of antibiotics in Protective yeast cultures (D. hansenii and Saccharomycopsis animal husbandry for decades, leading to a large pool of AR fibuligera) were recently shown to inhibit OTA producing genes present in the microbial population, spreading also to fungi in a fermented meat product [190]. It is unclear if bacteria in the food chain [205]. To minimize the potential this technique can be applied to fermented sausages where risks associated with the intentional use of microorganisms a mold coat is desired. Another biocontrol approach is the in food (e.g., starter cultures and/or probiotics), including use of nontoxigenic molds producing small, cysteine-rich transferofAR,EFSAhasregulatedtheindustrialuseof antifungal proteins (AFPs). These strains, or the purified bacteria as starter cultures through the QPS system [196]. In AFPs, have been suggested as useful for controlling growth addition, guidelines have been developed for assessing AR in and mycotoxin production by toxigenic fungi on dry-ripened relevant strains [206]. 12 Journal of Food Quality

6. Reduction of Microbial Hazards to the broad range of DFS products varying in pH, salt content, 𝑎𝑤, recipe, and production process like fermentation Reported outbreaks and disease history have shown that main temperature and maturation time. Parameters important for microbial pathogens in DFSs include VTEC and Salmonella. VTEC reductions have been reviewed previously [6, 7]. As a food safety hazard in DFS, L. monocytogenes is regarded Reduction of VTEC in traditional production processes of less relevant although their presence throughout the man- salami, pepperoni, and some other types of DFS was generally ufacturing processes of DFS is well documented [207–210]. 1-2 log, although some higher reductions were also reported Nevertheless, L. monocytogenes is a significant pathogen [6]. Comparable reductions are often reported for Salmonella where its presence in ready-to-eat products is troublesome. while inactivation of L. monocytogenes is generally lower, typ- Its elimination from DFS products is therefore important. ically <1log[103,116,152,219–222].ReducedinactivationofL. Strategies for control and elimination of pathogens in DFS monocytogenes is probably due to their overall high tolerance include optimization of recipe and process parameters and to acid, high salt, and low 𝑎𝑤 environments [223]. In several eventually use of postprocess treatments of finished sausages studies, ingredients or production parameters (i.e., nitrite to ensure safe products. Several outbreaks caused by VTEC concentration, fermentation temperature, final pH, degree of contaminated fermented sausages lead the US Food Safety drying, and ripening time) have been varied systematically and Inspection Service to establish a lethality performance to enhance the safety of DFS [6]. Our group studied the standard requiring 5-log reduction of E. coli during DFS potential for VTEC reductions by combining recipe and processing. In Canada, a 5-log reduction is recommended, process parameters within limits that would give acceptable while in Australia, the required reduction is 3-log units [211]. products of two types of DFS, salami, and Morr [104, 224]. There are limitations in how much different parameters in The factorial designed experiments showed that high levels recipe and process can be varied without negatively affecting of salt and curing salt (NaCl and NaNO2)andglucose the characteristics and sensory quality of these products. (lower final pH in the sausages) along with fermentation at Combination of parameters in recipe and process according elevatedtemperatureprovidedenhancedVTECreductions. to the “hurdle concept” for optimal reduction of pathogens High fat and large casing diameters gave the opposite effect. while maintaining the sensory quality of the products has The importance of 𝑎𝑤 for VTEC reductions in DFS was been one approach. More recently, the effects of more novel documented. High and optimal fermentation temperature technologies for, for example, meat batter decontamination were important to ensure growth and activity of the starter and postprocess treatments of finalized DFS have been culture, with subsequent lactic acid production, pH drop, evaluated [212]. moisture loss, and 𝑎𝑤 reduction over time. In line with An overview of reported processing and postprocess other studies, approximately 3-log reductions were obtained strategies for elimination of pathogens in DFS, with particular compared to 1.5-log reductions for standard recipe DFS [104]. focus on VTEC, is provided below. Higher reductions have been reported but seem difficult to obtain within levels relevant to producing high quality DFS 6.1. Reductions of Pathogens in Raw Meat Ingredients. Con- [6]. taminated raw meat and possibly nonmeat ingredients can A meta-analysis of 44 separate studies investigated the provide important sources of VTEC and Salmonella. Freezing relative effects of temperature, pH, and 𝑎𝑤 on the survival of raw meat prior to be used in DFS production is not of E. coli during manufacture of fermented meats. The study uncommon. Bacteria in the meat can be damaged by a indicated that temperature (fermentation, maturation, and freeze/thaw process, and this has been shown to provide an storage) accounted for 61% of the variability in the data while extra 0.5-1-log reduction of E. coli O157:H7 in the final salami pH and 𝑎𝑤 accounted for less than 8% [225]. Similarly, in product [213]. Another strategy, commercially used in the a meta-analysis including 13 studies on inactivation of L. USA, is heat treatments of raw meat ingredients by lactic ∘ monocytogenes in fermented sausages, temperature explained acid–hot water (80–90 C). The process provided 3.6–3.9- 60% of the data variability while pH and 𝑎𝑤 explained only a log reductions of Salmonella and E. coli O157 in final DFS, small part [226]. though with some negative sensory influences [214]. Use of The above studies show that elevated temperatures in ∘ high pressure processing (HPP) of meat trimmings for DFS the range 25–47 C, although not lethal to E. coli and L. affected the physiochemical properties of the meat batters monocytogenes perse,wouldbeeffectiveforpathogeninac- and had negative effect on the sensory properties of the tivation in the processing of DFS under conditions where DFS [215]. Irradiation in the range 1.5–4 kGy of raw meat/fat the bacteria are unable to grow. Increased inactivation of ingredients prior to production of DFS delivered a 5-log relevant pathogens, including VTEC, L. monocytogenes,and reduction of E. coli O157:H7 but was less effective in reducing Salmonella, with increasing temperatures has been shown L. monocytogenes [216, 217]. Irradiation resulted in products in several studies [102, 103, 222, 225–227]. For effective with quality indicators closely resembling those of traditional inactivation of pathogens it is crucial to obtain conditions dry sausage [216, 218]. preventing pathogen growth (low pH, 𝑎𝑤), but once these conditions have been reached, it is the factors of time and 6.2. Reductions of Pathogens through Changes in Recipe and temperaturethatmostdramaticallyimprovethemicrobial Process Parameters. There are large variations in the reduc- safety of the product. Overall, optimal combinations of tions of pathogenic E. coli, Salmonella,andListeria in dif- hurdles and control strategies during DFS processing could ferent processes and products of DFS. This is expected due enhance the safety of DFS but finished products could still Journal of Food Quality 13 contain surviving pathogens. No single parameter appears S. aureus,anddifferentclostridia.Theuseofbacteriocin- to enhance VTEC reduction enough to entirely eliminate producing LAB as starters for fermented sausages therefore pathogens. Consequently, application of several measures to shows potential for natural enhanced safety of these products reduce risk should be taken. [64, 87, 242]. The so-called class IIa bacteriocins, sometimes Changes in recipe or process parameters do not neces- referred to as “pediocin-like” (after the first discovery of this sarily lead to enhanced reduction of pathogens. For example, class, pediocin PA-1), are particularly potent against Listeria when semidry reduced fat (20% less than control) Italian species, including L. monocytogenes [245]. Class IIa bacteri- salami was spiked with E. coli, S. Typhimurium, and L. ocins are relatively small amphiphilic peptides of 3.5–5 kDa monocytogenes, the reductions during manufacturing were and the mode of action is permeabilization of the cell mem- similar to those of other typical Italian salami [228]. brane of susceptible cells, mediated via a membrane-located Application of novel technologies combined with tra- receptor protein [246]. Production of class IIa bacteriocins ditional hurdles (e.g., low pH, 𝑎𝑤, and temperature) in is a relatively common trait among LAB species relevant for the production process of DFS also presents an interesting fermented sausages, that is, L. curvatus and L. sakei [245, 247– venue for enhancing the quality and safety of fermented 251]. Bacteriocinogenic strains of these species have therefore meat products [212]. For optimal combinations of control been tested as starter cultures in several fermented sausage strategies, it is important to consider bacterial stress tolerance experiments and their antilisterial effect has been evaluated and cross-protection scenarios ranging from possible antago- [239, 252–259]. Generally, bacteriocinogenic L. curvatus and nistic to additive to synergistic effects that can be obtained by L. sakei starters could reduce the L. monocytogenes numbers combining different treatments and hurdles; see, for example, to some degree in the finished product compared to con- Gayan´ et al. [229]. trols with nonbacteriocinogenic cultures. However, the effect Overall, optimal combinations of hurdles and control varied between barely significant to a 2-log cfu/g reduction, strategies during DFS processing could enhance the safety of depending on strain and recipe. This rather modest effect DFS; however finished products may still contain surviving compared to the promising inhibitory potential as measured pathogens. in in vitro experiments can be explained by interaction of the bacteriocin with the sausage matrix, for example, fat 6.3. Importance of Starter Cultures for Safety. The importance adsorption or proteolytic degradation [260]. Moreover, the of using starter cultures for effective reduction and inactiva- potential for bacteriocin production by the producer strain tion of pathogens of E. coli, Salmonella,andListeria in DFS is maybeinhibitedtosomedegreeinthesausageenvironment well documented [7, 64, 105, 230]. Different starter cultures [64]. The most common LAB bacteriocins used for sausages, may vary in their abilities to reduce these pathogens [64, such as those of class IIa, also have some general drawbacks. 153, 231, 232]. Combinations of starters may give increased Theyhavenoactivitywhatsoeveronsomeofthemain reduction in E. coli during sausage production [233, 234]. The pathogens relevant for the product, Salmonella and EHEC performance of Lactobacillus sakei in sausage fermentation [239]. In addition, L. monocytogenes strains may develop was shown to be improved by heat, cold, and salt stress resistance to some bacteriocins, especially class IIa, at rela- prior to inoculation [235]. Selection criteria for lactic acid tively high frequencies in vitro [241]. Whether this occurs in a bacteria used as starter cultures in fermented sausage were food product is currently unclear. In conclusion, bacteriocin- reviewed by Ammor and Mayo [236]. The growing interest in producing starters may enhance food safety to some degree artisanal products of fermented sausages has also identified a but can never replace good manufacturing practices [64]. need for the isolation and use of appropriate starter cultures that could provide increased food safety and maintain the 6.4. Preservatives for Enhanced Safety. The addition of vari- characteristics of such products. These sausages are often ous compounds with antibacterial effects has been evaluated produced following traditional practice in small processing as ingredients in DFS for improved safety. Microencapsulated units, with no use of starter cultures and less control of allyl isothiocyanate (AIT) at 500 ppm gave 4.75-log reduc- temperature and humidity during fermentation and ripening tions of E. coli O157:H7 in DFS 28 days after processing, >3log compared to industrial production [237, 238]. more than control DFS [261]. Deodorized mustard powder, The main preservative effect of starter cultures for fer- containing AIT as an antimicrobial ingredient, provided 5- mented sausages is production of organic acids, mainly lactic log reduction of E. coli O157:H7 28 days after processing acid, by LAB [239]. It has long been recognized that LAB when used at 4% in DFS [262–266]. However, mustard levels may produce additional antimicrobial compounds [240, 241]. needed to cause the required inhibition of E. coli O157:H7 Of these, the bacteriocins have received the most attention. reduced consumer acceptability of the sausages [265]. Other Bacteriocins are antibacterial peptides or proteins that kill or ingredients tested include the use of lactoferrin [234] and inhibit the growth of closely related bacteria. For many LAB diacetyl [267]. The former was shown to provide mainly bacteriocins, the inhibitory spectrum includes only other nonlethal injury of E. coli O157:H7, while an extra 1-log LABlikelytobepresentinthesameecologicalniche,thusgiv- reduction was obtained by addition of 300 ppm diacetyl to ing the bacteriocin producer a competitive advantage [242– the sausage batter. The antibacterial activity of essential oils 244]. However, some LAB bacteriocins have a somewhat from herbs and spices were recently demonstrated in DFS. At larger spectrum of inhibition and may be active towards concentrations of 0.005% and 0.05%, decreases of Salmonella a broader panel of Gram-positive bacteria, including food- and L. monocytogenes were >2 log and significantly higher borne pathogens, such as L. monocytogenes, Bacillus cereus, than in control sausages. However, the sensory impact of 14 Journal of Food Quality essential oils is a factor limiting their application in DFS at low temperatures. Several consumer trials have revealed [268]. that the sensory quality of HPP treated RTE products is maintained after a storage period [280–282]. However, there 6.5. Postprocessing Treatments. Storage of DFS at elevated can be some differences between HPP treated and nontreated ∘ temperatures (≥20–25 C), short-term heat treatments, and DFS during the storage period. Raw meat ingredients are freezing/thawing regimes are the most widely applied post- less suited for HPP treatments. Omer et al. found that the process measures. In the review of Holck et al., reductions organoleptic properties of DFS made from HPP treated meat rates of E. coli O157:H7 showed large variations but generally trimmings changed substantially and were less favored after increased with lower pH, lower 𝑎𝑤, and higher storage tem- 2 weeks of storage compared with the nontreated ones [215]. ∘ peratures [6]. Storage at low temperatures (4 C) for up to two When frozen raw materials were used, the sensory differences months usually gives marginal reductions [105, 227], whereas between treated and nontreated samples were reduced. ∘ storage at 20–25 C may result in considerable reductions. Very high pressure levels, up to 600 MPa, are often used Including a storage step at ambient temperatures in forDFS.Severalstudieshaveshownhighinitialreductionsof addition to the production process itself may not be enough microorganisms after HPP [280, 282]. Gill and Ramaswamy to achieve the 5-log reduction required in some countries. showed that the E. coli O157 numbers were reduced by greater Heat treatments may be effective to reduce the numbers of than4-logcfu/gbyHPP(600MPa,3min)andremained pathogens in sausages, also taking into account the fact that static after processing in Hungarian salami but increased ∘ E. coli O157:H7 show reduced tolerance to heat in low pH in All Beef salami during storage at 15 C[280].Theyalso meat products compared to higher pH meat products [269– showed that increasing the holding time to up to 9 min 271]. Total reductions of >5log were obtained for several did not give additional reductions. In a HPP study of combinations of products and storage/mild heat treatment NorwegiantypeDFS,treatmentat600MPafor10mingave regimes. More recent data from our group have shown heat reductions of 2.9-log cfu/g of E. coli O103:H25, and treatment ∘ treatments of 43 C, 24 h to provide >5-log total reductions in cycles (600 MPa for 200 s, 3 cycles) gave a somewhat higher for 11 E. coli strains including different VTEC serotypes. reduction of 3.3-log cfu/g [282]. The same study showed ∘ Similar reductions were obtained by freezing at −20 Cfor that elevated levels of dextrose, NaCl, and nitrite gave lower ∘ 24 h combined with 1 month of storage at 20 C[272].Higher reduction (2.7-log cfu/g) compared with the standard recipe. resistance to heat has been observed for L. monocytogenes Porto-Fett et al. tested treatments of DFS added pathogens compared to E. coli and Salmonella in DFS [221]. Others have with several pressure levels between 483 and 600 MPa for reported that heat treatments providing >5-log reductions 1–12 min [220]. The reduction varied from 1.6 to 5.8-log cfu/g of E. coli in Lebanon Bologna were sufficient for similar depending on pressure conditions and bacteria (Listeria, E. reductions of L. monocytogenes [273]. The studies illustrate coli,andSalmonella). During storage, additional reductions that inactivation of L. monocytogenes is dependent on the were observed for all bacteria tested. same parameters as inactivation of E. coli and Salmonella but Differences in pathogen reductions obtained in the var- that lethal effects on pathogens are product dependent. ious studies of pressurizing DFS can be related to variation Different freezing/thawing and storage/mild heat treat- in the recipe, fermentation regime, and water activity level. ment regimes of DFS showed negligible sensory effects on The production process of DFS is shown to give a reduction treated DFS [274]. Other studies have reported variable ofabout2-logcfu/gofVTEC[282].Withtheadditional quality and sensory effects, ranging from unacceptable to reduction of 3-log cfu/g due to HPP, this will provide the improved sensory scores, due to heat treatments at higher desired 5-log reduction that is often required. ∘ temperatures (≥approx. 50 C)[105,221,275,276].Combina- tions of high temperature and reduced treatment times may 7. Mathematical Models for Predicting be regarded as most feasible in industrial production. Opti- Survival of Pathogens in DFS mal treatment regimes are likely to differ between products with different characteristics [102, 221, 273, 274, 276]. Predictive modeling has developed as an adjunct to tradi- High pressure processing (HPP) has been employed in tional microbiological techniques. Essentially, the survival many areas of food production [277]. In DFS products, HPP and/or growth of an organism of concern may be pre- has potential for postprocess reduction or elimination of dicted on the basis of a mathematical relationship between L. monocytogenes inthefinalproductsincompliancewith microbial growth rate and environmental conditions [283]. the requirements (9 CFR part 430; the Listeria Rule) for A large number of mathematical models to predict the L. monocytogenes control of such RTE products, as issued population kinetics of E. coli and other bacteria in foods by FSIS. HPP is recognized by the FDA as a method for are publically available, such as the ComBase Predictor achieving the 5-log VTEC reduction in DFS processing that (CP) [284], the Pathogen Modelling Program (PMP) [285], are required in USA [278] and Canada [279]. DFS products and Meat and Livestock Australia (MLA) E. coli inactiva- havingatexturethatislesssusceptibletochangesduringHPP tion model in fermented meat [286]. These models have compared to raw meat products are suitable for HPP. The limitations as they primarily focus on the static effect of DFS color is barely affected even at very high pressure levels, 𝑎𝑤, NaNO2, pH, and temperature. The MLA model con- and the in-package pasteurization by pressure is an advantage siders dynamic changes, however only those related to as possible recontamination is avoided. As a postprocessing temperatureinthesausageenvironmentduringproduction method, it also has the advantage that it can be performed [225, 286]. A simpler version of the MLA model calculates Journal of Food Quality 15 the reduction of E. coli as a function of temperature and “carcinogenic to humans” and red meat as “probably carcino- time during fermentation and maturation, available at genic to humans” for colorectal cancer, appealing to critically http://www.foodsafetycentre.com.au/fermenter.php. Specifi- consider the future role of meat in a healthy diet. Consid- cally, the inactivation of E. coli O157:H7 has been modeled erations around meat and cancer and possible mitigation asafunctionofpHand𝑎𝑤 in Soudjouk-style fermented sau- strategies have been summarized previously [291]. Groups sages during the process of fermentation and drying, avail- of consumers claim personal health motives for reducing able at https://pmp.errc.ars.usda.gov/PMPOnline.aspx [222]. or banning the consumption of meat [292]. A response to The software THERM predicts growth of E. coli O157:H7, negative perception related to meat products embraces an Salmonella,andS. aureus as a function of the time- innovation agenda [293]. However, the borderline between temperature history of raw meat products [287]. innovation and tradition appears complex since traditional A dynamic model to predict VTEC concentration products tend to be perceived more basic and natural [294]. throughout manufacturing and storage of fermented raw The benefits and risks associated with red and processed meat meat sausages has been developed by Quinto et al. [288]. consumption should not necessarily cause dilemmas, if these The model is implemented in a tool called E. coli SafeFerment meats are produced to ensure optimal microbial safety and (EcSF), available at http://www.ifr.ac.uk/safety/EcoliSafeFer- consumed in moderate amounts as part of balanced diets ment. EcSF integrates growth, probability of growth, and [291]. thermal and nonthermal inactivation models to give the predictions of VTEC concentration under constant or fluctu- Conflicts of Interest ating environmental conditions. The tool can be applied for the evaluation of the impact of modifications, interventions, The authors declare that there are no conflicts of interest or unexpected events during the manufacturing process regarding the publication of this paper. and/or storage period on VTEC survival. Recently, Gunvig et al. developed three models for predicting survival of Acknowledgments VTEC, L. monocytogenes and Salmonella, taking into account the dynamics of the sausage environment and maturation The preparation of this paper was funded by grants financed of fermented sausages [289]. Based on challenge experi- by the Norwegian Research Council (Project 221663) and the ments under production conditions of dried and semidried Research Levy on Agricultural Products (Project 262306). sausages, the models covered dynamic changes related to various pH decreases, weight losses during maturation, NaNO2 References concentrations, and 𝑎𝑤. 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Research Article Effects of Ozone Treatments on the Physicochemical Changes of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix) during Frozen Storage

Rongrong Zhang,1,2 Shanbai Xiong,1,3,4 Juan You,1,3 Yang Hu,1,3 Ru Liu,1,3 and Tao Yin1,3

1 College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, China 2School of Agriculture and Food Sciences, Faculty of Science, University of Queensland, Brisbane, QLD 4072, Australia 3National R&D Branch Center for Conventional Freshwater Fish Processing, Wuhan 430070, China 4Collaborative Innovation Center for Efficient and Health Production of Fisheries, Changde, Hunan Province 415000, China

Correspondence should be addressed to Tao Yin; [email protected]

Received 12 January 2017; Revised 24 February 2017; Accepted 16 March 2017; Published 13 April 2017

Academic Editor: Marta Laranjo

Copyright © 2017 Rongrong Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Physicochemical changes of myofibrillar proteins from silver carp surimi during frozen storage as affected by two manners of ozone treatments were investigated. For preparation of surimi treated with ozone, ozone water (8 mg/L) 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 proteins from To1 surimi showed slightly lower free sulfhydryl contents and higher surface hydrophobicity throughout frozen storage and 2+ lower Ca -ATPase activities after 30 d. To2 did not significantly (𝑃 > 0.05) affect these physicochemical properties, indicating that myofibrillar proteins structure was well maintained. Consequently, To1 significantly (𝑃 > 0.05) decreased breaking force of surimi gels while To2 did not significantly (𝑃 > 0.05) affect gel breaking force. In addition, the whiteness of surimi gels was increased more obviously by To2 than by To1. The results indicate that To2 could be used as a mild oxidation treatment for improving white color of silver carp surimi without negatively affecting gel texture.

1. Introduction Ozone, which has regulatory approval and is generally recognized as environmentally friendly, has been broadly Silver carp (Hypophthalmichthys molitrix) is one of the main used in water treatment, sanitization, cleaning, and disin- freshwater fish species farmed in China. In 2014, total fection of equipment, in off-odour removal, and for pro- production output was 4.23 million t [1]. Utilization of cessing various food products [7]. Ozone also shows great silver carp has increased in recent years due to massive potential for improving the quality of aquatic products with overexploitation of sea-water fish and the resulting shortage regard to shelf life, sensory, and so forth [8]. Zhang et al. of raw material for frozen surimi. The output of silver carp surimi was estimated at about 30,000 t in 2013 [2] and has [9]reportedthatabout42–69.19%GEOinbigheadcarp rapidly grown; reaching over 40,000 t in 2015 [3]. However, (Hypophthalmichthys nobilis) meat was removed by ozone silver carp surimi possesses an earthy-musty off-odour [4], water (3.3–7.6mg/L) rinsing for 5–20 min. According to the which is generally thought to be associated with geosmin study by Wang et al. [4], the muddy flavours of silver carp (GEO) and 2-methylisoborneol (MIB) [5]. Furthermore, surimi were effectively eliminated after washing for 20 min whitenessofsilvercarpsurimiisinferiortothatoffishspecies usingozonewaterwithaninitialconcentrationof0.96mg/L. that are traditionally used for high-quality surimi products In addition, ozone is an oxidant that possesses a bleaching [6]. Consequently, these defects negatively affect consumer effect, which helps to increase the whiteness of surimi and perception of surimi products made from silver carp. other aquatic products [10, 11]. Accordingly, the defects of 2 Journal of Food Quality silver carp surimi in sensory quality and color may be rinsing on the physicochemical changes of fish myofibrillar alleviated by applying appropriate ozone treatment. proteins during frozen storage and subsequent gelation prop- Myofibrillar proteins are the major components of surimi erties. and are responsible for the formation of gel texture upon heating. During the rinsing process of surimi production, 2. Materials and Methods oxidation of myofibrillar proteins by ozone treatment may cause the formation of intra- and/or intermolecular disulfide 2.1. Materials. Silver carp (Hypophthalmichthys molitrix), bonds, peptide bond cleavage, amino acid residue modifica- approximate 1.5 kg, was obtained from a local fish farm tion, unfolding of protein molecules, and alternation in pro- (Wuhan, China). Reagents used for SDS-PAGE were pur- tein functionality [7]. These changes to protein functionality chased from Bio-Rad (Hercules, CA, USA). Adenosine tri- ultimately affect the textural properties of surimi gels. phosphate(ATP), 5, 5-dithiobis(2-nitrobenzoic acid) (DTNB), Recently, researchers have begun to investigate the effects and 1-anilino-8-napthalenesulfonate (ANS) were purchased of ozone treatment on the properties of surimi and surimi from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). products[4,9,12,13].Zhangetal.[9]reportedthatozone Sugar and sodium tripolyphosphate were purchased from water treatment was a mild oxidation protocol to enhance Guangshengyuan Food Co., Ltd. (Wuhan, China) and Xingfa the functionality of myofibrillar proteins from bighead carp. GroupCo.,Ltd.(Wuhan,China),respectively.Allother Ozone treatment was found to significantly (𝑃 < 0.05) chemicals were of analytical grade. 2+ increase salt solubility, Ca -ATPase activity, carbonyl con- 2.2. Preparation of Surimi Treated with Ozone. Silver carp was tent, sulfhydryl content, and gel textural values of proteins headed, gutted, and thoroughly cleaned prior to deboning without increasing peroxide values too much. However, the carcass by a roll-type fish meat separator (YBYM-6004- deformation of mackerel surimi gels with ozone treatment B, Yingbo Food Machinery Co., Ltd., Xiamen, China). The was found to be significantly (𝑃 < 0.05) lower than that obtained fish mince was subjected to two rinsing cycles with of control samples (without ozone treatment) and gradually a water : mince ratio and rinsing time at 4 : 1 and 10 min, decreased with increased ozonation time [14]. Xie et al. [12] respectively. A total of 3 rinsing treatments were conducted: also found that textural values of silver carp surimi gels (1) two washing cycles using ice water only (Tc); (2) 1st and significantly𝑃 ( < 0.05) decreased after rinsing with ozone 2nd cycle using ice water containing 8 mg/L ozone and ice water. Textural properties of surimi gels as affected by ozone water, respectively (To1); (3) 1st and 2nd cycle using ice water water treatment, therefore, may be influenced by differences and ice water containing 8 mg/L ozone, respectively (To2). between fish species and/or ozonation conditions. Ozone water, containing an initial concentration of 8 mg/L, Studies on the properties of fish myofibrillar proteins was prepared according to the method by Zhang et al. [11] or mince as affected by ozone water rinsing are limited. In using a corona discharge ozone generator (SY-SB40, Sheng the majority of published literature, fish myofibrillar proteins Ya Co., Ltd., Xuzhou, China). After rinsing, fish mince was wereimmediatelyheatedtoformagelafterbeingwashed wrapped in cheesecloth and centrifuged (SS-300, Runxin with ozone water and then subjected to penetration test for Machinery Works, Zhangjiagang, China) at 15,000 rpm to evaluation of the ozone treatment on gel texture [4, 9, 12]. remove excess water. The concentrated myofibrillar proteins Ozone treatment enhances unfolding of fish myofibrillar were mixed with cryoprotectants (6% sucrose and 0.3% proteins, which may contribute to stronger gel formation tripolyphosphate), vacuum packaged (∼600 g each bag), and ∘ during the heating step [11]. However, surimi is an interme- stored in a freezer (−18 C) until used (0, 7, 15, 30, 60, and 90 diate product that is typically mixed with cryoprotectants days). Room temperature during all of the aforementioned ∘ andthensubjectedtoaperiodoffrozenstorageprior operations was maintained below 10 C. to being manufactured into different products. Therefore, partially unfolding proteins with ozone treatment before 2.3. Extraction of Myofibrillar Proteins. Myofibrillar proteins freezing may result in promoting protein aggregation during were extracted from surimi according to the method of storage, which ultimately results in a weaker surimi gel being Poowakanjana and Park [18] with slight modification. Briefly, formed in the finished product [26]. The impacts of ozone- 1 g surimi was add to 29 mL buffer (0.6 M KCl, 20 mM induced oxidation on the physicochemical changes of fish Tris-HCl, and pH 7.0) and homogenized (FJ-200, Shanghai myofibrillar proteins during frozen storage, however, have Specimen and Models Factory, China) at 8,000 rpm for not been reported. 1 min. The homogenate was centrifuged at 15,000 ×g(J- ∘ Currently, in the production of silver carp surimi, it is 26XP, Beckman Coulter Inc., Fullerton, CA, USA) at 4 C common to use water : mince ratios of 5 : 1 to 3 : 1 with two for 30 min. After centrifugation, the supernatant was filtered 2+ rinsing cycles. The majority of the water-soluble proteins, and used for analyzing free sulfhydryl content, Ca -ATPase primarily sarcoplasmic protein, and lipids are removed after activity, and surface hydrophobicity as detailed below. Protein the first rinsing cycle [15]. Oxidization of fish myofibrillar concentration of the supernatant was measured using the proteins may be influenced by the presence or absence Lowry method [19]. of sarcoplasmic proteins and lipids [16, 17]. In order to determine appropriate application of ozone treatment for 2.4. Determination of Free Sulfhydryl Content. Free sulfhydryl improving silver carp surimi quality, this study investigated content was determined according to the method of Jiang the application of ozone water in the first or second cycle of et al. [13] using Ellman’s reagent (DTNB) with some Journal of Food Quality 3 modifications. Protein concentration of the myofibrillar using 5% (w/v) and 12% (w/v) acrylamide, respectively. Each protein sample, as described above, was diluted to 0.5 mg lane was loaded with 10 𝜇gprotein.Afterrunning,gelswere protein/mL using 0.6 M KCl in 20 mM Tris-HCl buffer (pH fixed and stained with 0.125% Coomassie brilliant blue R-250 7.0). The diluted sample (0.5 mL) was mixed with 2 mLof and destained in DI water containing 50% methanol and 10% 8 M urea in 0.2 mM Tris-HCl buffer (pH 7.0) and 50 𝜇Lof acetic acid. 0.1 M sodium phosphate buffer (pH 7.0) containing 10 mM DTNBand0.2mMEDTA.Themixturewasincubatedat ∘ 2.8. Preparation of Surimi Gel. Vacuum-packaged frozen 40 C for 15 min before measuring absorbance at 412 nm surimiwasremovedattherespectivestoragetime(0,7,15, (722 s, Shanghai Precision and Scientific Instrument Co., Ltd., 30, 60, or 90 days) and partially thawed at room temperature China). Free sulfhydryl content was calculated using the × × −1 −1 for 40 min before being cut into approximately 2 cm 2cm extinction coefficient of 13600 M cm and expressed as 5 4 cm cubes. The cubes were comminuted using a silent cutter mol per 10 gprotein. (Multiquick 3, Braun, Germany) at speed 3 for 30 sec. Sodium chloride (2%) was added to extract myofibrillar proteins. 2+ ∘ 2.5. Determination of Ca -ATPase Activity. Determination Moisture content was adjusted to 78% using ice water (0 C). 2+ of Ca -ATPase activity was performed according to the The mixture was blended and ground in a stainless steel method of Benjakul et al. [20] with some modifications. The mortarusingtwinpestles(CA1,KinnShangHooIronWorks, Taiwan) at an agitation speed of 45 rpm for 30 min. Final myofibrillar protein sample (1 mL) was mixed with 0.5 mL of ∘ temperature of the paste was below 10 C. The paste was then 0.5 M Tris-maleate buffer (pH 7.0)and 0.5 mL of 0.1 M CaCl2. Deionized water was added to a total volume of 9.5 mL. stuffed into a polyethylene sausage casing (2.5 cm diameter) Subsequently, 0.5 mL of 20 mM ATP was added to initiate using a sausage stuffer (Tre-mss7kh, Trs Spade, Italy). Both ∘ the reaction. The mixture was incubated at 25 Cfor8min ends were sealed with U-shaped aluminum wire clips using a clipper (Hk12, Hakanson, Sweden). The sample was then andthenterminatedbyadding5mLofchilledtrichloroacetic ∘ acid (15 g/100 mL). The reaction mixture was centrifuged at heated at 90 Cfor30min.Cookedgelswereimmediately submerged in ice water and then stored overnight in a 3,500 ×g for 5 min and filtered. Inorganic phosphate liberated ∘ in the filtrate was measured by the method of Fiske and Sub- refrigerator (4 C). barow[21].Specificactivitywasexpressedas𝜇Minorganic phosphate (Pi) released/mg protein/min. 2.9. Texture Analysis. Gel strength of surimi gels was determined by the method described by Yin and Park [23]. Chilled ∼ ∘ 2.6. Determination of Surface Hydrophobicity. Surface hydro- surimi gels were equilibrated at room temperature ( 25 C) phobicity was measured using ANS probe according to the for 2 h. Samples were then cut into 2.5 cm cylinders and method of Poowakanjana and Park [18] with slight modifica- subjected to the penetration test using a TA-XT texture tion. Protein concentration of the myofibrillar proteins was analyzer (Stable Micro Systems, Surrey, UK) equipped with diluted to 0.1, 0.2, 0.3, and 0.5 mg protein/mL using 0.6 M a spherical probe (diameter 5.0 mm and crosshead speed of KClin20mMTris-HClbuffer(pH7.0).Then4mLofsample 1 mm/s). 𝜇 with different protein concentrations was mixed with 20 Lof ∗ 2.10. Color Measurement. Color parameters, L (lightness), 0.1 M phosphate buffer (pH 7.4) containing 8 mM ANS and ∗ ∗ left at room temperature for 10 min. Fluorescence intensity a (redness to greenness), and b (yellowness to blueness), of was immediately measured using a spectrofluorometer (RF- the surimi gels were measured using a CR-400 colorimeter (Konica Minolta, Osaka, Japan). Whiteness was calculated 1501, Shimadzu, Kyoto, Japan) with excitation and emission ∗ wavelengths of 390 nm and 470 nm, respectively. The surface according to the equation (L-3b ) developed by Park [24] for hydrophobicity was calculated from the initial slope of the net surimi gel. relative fluorescence intensity versus the myofibrillar proteins concentration. 2.11. Statistical Analysis. Analysis of variance (ANOVA) was conducted using the SAS program (V8, SAS Institute Inc, 2.7. Protein Patterns. The protein pattern of myofibrillar Carry, NC, USA). Differences among mean values were proteins at different storage times (0, 7,15, 30, 60, and 90 days) evaluatedbytheDuncanmultiplerangetest(DMRT)usinga was revealed using SDS-PAGE according to Laemmli [22] 95% confidence interval. with some modifications. The sample was homogenized (Ika T18, Cole-Parmer, Co., Ltd., Shanghai, China) at 10,000 rpm 3. Result and Discussion for 1 min and solubilized using 5% sodium dodecyl sulfate ∘ solution (90 C). Solubilized proteins were centrifuged at 3.1. Free Sulfhydryl Content. Conversion of sulfhydryl groups 17,000 ×g for 20 min at room temperature. Protein content (R-SH) into disulfide covalent bonds (RS-SR) and other of the supernatant was measured using the Lowry method oxidized species through oxidation of sulfhydryl groups [19]. Protein sample (2.5 mg/mL) was dissolved in Laemmli or disulfide interchanges is generally considered a good 5x sample buffer with or without 𝛽-mercaptoethanol and indicator for analyzing the radical-mediated oxidation of ∘ followed by heating at 100 Cfor3min.𝛽-ME, as a reducing proteins [7]. As shown in Figure 1, the free sulfhydryl contents agent,wasusedtocleavetheRS−SRbondsofproteinsinthe of silver carp myofibrillar proteins with and without ozone SDS-PAGE analysis. Stacking and separating gels were made treatments decreased significantly𝑃 ( < 0.05)afterstoring 4 Journal of Food Quality

12 Although cryoprotectants are mixed into surimi to maintain A A A protein structure during frozen storage, fish myofibrillar g pro)

5 10 A proteins continue to gradually unfold and subsequently 10 A A A A A A A 8 A aggregateduringfrozenstorage[26]. Before frozen storage (0 d), the free sulfhydryl content 6 A of the samples in the descending order was TC,To2,and A A A 𝑃> A A To1, respectively. But the differential was not significant ( 4 0.05). However, Zhang et al. [11] reported that the free sulf- 2 hydryl content of myofibrillar proteins recovered from bighead carp decreased about 12% after rinsing with 7.6 mg/L 0 ozonewater.Thereasonmightbeduetoamuchlonger Free sulfhydryl content (mol/ sulfhydryl content Free 0 7 15306090 rinsing time (20 min) used in their study. This order of the Storage time (day) threesamples(Tc,To2,To1),withregardtofreesulfhydryl Tc content,wasmaintainedthroughoutfrozenstorage.Freesulf- To1 hydryl content of To1 was lower than that of To2; however To2 the difference was not significant𝑃 ( > 0.05). It might be related to the formation of lipid radicals and peroxide during Figure 1: Changes in free sulfhydryl content of myofibrillar proteins extracted from silver carp surimi during frozen storage. Light grey: the ozone water rinsing which enhanced the oxidation of two washing cycles using ice water only (Tc), dark grey: first and the myofibrillar proteins [16, 17]. It has been reported that second washing cycle using ice water containing 8 mg/L ozone and peroxyl radicals from lipids abstracted hydrogen atoms from ice water, respectively (To1); black: first and second washing cycle molecules of protein leading to a radical-mediated chain using ice water and ice water containing 8 mg/L ozone, respectively reaction similar to that of lipid oxidation [27]. And Lund et (To2). Different letters indicate significant difference among samples al. [28] reported that oxidation of lipids in meat systems took (Tc, To1, and To2). place faster than that of myofibrillar proteins and, hence, it was more likely that lipid derivatives (radicals and peroxides) promoted proteins oxidation than the other way round. For the ozone treatment, the To1 sample was rinsed in ozone ∘ (−18 C) for 7 d. The sulfhydryl contents remained constant water with the presence of a high concentration of lipids. As (𝑃 > 0.05) during frozen storage from day 7 to day 30, and fortheTo2samplelipidsweremajorlyremovedpriortothe then continued to decrease (𝑃 < 0.05)atday60.Reduction second cycle of rinsing using ozone water. of free sulfhydryl content resulted from the formation of 2+ 2+ disulfide covalent bonds (RS-SR), as evidenced by changes 3.2. Ca -ATPase Activity. Ca -ATPase activity is widely of the protein patterns (Figure 4(a)). Myofibrillar proteins used as an index of the denaturation of fish myofibrillar exhibited a reduction of about 23%, 24%, 24%, and 62%, proteins during storage and processing [29]. As shown in 2+ respectively, in the sulfhydryl contents after 7 d, 15 d, 30 d, and Figure 2, Ca -ATPaseactivityofsilvercarpmyofibrillarpro- 60 d of storage. teins with and without ozone treatment gradually declined Changes in the sulfhydryl content of silver carp pro- (𝑃 < 0.05) during frozen storage. These results were 2+ teins during frozen storage were similar to that of croaker, consistent with the report by Cao et al. [30] that Ca - threadfin bream, and bigeyes snapper as reported by Benjakul ATPase activity of silver carp surimi with various types of ∘ et al. [20]. Myofibrillar proteins are mainly composed of cryoprotectants decreased with frozen storage (−80 C) up to ∼ ∼ myosin ( 55%) and actin ( 20%), which contain about 42 90 d. The oxidation of sulfhydryl groups, especially in the 2+ and 12 sulfhydryl groups, respectively [25]. The sulfhydryl head region (SH1 and SH2), caused Ca -ATPase activity to groupsincludeactivesulfhydrylgroupsonthesurfaceandthe decline. Moreover, inter- and/or intramolecular interactions hidden sulfhydryl groups in the protein interior. Oxidization of myofibrillar proteins during frozen storage could also oftheactivesulfhydrylgroupsonthesurfacereduced 2+ < contribute to decreased Ca -ATPase activity [26]. sulfhydryl content during early storage ( 7 d). Subsequently, 2+ myofibrillar proteins unfolded during extended storage; thus, Within 15 d of frozen storage, Ca -ATPase activity of To1 some of the original hidden sulfhydryl groups were exposed was higher (𝑃 < 0.05) than the control (Tc) (Figure 2). The results generally coincided with the findings of Zhang et al. to the surface. These exposed sulfhydryl groups were then 2+ activated, which caused sulfhydryl content to decrease fur- [9, 11], in which the Ca -ATPase activity of bighead carp ther (>30d).Thedecreaseinthesulfhydrylcontentcoincided proteins increased after rinsing with 5.1 mg/L ozone water for with an increase in surface hydrophobicity (Figure 3), which 20 min followed by rinsing with distilled water. A change in represents change in the tertiary structure of the protein. thetertiarystructureofthemyosinheadregionoranincrease Surprisingly, the surface hydrophobicity significantly𝑃< ( in its flexibility, owing to a light extent of denaturation, may 2+ 0.05) increased (Figure 3) at day 90 while the sulfhydryl result in increased Ca -ATPase activity [31]. The results of contentwasnotsignificantly(𝑃 > 0.05)changed.Thisobser- Zhang et al. [9] also indicated that the tertiary structure of the vation may be due to the masking of sulfhydryl groups by myosin head region was slightly influenced by ozone water aggregation of partially unfolded myofibrillar proteins [20]. rinsing. Rinsing with ozone exposed the globular myosin Journal of Food Quality 5

1.6 60 AB A 1.4 A A A A 50 B A A A A 1.2 B B C B B A C A A A A 1.0 B C 40 A A A A A A A A A 0.8 30 A A A 0.6 20 0.4 Surface hydrophobicity Surface -ATPase (umol Pi/mg pro/min) Pi/mg (umol -ATPase 0.2

2+ 10

Ca 0.0 0 7 15306090 0 0 7 15306090 Storage time (day) Storage time (day) Tc To1 Tc To2 To1 To2 2+ Figure 2: Changes in Ca -ATPase activity of myofibrillar proteins extracted from silver carp surimi during frozen storage. Light grey: Figure 3: Changes in surface hydrophobicity of myofibrillar pro- two washing cycles using ice water only (Tc), dark grey: first and teins extracted from silver carp surimi during frozen storage. Light second washing cycle using ice water containing 8 mg/L ozone and grey: two washing cycles using ice water only (Tc), dark grey: ice water, respectively (To1); black: first and second washing cycle first and second washing cycle using ice water containing 8 mg/L using ice water and ice water containing 8 mg/L ozone, respectively ozone and ice water, respectively (To1); black: first and second (To2). Different letters indicate significant difference among samples washing cycle using ice water and ice water containing 8 mg/L ozone, (Tc, To1, and To2). respectively (To2). Different letters indicate significant difference among samples (Tc, To1, and To2). head,whichistypicallyburiedwithintheproteinstructure, outside the tertiary structure [9]. Surface hydrophobicity is an effective indicator for 2+ After 30 d of frozen storage, our results showed the Ca - reflecting the conformational change of protein from its ATPase activity of the To1 sample was lower (𝑃 < 0.05)than native structure [26]. Surface hydrophobicity of To1 was that of Tc (Figure 2). As storage duration extended, negative higher than that of Tc, but not significantly𝑃 ( > 0.05), effects (sulfhydryl oxidation and/or protein interactions) during 90 d of frozen storage (Figure 3). In addition, the 2+ dominated and Ca -ATPase activity continued to decline. surface hydrophobicity of To2 was comparable to Tc. Results 2+ Before frozen storage, Ca -ATPase activity of To2 was lower demonstrated that the oxidization in this study is mild. (𝑃 < 0.05) than To1 (Figure 2). However, after 30 d of frozen Changes of the surface hydrophobicity could be used to 2+ 2+ storage the Ca -ATPaseactivityofTo2washigher(𝑃 < 0.05) explain changes of sulfhydryl content (Figure 1) and Ca - than To1 (Figure 2). This result might be due to the lesser ATPase activity (Figure 2) as affected by ozone water rinsing extent of oxidation in To2, which minimally influenced the andfrozenstorage. physiological activity of myosin. 3.4. Protein Patterns. SDS-PAGE was performed to monitor 3.3. Surface Hydrophobicity. The changes in surface hydro- polymerization or degradation of the myofibrillar proteins phobicity of silver carp myofibrillar proteins with different as affected by ozone oxidation and frozen storage. Bands of ozone treatments during frozen storage are illustrated in myosin heavy chain (MHC 200 kDa) and actin (AC 45 kDa) Figure 3. The surface hydrophobicity of the three samples with high densities were clearly visible on all SDS-PAGE (Tc, To1, and To2) increased significantly (𝑃 < 0.05)after gels (Figure 4). In addition, bands assigned to troponin- storing for 7 d, remained unchanged (𝑃 > 0.05)forupto T (TN T 35 kDa), myosin light chain 1 (MLC 1 21 kDa), 30 d, and then subsequently increased (𝑃 < 0.05)forup tropomyosin (TM 40 kDa), and several other proteins were to 90 d. After storing for 90 d, the surface hydrophobicity of also observed, but with relatively lower densities. In the the control sample increased by approximately 87%. Similar absence of 𝛽-mercaptoethanol, bands with molecular weight tendencies were also found in the surface hydrophobicity of above 200 kDa were noticed on the SDS-PAGE gel (Fig- croaker, threadfin bream, and bigeye snapper myofibrillar ure 4(a)). However, those bands (>200 kDa) seemed to proteins during frozen storage [16]. The increase of surface disappear from SDS-PAGE gel when 𝛽-mercaptoethanol was hydrophobicity during extended frozen storage is connected present (Figure 4(b)). to the exposure of the hydrophobic bonds of myofibrillar 𝛽-mercaptoethanol is a reducing agent that possesses the proteins, which are located in the interior of the protein ability to cleave disulfide covalent bonds (RS−SR) of proteins. structure [20]. Frozen storage directly altered the tertiary Therefore, the obvious difference in the protein patterns structure of protein molecules, which results in functionality caused by 𝛽-mercaptoethanol could be mainly attributed loss as observed by a decline in gelling ability (Figure 5). to myosin heave chain (MHC) polymer formation through 6 Journal of Food Quality

STD Tc To1 To2 Tc To2To1 Tc To1 To2 STD Tc To1 To2 Tc To2To1 Tc To1 To2 250 KD MHCXL 150 KD MHC 100 KD 75 KD 50 KD AC 37 KD TM TN T 25 KD

20 KD MLC 1

0 d 7 d 15 d 30 d 60 d 90 d (a) STD Tc To1 To2 Tc To2To1 Tc To1 To2 STD Tc To1 To2 Tc To2To1 Tc To1 To2 250 KD MHC 150 KD 100 KD 75 KD 50 KD AC 37 KD TM TN T 25 KD

MLC 1 20 KD

0 d 7 d 15 d 30 d 60 d 90 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 and second washing cycle using ice water containing 8 mg/L ozone and ice water, respectively; To2: first and second washing cycle using ice water and ice water containing 8 mg/L ozone, respectively. (a) Protein sample was dissolved in loading buffer without 𝛽-mercaptoethanol; (b) protein sample was dissolved in loading buffer with 𝛽-mercaptoethanol; STD: kaleidoscope protein standard; MHCXL: cross-links of myosin heavy 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 three ozone-flotation) and various treatment times on myofibrillar kinds of active sulfhydryls, including SH1, SH2, and SHa. proteins from bighead carp. They found that protein patterns SH1 and SH2 are located in the globular myosin head and among all samples did not behave differently under ozone 2+ are closely related to Ca -ATPase activity. SHa is distributed water rinsing of different time. However, densities of bands in the light meromyosin chain (LMM) and is related to the with molecular weights between 80 and 200 kDa clearly oxidation of the myosin heavy chain (MHC) and polymer increased with ozone-flotation time. Results in this study formation [11]. The number of bands (>200 kDa) increased confirmed, once again, that rinsing silver carp myofibrillar with frozen storage time up to 90 d (Figure 4(b)). Results proteinswith8mg/Lozonewaterfor10minwasamild confirmed the formation of disulfide covalent bonds during oxidation process. frozen storage, which also coincided with the reduction of free sulfhydryl content (Figure 1). 3.5. Gel Texture. Gel-forming ability is an important index Regardless of 𝛽-mercaptoethanol, there was no consider- forsurimiquality.Theintegrityofmyofibrillarproteinsis able difference among samples rinsed with and without ozone essential to form a strong gel. Breaking force and penetration water when compared at the same frozen storage period. This distance of silver carp surimi gels with and without ozone indicates that the ozone treatments used in this study did not treatments decreased (𝑃 < 0.05) gradually during frozen induce detectable polypeptide chain breakage or RS-SR cross- storage (Figure 5). After 90 d of frozen storage, breaking linking. Zhang et al. [9] compared the effects of two manners force and penetration distance significantly𝑃 ( < 0.05) of ozone treatments (washing with ozonized water and declined by 7–18% and 13–21%, respectively. The decrease Journal of Food Quality 7

350 14 A A A A 300 B A 12 ABB A A A A A A AB A B A A B A A A B B B B B A B A A 250 ABB A 10 A

200 8

150 6

Breaking force (g) force Breaking 100 4

50 (mm) distance Penetration 2

0 0 0 7 15306090 0 7 15306090 Storage time (day) Storage time (day)

Tc Tc To1 To1 To2 To2 (a) (b)

Figure 5: Changes in breaking force (a) and penetration distance (b) of silver carp surimi gels during frozen storage. Light grey: two washing cycles using ice water only (Tc); dark grey: first and second washing cycle using ice water containing 8 mg/L ozone and ice water, respectively (To1); black: first and second washing cycle using ice water and ice water containing 8 mg/L ozone, respectively (To2). Different letters indicate significant difference among samples (Tc, To1, and To2).

2+ in textural values was in accordance with decreased Ca - 70 A ATPase activity (Figure 2), which can be used as an indicator 68 for the integrity of the myosin molecules. A A 66 A B A A A A B Before storage, breaking force and penetration distance of B B samples with ozone rinsing (To1 and To2) were significantly 64 B B C B 𝑃 < 0.05 C lower ( ) than Tc, which was consistent with results 62 C reported by Xie et al. [12]. Myofibrillar proteins unfold and then aggregate to form three-dimensional gel networks Whiteness 60 through intermolecular interactions (hydrophobic interac- 58 tions, disulfide covalent bonds, ionic bonds, etc.) of exposed 56 functional groups. Conversion of sulfhydryl groups into 54 disulfide covalent bonds before the myofibrillar proteins are 0 7 15306090 well unfolded may result in a weak gel [12]. Breaking force of Storage time (day) To1 was significantly lower (𝑃 < 0.05) than that of Tc during frozen storage. However, breaking force of To2 after 15 d was Tc not significantly (𝑃 > 0.05) different from that of Tc. To1 To2

3.6. Whiteness. Whiteness is an important factor affecting Figure 6: Changes in whiteness of silver carp surimi gel during costumer acceptability of the end surimi products. As shown frozen storage. Light grey: two washing cycles using ice water only (Tc), dark grey: first and second washing cycle using ice in Figure 6, whiteness of surimi rinsed with ozone water (To1 water containing 8 mg/L ozone and ice water, respectively (To1); and To2) was significantly higher than that of Tc, which could black: first and second washing cycle using ice water and ice be attributed to the bleaching function of ozone. During water containing 8 mg/L ozone, respectively (To2). Different letters ozone water rinsing, the porphyrin structure of the heme indicate significant difference among samples (Tc, To1, and To2). pigment is destroyed and consequently discolored [26]. To2 showed a better effect on increasing whiteness than To1. InTo2,lipidsandhemepigments(mainlymyoglobinand hemoglobin) were partially removed after the first cycle of cookedgelsandresultedin“whitening”oftheproteins. rinsing. Thus, ozone more effectively discolored the reduced However, Benjakul et al. [16] reported that whiteness of amount of remaining pigments. surimi made from four kinds of fish species harvested in The whiteness of all samples (Tc, To1, and To2) continu- Thailand gradually decreased with increased frozen storage ouslyincreasedasfrozenstoragetimeincreased.Thedecrease time. This might be due to different surimi processing of gel-forming ability during frozen storage (Figure 5) might methods. In their study, whole fish were subjected to different contribute to increased free water contained in the surimi periods of frozen storage prior to being manufactured into gels, which led to increased reflectivity on the surface of surimi. Denaturation of heme proteins during frozen storage 8 Journal of Food Quality can result in their irreversible binding to myofibrillar proteins [4] Y. Wang, L. Liu, S. Liu, X. N. Li, and L. Z. Liu, “Effects of ozone and thus decreased whiteness of surimi [26]. on deodorization process and gel strength of surimi from silver carp,” JournalofWuhanPolytechnicUniversity,vol.32,pp.15–19, 2013. 4. Conclusion [5] P. Howgate, “Tainting of farmed fish by geosmin and 2- The results demonstrated that physicochemical properties of methyl-iso-borneol: a review of sensory aspects and of uptake/depuration,” Aquaculture,vol.234,no.1–4,pp.155–181, myofibrillar proteins from silver carp surimi during frozen 2004. storage were affected by the ozone treatment protocol (Tc, To1, or To2). As compared to only water rinsing (Tc), addition [6]Y.K.Luo,R.Kuwahara,M.Kaneniwa,Y.Murata,andM. Yokoyama, “Comparison of gel properties of surimi from of 8 mg/L ozone in the first cycle of rinsing (To1) enhanced Alaska pollock and three freshwater fish species: effects of oxidation and denaturation of myofibrillar proteins during thermal processing and protein concentration,” Journal of Food frozen storage, resulting in a gel with lower breaking force. Science, vol. 66, no. 4, pp. 548–554, 2001. Addition of ozone of the same concentration in the second [7]C.O’Donnell,B.K.Tiwari,P.J.Cullen,andR.G.Rice, cycle of rinsing (To2) minimally affected the physicochemical “Status and trends of ozone in food processing,” in Ozone in properties of myofibrillar proteins, including free sulfhydryl Food Processing,C.O’Donnell,Ed.,pp.1–18,Wiley-Blackwell, 2+ content, Ca -ATPase activity, surface hydrophobicity, and Oxford, UK, 2012. gel textural values. In addition To2 treatment significantly [8] C. O. R. Okpala, “Investigation of quality attributes of ice- increased whiteness of the surimi gel. Addition of ozone in stored Pacific white shrimp (Litopenaeus vannamei)asaffected the second rinse cycle is therefore a promising technology by sequential minimal ozone treatment,” LWT—Food Science to upgrade freshwater fish surimi in color without negatively and Technology,vol.57,no.2,pp.538–547,2014. affecting gelation properties or gel texture. [9]T.Zhang,Y.Xue,Z.J.Li,Y.M.Wang,W.Yang,andC. H. Xue, “Effects of ozone on the removal of geosmin and the physicochemical properties of fish meat from bighead Additional Points carp (Hypophthalmichthys nobilis),” Innovative Food Science and Emerging Technologies,vol.34,pp.16–23,2016. Practical Applications. Ozone has regulatory approval and is recognized as being environmentally friendly; therefore [10] L. Feng, T. Jiang, Y. Wang, and J. Li, “Effects of tea polyphenol coating combined with ozone water washing on the storage thereisgreatpotentialtouseozoneinaquaticprocess- quality of black sea bream (Sparus macrocephalus),” Food ing industries. The present results indicated that applying Chemistry,vol.135,no.4,pp.2915–2921,2012. ozonetreatment(8mg/Land10min)inthesecondcycleof [11] T. Zhang, Y.Xue, Z. J. Li, Y.M. Wang, W.G. Yang, and C. H. Xue, rinsing minimally affected the physicochemical properties “Effects of ozone-induced oxidation on the physicochemical of myofibrillar proteins during frozen storage. This study properties of myofibrillar proteins recovered from bighead carp provides scientific evidence for using ozone treatment as a (Hypophthalmichthys nobilis),” Food and Bioprocess Technology, mild oxidation treatment to improve the white color of silver vol.8,no.1,pp.181–190,2014. carp surimi without negatively affecting gel texture. [12] S. D. Xie, L. H. Chen, Y. Zhang, and B. D. Zheng, “Effects of ozone on the quality of fish-ball made from silver carp,” Journal Conflicts of Interest of Fujian Agriculture and Forestry University,vol.38,pp.552– 557, 2009. The authors declare that there are no conflicts of interest [13]W.X.Jiang,Y.F.He,S.B.Xiongetal.,“Effectofmildozoneoxi- regarding the publication of this paper. dation on structural changes of silver carp (Hypophthalmichthys molitrix) myosin,” Food and Bioprocess Technology,vol.10,no.2, pp. 370–378, 2017. Acknowledgments [14]S.T.Jiang,M.L.Ho,S.H.Jiang,L.Lo,andH.C.Chen,“Effects of ozone on the quality of fish-ball made from silver carp,” Authors gratefully acknowledge financial support from The Journal of Fujian Agriculture and Forestry University,vol.63,pp. National Natural Science Foundation of China (31501517) 652–655, 1998. and China Agriculture Research System (CARS-46-23). A [15] C. A. M. Dewitt, J. T. M. Lin, and A. Ismond, “Waste manage- special thank you is extended to OSU Senior Faculty Research ment,utilization,andchallenges,”inSurimi and Surimi Seafood, Assistant Ms. Angela Hunt for her help in revising this article J.W.Park,Ed.,pp.314–335,CRCPress,BocaRaton,Fla,USA, in English. 2014. [16] S. Benjakul, W. Visessanguan, C. Thongkaew, and M. Tanaka, References “Effect of frozen storage on chemical and gel-forming properties of fish commonly used for surimi production in Thailand,” [1] China Fishery Ministry, Fishery Yearbook, China Agriculture Food Hydrocolloids,vol.19,no.2,pp.197–207,2005. Press, Beijing, China, 2015. [17] S. Saeed and N. K. Howell, “Effect of lipid oxidation and frozen [2] P. Gueneeugues and J. Ianelli, “Surimi resources and market,” storageonmuscleproteinsofAtlanticmackerel(Scomber in Surimi and Surimi Seafood,J.W.Park,Ed.,pp.25–62,CRC scombrus),” Journal of the Science of Food and Agriculture,vol. Press, Boca Raton, Fla, USA, 2014. 82,no.5,pp.579–586,2002. [3] S. S. 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as affected by comminution conditions,” Food Chemistry,vol. 138, no. 1, pp. 200–207, 2013. [19]O.H.Lowry,N.J.Rosebrough,A.L.Farr,andR.J.Randall, “Protein measurement with the Folin phenol reagent,” The JournalofBiologicalChemistry,vol.193,no.1,pp.265–275,1951. [20] S. Benjakul, W. Visessanguan, C. Thongkaew, and M. Tanaka, “Comparative study on physicochemical changes of muscle proteins from some tropical fish during frozen storage,” Food Research International,vol.36,no.8,pp.787–795,2003. [21] C. H. Fiske and Y. Subbarow, “The colorimetric determination of phosphorus,” TheJournalofBiologicalChemistry,vol.66,pp. 375–400, 1925. [22] U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature,vol.227,no. 5259, pp. 680–685, 1970. [23] T. Yin and J. W. Park, “Effects of nano-scaled fish bone on the gelation properties of Alaska pollock surimi,” Food Chemistry, vol.150,pp.463–468,2014. [24] J. W.Park, “Functional protein additives in surimi gels,” Journal of Food Science,vol.59,no.3,pp.525–527,1994. [25] K. Hofmann and R. Hamm, “Sulfhydryl and disulfide groups in meats,” Advances in Food Research, vol. 24, pp. 1–111, 1978. [26] T. C. Lanier, J. Yongsawatdigul, and P. Carvajal-Rondanelli, “Surimi gelation chemistry,” in Surimi and Surimi Seafood,J.W. Park,Ed.,pp.101–131,CRCPress,BocaRaton,Fla,USA,2014. [27] E. R. Stadtman and R. L. Levine, “Free radical-mediated oxidation of free amino acids and amino acid residues in proteins,” Amino Acids,vol.25,no.3-4,pp.207–218,2003. [28] M. N. Lund, M. Heinonen, C. P.Baron, and M. Estevez,´ “Protein oxidation in muscle foods: a review,” Molecular Nutrition and Food Research,vol.55,no.1,pp.83–95,2011. [29] G. A. M. Donald and T. C. Lanier, “Actomyosin stabilization to freeze-thaw and heat denaturation by lactate salts,” Journal of Food Science,vol.59,no.1,pp.101–105,1994. [30]L.Cao,Y.An,S.Xiong,S.Li,andR.Liu,“Conformational changes and kinetic study of actomyosin from silver carp surimi with modified starch-sucrose mixtures during frozen storage,” Journal of Food Quality,vol.39,no.1,pp.54–63,2016. [31] T. Watanabe, N. Kitabatake, and E. Dol, “Protective effects of non-ionic surfactantsagainst denaturation of rabbit skeletal myosin by freezing and thawing,” Agricultural and Biological Chemistry, vol. 52, no. 10, pp. 2517–2523, 1988. Hindawi Journal of Food Quality Volume 2017, Article ID 8078062, 8 pages https://doi.org/10.1155/2017/8078062

Research Article Effects of Micron Fish Bone with Different Particle Size on the Properties of Silver Carp (Hypophthalmichthys molitrix) Surimi Gels

Tao Yin,1,2,3 Jae W. Park,3,4 and Shanbai Xiong1,2

1 College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, China 2National R&D Branch Center for Conventional Freshwater Fish Processing, Wuhan 430070, China 3Oregon State University Seafood Research and Education Center, 2001 Marine Drive, Room 253, Astoria, OR 97103, USA 4Department of Food Bioscience and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, Republic of Korea

Correspondence should be addressed to Shanbai Xiong; [email protected]

Received 30 November 2016; Accepted 10 January 2017; Published 28 February 2017

Academic Editor: Andrea Laukova´

Copyright © 2017 Tao Yin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Qualities of silver carp surimi (SCS) gels incorporated with micron fish bone of different particle size (22 to 0.12 𝜇m) were evaluated. Textural values, whiteness, and water holding capacity of the SCS gels with setting significantly increased𝑃 ( < 0.05) as the micron fish bone particle size decreased. As the particle size decreased, more calcium ion was apparently released from the fish bone (𝑃 < 0.05). Consequently the released calcium ion increased the activity of endogenous transglutaminase (TGase) and resulted in the formation of more myosin heavy chain (MHC) cross-links in the SCS gel with setting. Fish bone with particle size below 0.48 𝜇m was steadily trapped in the three-dimensional SCS gel network without disrupting the matrices. Results indicated that size reduction of the incorporated micron fish bone improved qualities of the SCS gel with setting by the means of releasing more calcium ion and maintaining better gel matrices.

1. Introduction calcium release as the particle size decreased from micron to submicron range [5, 6]. Size reduction improved properties Fish bone in some cuisines or processed products is tradi- of fish bone particle in solubility, water holding capacity tionally eaten and regarded as an important calcium sources [3], calcium bioavailability [7], and sensory quality (i.e., in Southeast Asia [1]. Fish bone is rich in calcium (234 g/kg grittiness) as well. Recently, researchers have paid attention dry bone), which is mainly in the form of hydroxyapatite to developing calcium-fortified food products using the (HA) and calcium carbonate [2]. However, so far fish bone downsized fish bone [8, 9]. generated from production process of numerous aquatic Surimi is an intermediate product of the concentrated products (fillet and surimi, etc.) is conventionally used for myofibrillar proteins produced through several times of fishmeal and fertilizer production or directly discarded into washing and dewatering which inevitably remove the major- the sea, river, and estuaries, resulting in environmental pol- ity of minerals contained in the original fish flesh. Gelling lution.Thefishbonecanbeconvertedintonutritivefoodsor of the myofibrillar proteins is a vital process of forming additives for humans by reducing its particle size. According desired texture for surimi products. During gelling process, to the reports, micron fish bone powders with a minimum functional groups imbedded inside of the protein molec- averageparticlesizeof7.65and1.75𝜇m were prepared using ular are exposed, which subsequently form intra and/or superfine grinding and dry media milling, respectively [3, 4]. intermolecular bonds, resulting in a three-dimensional gel Particle size of fish bone was further reduced to submicron network [10]. Addition of calcium compounds has been range (1 to 0.1 𝜇m) using high-energy wet media milling reported to improve gel functionality of surimi [11–13]. Gen- [5]. Consequently, there was about an eightfold increase in erally, calcium ion released from those compounds induces 2 Journal of Food Quality endogenous transglutaminase (TGase), which catalyzes the was determined to be about 22 𝜇m using a Mastersizer 2000 formation of 𝜀-(𝛾-glutamyl) lysine cross-links (isopeptide analyzer (Malvern Instruments Ltd., Worcestershire, UK). covalent bonds) between myofibrillar proteins during setting, The fish bone powder was mixed with deionized water (DI) and thus improves the texture of surimi gel [10]. Furthermore, and further diminished using a high-energy wet bead mill calcium ion enhances the unfolding of myosin and forms (MiniZeta 03, Netzsch, Selb, Germany) according to the “calcium bridge” among the negatively charged myofibrillar method as described by Yin et al. [5]. Average size of the proteins [10, 14], which may contribute to the improvement fishboneparticlesintheemulsionaftermillingfor1,2,4, of gel texture as well. and 6 h was about 0.48, 0.30, 0.18, and 0.12 𝜇m, respectively, Silver carp is one of the main aquacultured freshwater which were analyzed using a Nano ZS90 analyzer (Malvern fish in China with a total production of 4.23 million t in 2014 Instruments Ltd., Worcestershire, UK). Morphologies of the [15]. In recent years, surimi production from marine fish fish bone particles observed using field emission scanning in China has been limited possibly due to overfishing. The electron microscope (ULTRA PLUS-43-13, Zeiss, Germany) Chinesesurimiproducershavecompensatedfortheshortage were shown in Figure 1. ofmarinefishbyusingsilvercarp.Theproductionofsurimi from silver carp was estimated at around 30,000 t in 2013 2.3. Preparation of Surimi Gel. The vacuum-packaged frozen [16] and has grown fast; reaching over 40,000 t in 2015 [17]. surimi was tempered at room temperature for 40 min before Calcium compounds from fish bone with a particle size of being cut into approximately 2 cm × 2cm × 4cm cubes. 0.28 𝜇mhavebeenreportedtoimprovegeltextureofAlaska Surimi cubes were comminuted using a silent cutter (Mul- pollock surimi [18]. According to the reports, impacts of tiquick 3, Braun, Germany) at speed 3 for 30 sec. Sodium adding calcium compounds on gel properties vary to fish chloride (2%) was added to extract myofibrillar protein. Fish species used for preparing surimi [13, 19]. Micron fish bone bone of different size (22, 0.48, 0.30, 0.18, and 0.12 𝜇m), at with an appropriate particle size may be used for texture 1 g dried fish bone/100 g surimi paste, was added. Moisture ∘ enhancement of silver carp surimi products while providing content was adjusted to 78% using ice water (0 C). The additional dietary minerals. However, there have been no mixture was blended and ground in a stainless steel mortar studies investigating the effect of micron fish bone on the using twin pestles (CA 1, Kinn Shang Hoo Iron Works, gelation properties of silver carp surimi. The production cost, Taiwan) at an agitation speed of 45 rpm for 30 min. Final ∘ properties, and applications of fish bone products highly temperatureofthepastewasbelow10C. The paste was depend on its particle size. Therefore, the objective of this stuffed into a polyethylene sausage casing (2.5 cm diameter) study is to investigate the effects of micron fish bone with withoneendpresealedusingasausagestuffer(Tre-mss7kh, different particle size on the qualities of silver carp surimi Trs Spade, Italy). After stuffing the other end was sealed gels. with U-shape aluminum wire clips using a clipper (Hk12, Hakanson, Sweden). The samples were heated with two ∘ different thermal treatments: (1) 90 Cfor30min(direct 2. Material and Methods ∘ ∘ cooking); (2) 40 Cfor1hsettingfollowedby90 Ccookingfor 2.1. Materials. Silver carp (Hypophthalmichthys molitrix) 30 min. Cooked gels were submerged in ice water for 15 min ∘ surimi (AAA grade) with cryoprotectants (6% sucrose and and stored overnight in a refrigerator (4 C). 0.3% sodium tripolyphosphate) was obtained from a local surimi plant (Jingli Aquatic Product Co., Ltd., Honghu, 2.4. Determination of Gel Strength. Gel strength of the silver China). Frozen surimi was cut into about 800 g blocks, carp surimi gels incorporated with MFB of different particle ∘ vacuum-packaged, and stored in a freezer (−18 C) through- sizes was determined by the method as described by Cao et out the experiments. Silver carp backbone was collected after al. [20]. The chilled surimi gels were equilibrated at room ∘ the deboning process of surimi production. It was cleaned temperature (∼25 C) for 2 h. Samples were cut to 2.5 cm cylin- ∘ andstoredinafreezer(−18 C) before use. der and subjected to fracture by penetration using a TA-XT 󸀠 N,N -Dimethylated casein (DMC), monodansylcadav- texture analyzer (Stable Micro Systems, Surrey, UK) equipped erine (MDC), and glutaraldehyde used for TGase activity with a spherical probe (diameter 5.0 mm and crosshead test were purchased from Sigma Chemical Company (St. speed of 1 mm/s). Breaking force (g) indicating gel strength Louis, MO, USA). Reagents used for gel electrophoresis and penetration distance (mm) denoting deformability were were obtained from Bio-Rad (Hercules, CA, USA). All other recorded. chemicals were of analytical grade. 2.5. Determination of Calcium Ion Concentration. Calcium 2.2. Preparation of Micron Fish Bone (MFB). Frozen fish ion concentration in the surimi paste incorporated with MFB bonewasthawedwithrunningtapwaterandthenheatedat of different particle sizes was measured according to the ∘ 121 C for 60 min (ZM-100, GBPI Packaging Test Instruments method as described by Yin et al. [5] with some modification. Co., Ltd., Guangzhou, China). The heated fish bone was Surimi paste prepared as described above was added with 4 rinsed with tap water 5 times and drained off before grinding times the volumes of DI water and homogenized at 5,000 rpm (MKCA6-2, Masuko Co., Tokyo, Japan). The bone paste was for 1 min (IKA T18, Cole-Parmer, Shanghai, China). The ∘ dried at 105 Cfor6handthencoarselymilled(RT-08HK, homogenate was then centrifuged at 10,000 ×gfor30min KaichuangtongheTechnologyDevelopmentCo.,Ltd.,Beijing (J-26XP, Beckman Coulter Inc., Fullerton, CA, USA). After China). Average particle size of the obtained fish bone power centrifugation, the supernatant was filtrated (Number 1 Journal of Food Quality 3

(a) (b) (c)

Figure 1: Morphologies of fish bone particles. (a) Micro fish bone powder, (b) micron fish bone particles in the emulsion with 1hofhigh- energy wet media milling, and (c) micron fish bone particles in emulsion with 6 h of milling.

Waterman,Xinhua,FilterPaperCo.,Ltd.,Hangzhou,China) temperature. The sample was rinsed three times using 0.2 M and diluted with DI water. The concentration of calcium phosphate (pH 7.2). The fixed sample was dehydrated in ion in the dilution was analyzed using an atomic absorption graded ethanol solution with serial concentrations of 30%, spectrophotometer (AA-6300c, Shimadzu, Kyoto, Japan). 50%, 70%, 80% 95%, and 100%. Samples were submerged in acetic acid isopropyl ester (substituting ethanol) and then 2.6. Determination of TGase Activity. TGase activity was critical-point-dried (HCP-2, Hitachi Koki Co., Ltd., Tokyo, measuredbythemethodofYinandPark[18]withslight Japan) using CO2 as the transition fluid. Dried sample was modifications. Silver carp surimi was added with 4 volumes mountedonabronzestubandsputter-coatedwithgold.The of extraction buffer (10 mM NaCl and 10 mM Tris-HCl, specimen was observed using a scanning electron microscope pH 7.5) and homogenized (Ika T18, Cole-parmer, Co., Ltd., (Quanta 3D Dual Beam, FEI Co., Tokyo, Japan) at an Shanghai, China) at 5,000 rpm for 1 min. The homogenate acceleration voltage of 15 kV. was centrifuged (J-26XP, Beckman Coulter Inc., Fullerton, ∘ CA, USA) at 16,000 ×g for 30 min under 4 C. The supernatant 2.9. Determination of Water Holding Capacity (WHC). WHC was filtrated and used as crude extract. Fish bone emulsion of gel sample was measured according to the method of prepared as described above was centrifuged at 10,000 ×g Shi et al. [22]. Cylindrical gel samples were cut into a for 30 min (Beckman Coulter Inc., Fullerton, CA, USA) thickness of about 5 mm, weighed accurately, and placed and filtrated. Filtrate was added to an assay mixture (15 𝜇M between two layers of filter paper (Number 1 Waterman paper, MDC, 1.0 mg/mL DMC, 3 mM DDT, and 50 mM Tris-HCl, Xinhua, Filter Paper Co., Ltd., Hangzhou, China). Sample pH 7.5) at a volumetric ration of 1 to 4. For the control was then placed at the bottom of a centrifuge tube (50 mL) sample, calcium chloride was added to the assay mixture and centrifuged at 3,000 ×g for 15 min (TDL-5A, Fulgor and reached a concentration of 0.17 mM. The crude enzyme Instruments Ltd., Shanghai, China). WHC was calculated as was added and vortexed. After incubating the mixture at percentage of water retained after centrifugation. ∘ 40 C for 10 min, EDTA solution was added to terminate ∗ ∗ the catalytic reaction. Fluorescence intensity of the mixture 2.10. Color Evaluation. Color parameters, 𝐿 (lightness), 𝑎 ∗ was immediately measured (RF-1501, Shimadzu Co., Kyoto, (redness to greenness), and 𝑏 (yellowness to blueness), Japan). were measured using a CR-400 colorimeter (Konica Minolta, Osaka, Japan). Whiteness was calculated according to an 2.7. SDS-PAGE. Protein patterns of all surimi gel samples equation developed by Park [23] for surimi gel. were revealed using SDS-PAGE according to Laemmli [21] with some modification. Surimi gel samples were homog- 2.11. Statistical Analysis. All the data were obtained from at enized (Ika T18, Cole-Parmer, Co., Ltd., Shanghai, China) least triplicate measurements. Analysis of variance (ANOVA) at 10,000 rpm for 1 min and solubilized using 5% sodium was carried out using the SAS program (V8, SAS Institute ∘ dodecyl sulfate solution (90 C). Stacking and separating Inc., Carry, NC, USA). Differences among mean values were gels were made using 5% (w/v) and 12% (w/v) acrylamide, evaluatedbytheDuncanmultiplerangetest(DMRT)usinga respectively. Each lane was loaded with 10 𝜇gprotein.After 95% confidence interval. running,gelswerefixedandstainedwith0.125%Coomassie brilliant blue R-250 and destained in DI water containing 50% 3. Results and Discussion methanol and 10% acetic acid. 3.1. Gel Texture. Effects of added MFB with different particle 2.8. Scanning Electron Microscopy (SEM). Surimi gel was cut size on breaking force and penetration distance of silver into pieces (5 mm × 5mm × 1 mm) and fixed with 2.5% carp surimi gels prepared with two thermal treatments glutaraldehyde in 0.2 M phosphate (pH 7.2) for 2 h at room are illustrated in Figure 2. Breaking force and penetration 4 Journal of Food Quality

700 24

600 a 20 a bc b bc ab c d cd 500 d d e 16

400 f f f f 12 g f 300

Breaking force (g) force Breaking 8

200 (mm) distance Penetration e e e e e f 4 100

0 0 Con 22 0.48 0.30 0.18 0.12 Con 22 0.48 0.30 0.18 0.12 Particle size (𝜇m) Particle size (𝜇m)

Figure 2: Breaking force and penetration distance of surimi gels with different size of fish bone particles and thermal treatments. Con: control ∘ ∘ ∘ sample,withoutaddedfishbone.Blackbars:gelcookedat90C for 30 min. Grey bars: gel incubated at 40 C for 1 h followed by 90 Ccooking for 30 min. Different lowercases above the error bar indicate significant differences among samples with fish bone of different particlesize (𝑃 < 0.05).

∘ distance of directly cooked gel (90 C/30 min) containing 35 a 2 MFB were (𝑃 < 0.05) higher than the control. When ∘ 30 gels were prepared with setting before cooking (40 C/1 h + ∘ b 1.5 90 C/30 min), MFB with an average particle size between 25 𝜇 𝑃 < 0.05 0.48 and 0.12 msignificantly( )increasedbreaking c c 20 force and penetration distance. However, MFB with an A 1 𝜇 𝑃 > 0.05 B averageparticlesizeof22 mhadnoeffect( )on 15 d (mmol/kg) +

𝑃 < 0.05 2 breaking force while significantly ( )decreasing e C 10 D Ca penetration distance. 0.5 TGase activity (U/mL) activity TGase E Breaking force and penetration distance of gels with 5 setting increased as MFB particle size decreased (𝑃< F 0.05). More calcium ions released from smaller fish bone 0 0 particles increased the activity of TGase in silver carp surimi Con 22 0.48 0.30 0.18 0.12 (Figure 3). Increased calcium ion release contributed to the Particle size (𝜇m) formation of MHC cross-links in the surimi gel (Figure 4). Figure 3: Endogenous TGase activity and calcium ion concen- In addition to being an endogenous TGase activator, calcium ∘ tration from surimi paste as affected by added fish bone particle ions, in conjunction with setting (40 Cfor1h),possibly size. Column: TGase activity; line: calcium ion concentration. Con: enhanced the unfolding of silver carp myofibrillar proteins. control sample, without added fish bone. Different letters above the Consequently, more exposure of the reactive residues imbed- error bar indicate significant differences among samples with fish ded inside the myofibrillar proteins might contribute to the bone of different particle size (𝑃 < 0.05). formation of more 𝜀-(𝛾-glutamyl) lysine cross-links and a higher degree of hydrophobic interactions [14]. On the other side,reductionoffishboneparticlesizecontributedpositively fish species. Compared to silver carp, myosin from Alaska to maintaining better surimi gel matrices (Figure 5). The pollockisreportedtobemorereactiveandtendstoform addition of 1% MFB with an average particle size at 0.12 𝜇m larger polymers during cross-linking reaction [10]. resulted in increased breaking force and penetration distance Breaking force and penetration distance of surimi gel of gel with setting by approximately 19% and 8%, respectively, without setting increased gradually but not significantly𝑃> ( over the control (without added fish bone). The effectiveness 0.05) as MFB particle size decreased (Figure 2). This slight of MFB (0.28 𝜇m) addition on improvement of breaking increasemighthavebeenattributedtotheformationofa force and penetration distance was more pronounced with “calcium bridge” between negatively charged residues on two Alaska pollock surimi gel (25% and 14%) [18]. It might adjacent myofibrillar proteins. Generally, the strength of ionic be related to different endogenous TGase activity, myosin bonds in surimi gel is much weaker than that of hydrophobic reactivity, and endogenous calcium content from different interactions and covalent bonds [10]. Journal of Food Quality 5

250 KD 150 KD MHC 100 KD 75 KD 50 KD AC 37 KD 25 KD

20 KD

10 KD STDCon 22 0.48 0.30 0.18 0.12 STD Con 22 0.48 0.30 0.18 0.12 ∘ ∘ ∘ 90 C 40 C/90 C (a) (b)

Figure 4: SDS-PAGE patterns of silver carp surimi gels with different size of fish bone particles and thermal treatments. (a) Gel cooked at ∘ ∘ ∘ 90 C for 30 min; (b) gel incubated at 40 C for 1 h followed by 90 C cooking for 30 min. Numbers designate average fish bone particle size (𝜇m). STD: protein standard. Con: control sample, without added fish bone. MHC: myosin heavy chain. AC: actin.

Con 22 𝜇m 0.48 𝜇m 0.30 𝜇m 0.18 𝜇m 0.12 𝜇m

∗ ∗ ∗

(a) Con 22 𝜇m 0.48 𝜇m 0.30 𝜇m 0.18 𝜇m 0.12 𝜇m

∗ ∗

∗

(b)

∘ Figure 5: SEM images of silver carp surimi gels with different size of fish bone particles and thermal treatments. (a) Gel cooked at90 C ∘ ∘ for 30 min; (b) gel incubated at 40 C for 1 h followed by 90 C cooking for 30 min. Con: control sample, without added fish bone. Numbers designate average size of fish bone particles.

Results suggested that effects of MFB on surimi gel texture TGase extract incubated in assay with fish bone particle size might vary by fish bone particle size, thermal treatments, and at 22, 0.48, 0.30, 0.18, and 0.12 𝜇mwere12.66,17.19,18.23, fish species. 23.66, and 29.66 U/mL, respectively. Increased activity of endogenous TGase with decreased fish bone particle size was 3.2. TGase Activity. As shown in Figure 3, addition of micron due to the release of more calcium ions in the surimi paste fishboneobviouslyactivatedTGasefromsilvercarpsurimi. (Figure 3). Calcium compounds in the fish bone possess low It was consistent with the reports by Yin and Park [18] solubility. Furthermore, they are imbedded in the collagen and Hemung [24]. Activity of crude TGase extracted from matrix, which makes them even harder to dissolve in water. silver carp surimi without added fish bone was 8.23 U/mL During the wet milling process, specific surface area of fish ∘ extract at 40 C. It significantly𝑃 ( < 0.05) increased as the bone particle markedly increases, and the collagen matrix added fish bone particle size decreased. Activities of crude is destroyed, facilitating the release of calcium ion [6]. The 6 Journal of Food Quality calcium ion concentration in the surimi paste without fish 100 bone was 0.17 mmol/kg. Calcium ion concentration signif- a icantly increased with decreasing of fish bone particle size 80 b cd bc bc cd bc and reached the maximal at 0.95 mmol/kg. Optimal calcium cd d cd 60 e ion concentration for full activation of endogenous TGase f extracted from carp, however, depends on the purification procedure. It has been reported that optimal calcium ion 40 concentrations for crude TGase extract and purified TGase from carp were at 5 mM and 50 mM, respectively [25, 26]. 20 Water holding capacity (%) capacity holding Water

3.3. MHC Cross-Linking. Influence of fish bone particle size 0 on the cross-linking of MHC during gel formation was Con 22 0.48 0.30 0.18 0.12 𝜇 analyzed using SDS-PAGE. Compared to gel without setting Particle size ( m) (Figure 4(a)), MHC of silver carp surimi gel markedly Figure 6: Water holding capacity of the surimi gels with different disappeared after setting (Figure 4(b)). Reduction of MHC size of fish bone particles and thermal treatments. Black bars: gel ∘ ∘ after setting could be attributed to the formation of 𝜀-(𝛾- cooked at 90 C for 30 min. Grey bars: gel incubated at 40 Cfor1h ∘ glutamyl) lysine cross-links and/or proteolytic degradation. followed by 90 C cooking for 30 min. Different lowercases indicate Ogata et al. [27] reported that the degradation of MHC was significant differences among different treatments (𝑃 < 0.05). ∘ observed in the carp surimi sample incubated at 37 Cfor 10 min with addition of endogenous protease (cathepsin L) and accompanied by the appearance of the resultant product, discontinuous structures with large pores (asterisks in which had a molecular weight of ∼27 KD. No obviously Figure 5). In contrast to the porous and coarse networks visiblebandwithamolecularweightof∼27 KD was detected of directly heated gel (Figure 5(a)), networks of surimi on the SDS-PAGE after setting (Figure 4), implying that the gel prepared with two-step heating (setting and cooking) reduction of MHC in this study was mainly related to the were denser and more compact along with a concomitant cross-linking reaction. appearance of homogeneous surface (Figure 5(b)). Heat- Gradual reduction of MHC from the surimi gel with denatured surimi proteins align in an ordered fashion to setting was observed as MFB particle size decreased due to the develop a fine gel network when rapidly unfolded proteins activation of endogenous TGase (Figure 3), resulting in the are associated in slow heating fashion (i.e., setting) [29]. In formation of more 𝜀-(𝛾-glutamyl) lysine cross-links of MHC. addition, the formation of larger amount of 𝜀-(𝛾-glutamyl) MHC intensity of gel without setting remained constant as lysine cross-links after setting contributed to the stabilization theparticlesizeoffishbonechanged.Theseresultswere of the gel network. in agreement with the report by Wang et al. [28] that the intensities of MHC cross-links from silver carp surimi incu- ∘ bated at 35 C for various time significantly increased with 3.5. Water Holding Capacity (WHC). Higher WHC values calciumionconcentrationupto1.80mmol/kgsurimipaste. indicate less expressible water in the surimi gel. During the However, MHC cross-links of gel without setting were not heat-induced gelling process, the gel network formed while significantly affected by calcium ion concentration ranging binding water and entrapping other ingredients. WHC of the 𝑃 < 0.05 from 0 to 5.40 mmol/kg surimi paste. Changes of MHC on gels prepared with setting was significantly ( )higher the SDS-PAGE gel corresponded well with the changes of gel than that of gel without setting for all particle sizes (Figure 6) texture values (Figure 2). as setting induced more compact and denser gel networks The significant reduction of MHC band’s intensity was by the function of endogenous TGase (Figure 3). WHC noted when setting was employed; neither MHC cross-links increased from 46.20% to 64.96% and 60.84% to 73.93% for nor protease-induced small molecular bands were present the samples without and with setting, respectively, as MFB 𝜇 𝜇 (Figure 4). As discussed above, no visible band for protease- particle size decreased from 22 mto0.12 m. It might be induced ∼27 kDa [27] was observed. MHC cross-links, which attributed to the increased WHC contributed by fish bone arenotshownaboveMHContheSDS-PAGE,possiblycould addition [3]. As mud obviously holds more water than sand, not enter the polyacrylamide gel system because the size of the size of particle is a significant factor affecting WHC. cross-links was too large. 3.6. Color Attributes. Color parameters of gels from silver 3.4. Microstructure. Figure 5 shows scanning electron carp under different thermal treatments with various fish ∗ microscopy (SEM) images of the internal structures of bone particle size are shown in Table 1. 𝐿 value of control surimi gels added with different particle size of MFB and gel and gel with MFB of particle size below 0.48 𝜇m slightly prepared with two thermal treatments. Fibrous matrix, but significantly (𝑃 < 0.05)increasedaftersettingwhilethat which is a characteristic of heat-induced protein gel, was of gel with MFB of particle size at 22 𝜇m slightly decreased ∗ clearlyobservedinthesurimigels.Controlgels(without (𝑃 < 0.05). Yellowness value (+𝑏 ) decreased (𝑃 < 0.05) ∗ added fish bone) and gels with MFB of particle size between and whiteness value (𝐿 −3𝑏)increased (𝑃 < 0.05)after ∗ 0.48 and 0.12 𝜇m formed continuous structures (Figure 5), settingforallfishboneparticlesizes.𝐿 values of gels with while surimi with MFB particles size of 22 𝜇mformed MFB of particle size below 0.48 𝜇m, regardless of setting, Journal of Food Quality 7

∗ ∗ Table 1: Color parameters (lightness (𝐿), greenness (𝑎 ), yellowness (𝑏 ), and whiteness (𝑤)) of silver carp surimi gels with different size of fish bone particles (𝜇m) and thermal treatments.

∗ ∗ Color parameter 𝐿𝑎𝑏 𝑤 Con 74.27 ± 0.49g −2.05 ± 0.07a 2.44 ± 0.21g 66.95 ± 0.60b 22 74.79 ± 0.65f −2.71 ± 0.09cd 9.06 ± 0.43a 47.60 ± 1.28i 0.48 76.52 ± 0.48a −2.67 ± 0.06c 8.26 ± 0.21b 51.74 ± 0.54g Without setting 0.30 76.35 ± 0.23ab −2.74 ± 0.45de 7. 8 1 ± 0.20c 52.93 ± 0.61f 0.18 76.10 ± 0.35bc −2.81 ± 0.03f 7. 78 ± 0.37c 52.74 ± 0.93f 0.12 75.85 ± 0.43cd −2.80 ± 0.03ef 7. 3 6 ± 0.33d 53.77 ± 0.90e Con 75.49 ± 0.47de −2.36 ± 0.04b 1.91 ± 0.26h 69.76 ± 0.58a 22 75.11 ± 0.51ef −2.94 ± 0.07h 8.27 ± 0.18b 50.30 ± 0.32h 0.48 76.27 ± 0.48ab −2.88 ± 0.05g 7. 5 3 ± 0.26d 53.68 ± 0.59e With setting 0.30 75.81 ± 0.46cd −3.03 ± 0.05i 7. 31 ± 0.26d 53.88 ± 0.67e 0.18 75.70 ± 0.29cd −3.10 ± 0.05j 6.85 ± 0.32e 55.13 ± 0.93d 0.12 75.59 ± 0.27d −3.09 ± 0.05j 6.36 ± 0.22f 56.50 ± 0.56c The different lowercases in the same column indicate the significant differences (𝑃 < 0.05). Data are expressed as means ± standard deviations.

were significantly (𝑃 < 0.05) higher than that of gel with providing additional dietary calcium for silver carp surimi MFB of particle size at 22 𝜇m. As particle size decreased, products. ∗ 𝐿 values of gels with MFB of particle size below 0.48 𝜇m, 𝑃 < 0.05 regardless of setting, gradually decreased ( ). This Competing Interests might be due to the decreased light scattering effect of water molecular as a result of increased WHC of the gel (Figure 6). The authors declare that there is no conflict of interests Yellowness value of gels with and without setting decreased regarding the publication of this paper. gradually (𝑃 < 0.05) and whiteness value significantly𝑃< ( 0.05) increased with decreasing MFB particle size. Thus, both thermal treatment and fish bone particle size affected color Acknowledgments attributes of silver carp surimi gels. The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (no. 4. Conclusions 31601501) and the earmarked fund for China Agriculture Research System (no. CARS-46-23). Gel properties of silver carp surimi were significantly affected by thermal treatments and MFB particle size. Textural values, WHC, and whiteness of gels prepared with setting increased References (𝑃 < 0.05) as the MFB particle size decreased. Improvement [1]T.Larsen,S.H.Thilsted,K.Kongsbak,andM.Hansen,“Whole of gel texture and WHC resulted from reduced bone particle small fish as a rich calcium source,” British Journal of Nutrition, size which was primarily due to the function of endogenous vol.83,no.2,pp.191–196,2000. TGase and calcium ion from the MFB. In addition, reduction [2]J.Toppe,S.Albrektsen,B.Hope,andA.Aksnes,“Chemical of MFB particle size contributed positively to maintaining composition, mineral content and amino acid and lipid profiles integrity of the three-dimensional myofibrillar gel networks. 𝜇 in bones from various fish species,” Comparative Biochemistry MFB with an average particle size below 0.48 mpossesses and Physiology Part B: Biochemistry and Molecular Biology,vol. the potential to be used to improve gel quality while providing 146, no. 3, pp. 395–401, 2007. additional dietary minerals for silver carp surimi products. [3]G.-C.Wu,M.Zhang,Y.-Q.Wang,K.J.Mothibe,andW.-X. Chen, “Production of silver carp bone powder using superfine Additional Points grinding technology: suitable production parameters and its properties,” Journal of Food Engineering,vol.109,no.4,pp.730– Practical Applications. Fish bone is a natural calcium source. 735, 2012. Fish bone incorporated into silver carp surimi gels exhibited [4]T.Yin,H.Du,J.Zhang,andS.Xiong,“Preparationandcharac- different properties, which were affected by its particle size terization of ultrafine fish bone powder,” Journal of Aquatic Food and heating methods subjected to surimi paste. Improvement Product Technology,vol.25,no.7,pp.1045–1055,2016. of the surimi gel texture was achieved by addition of the fish [5] T. Yin, J. W. Park, and S. Xiong, “Physicochemical properties bone with particle size below 0.48 𝜇mwhencombinedwith of nano fish bone prepared by wet media milling,” LWT - Food setting. The practical application of this work is providing Science and Technology,vol.64,no.1,pp.367–373,2015. a theoretical foundation and basic data support for using [6]J.Zhang,T.Yin,S.B.Xiong,Y.J.Li,U.Ikram,andR. calcium-enriched fish bone to improve gel texture while Liu, “Thermal treatments affect breakage kinetics and calcium 8 Journal of Food Quality

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[19]Y.Q.Ding,Y.M.Liu,H.Yangetal.,“EffectsofCaCl2 on chemical interactions and gel properties of surimi gels from two species of carps,” European Food Research and Technology,vol. 233, no. 4, pp. 569–576, 2011. [20]L.Cao,Y.An,S.Xiong,S.Li,andR.Liu,“Conformational changes and kinetic study of actomyosin from silver carp surimi with modified starch-sucrose mixtures during frozen storage,” Journal of Food Quality,vol.39,no.1,pp.54–63,2016. [21] U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature,vol.227,no. 5259, pp. 680–685, 1970. [22] L. Shi, X. Wang, T. Chang, C. Wang, H. Yang, and M. Cui, “Effects of vegetable oils on gel properties of surimi gels,” LWT— Food Science and Technology,vol.57,no.2,pp.586–593,2014. [23] J.W.Park,“Surimigelcolorsasaffectedbymoisturecontentand physical conditions,” Journal of Food Science,vol.60,no.1,pp. 15–18, 1995. [24] B. O. Hemung, “Properties of tilapia bone powder and its calcium bioavailability based on transglutaminase assay,” Inter- national Journal of Bioscience, Biochemistry and Bioinformatics, vol. 3, pp. 306–309, 2013. Hindawi Journal of Food Quality Volume 2017, Article ID 8089135, 5 pages https://doi.org/10.1155/2017/8089135

Research Article Effects of Beeswax Coating on the Oxidative Stability of Long-Ripened Italian Salami

Marcello Trevisani, Matilde Cecchini, Daniela Siconolfi, Rocco Mancusi, and Roberto Rosmini Department of Veterinary Public Health and Animal Pathology, University of Bologna, Bologna, Italy

Correspondence should be addressed to Marcello Trevisani; [email protected]

Received 29 November 2016; Accepted 18 January 2017; Published 1 February 2017

Academic Editor: Marta Laranjo

Copyright © 2017 Marcello Trevisani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Beeswax coating of foods put a barrier to oxygen, light, and vapour that can help to prevent oxidation of fat and pigments and water loss. The amounts of 2-thiobarbituric acid reactive substances (TBARS) and water activity (Aw) were assessed in Italian salami coated with beeswax at 55 days of ripening and compared with controls at 5, 6, and 7 months of shelf life. The results were −1 correlated with sensory quality. TBARS levels were below 0.8 mg kg in the beeswax-coated salami until 6 months of aging (median 0.697,max 0.795) and significantly higher in the uncoated salami (median 1.176, max 1.227). A slight correlation between the amount of TBARS and Aw was observed in beeswax-coated salamis, whereas this effect was masked in controls by the large Aw variability observed at 7 months. Beeswax coating prevents case hardening and facilitated the peeling.

1. Introduction contamination by dirt and off-odour and protect the products fromoxygenandlossoruptakeofmoisture.Oxygenmoisture Beeswax is natural glazing agent that can be used in food barrier properties of beeswax are intermediate between low to prevent water loss and provide protection during storage. and high density polyethylene [4]. It is also a barrier to It is often used to prevent water loss and retard shrinkage photooxidation. Beeswax is also one of the most effective and spoilage in fruit and cheese. Refined beeswax coating materialsemployedtodecreasewatervapourpermeabilityof is a natural alternative to plastic envelopes that does not ediblefilmsduetoitshighhydrophobicityandsolidstateat harmtheenvironmentandmeetsthecriteriaforGRAS room temperature [5, 6]. These properties have been used to status defined by the FDA for food packaging materials preserve and improve the sensorial quality of salami during [1]. It is approved for food use in most countries and aging. Local producers in the area of Bologna (Italy) used in the European Union under the E number “E901” [2]. to coat salami with beeswax, after the products are shelf Colour changes occur due to oxidation phenomena involving stable (i.e., water activity has decreased to values below 0.92). myoglobin during ripening of salami. Moreover, shrinkage They use the natural beeswax to limit an excessive water loss due to dehydration results in aspect modifications, mainly during storage. Wax coating also prevents case hardening and ascribable to fat aggregation [3]. Even after salami has reached mould development and made the peelability easy. This study water activity (Aw) and pH values that make the product was aimed at assessing the oxidative stability and sensorial shelf stable without refrigeration, environmental conditions characteristics of salami after beeswax coating. for storage (relative humidity, relative air speed, temperature, and light) need to be controlled to prevent excessive water loss, product shrinkage and too hard consistency, 2. Materials and Methods oxidative changes, and excessive growth of moulds. During distribution salami is often wrapped in plastic film with 2.1. Reagents. The reagents were as follows: trichloroacetic reduced oxygen and water vapour permeability to prevent acid (TCA) 99%; 1,1,3,3-tetramethoxypropane (TMP) 99%; 2 Journal of Food Quality

−1 2-thiobarbituric acid (TBA) 98% (Sigma-Aldrich, Italy); rangeof0.15to4.26nmolmL were prepared. Nine TMP ethylenediaminetetraacetic acid disodium salt (EDTA) standards (from 20 to 560 𝜇L of the TMP working solution) (AnalR5 VWR); propyl gallate (Fluka); hydrochloridric acid were mixed with 2 mL of TBA reagent, 300 𝜇LofTCA100%, 37% (Merck); sodium hydroxide 20% water solution (Carlo and HCl 0.25 M up to 4 mL. A blank solution was made Erba, Italy); TCA 100% p/v (100 g TCA 99%, water up to as described before, but without TMP. Vials with the TMP −1 100 mL); TMP stock solution (1000 mg kg ); extracting standards and blank were incubated as described for the solution (75 mL TCA 100%, 20.8 mL HCl 0.25 M, 1 g EDTA, samples and the absorbance values at 532 nm (Abs532)were 1g propyl gallate, and water up to 1000mL); TBA reagent read (average of 5 replicates). Ten-point standard calibration 2 (TBA 80 mM in in NaOH 1 M, pH corrected at 4.0–4.2 with curves were designed. The coefficient R must be between −1 HCl 1 M); TMP (5 mg kg ) working solution (250 𝜇LTMP 0.995 and 1 in order to accept the curves for TBARS quan- stock solution, 3.75 mL TCA 100%, and HCl 0.25 M up to tification. The samples’ TBARS concentration was calculated 50 mL). by interpolation of their measured absorbance values (Abs). The resulting value was multiplied by the dilution factor of thesample(2.5gin25mL)andextract(1:2)tocorrectfor 2.2. Samples. Twelve salamis “Felino IGP” were provided by −1 a local producer at approximately 55 days of ripening. The the final concentration. Results are expressed as mg kg of salamis had the typical characteristics (length 40–45 cm, MDA equivalents TBARS. diameter approximately 6 cm, weight 1.0–1.2 Kg, weight loss 35–37.5%, and water activity 0.90–0.92). The salamis derived 2.4. Water Activity. The water activity (Aw) was assessed with from a single lot were divided into two groups. Six were used a dew point water activity meter (Aqualab Series 3, Decagon, as control. The remaining were brushed, washed, and put US) using the procedures recommended by the producer. back to dry in ventilated cells. Then they were wrapped in a cotton gauze and tied and covered with beeswax (yellow ∘ 2.5. Sensory Test. A hedonic test was conducted with eight organic beeswax melted and held at 62–64 C) for 3–5 sec- untrained assessors who scored the acceptability of 3 attrib- onds. The resulting wax layer had a thickness of 2-3 mm. utes (texture, taste, and flavour) using the following 1–10 point Therefore, all salamis were hang up to dry in well-aired cellars scale: texture (1 = hard; 10 = soft); presence of acid taste (1 = up to 5–7 months, packed in cartoons, and stored in a dark ∘ ∘ sharp burning; 10 = mild acidic); rancid off-flavour (1 = none; room at 5 C ± 1 C (relative humidity 85–90%) until analyses. 3 = slightly perceived; 10 = strong). With this aim, the salamis Comparative assessments were made on two salamis for each analysed for the TBARS at 6 and 7 months of storage were cut treatment group (wax coated and uncoated) at 5, 6, and 7 in slices and the panel was asked to comparatively assess the months. quality of the salami (blind test between beeswax-coated or noncoated salami). 2.3. Quantification of TBARS. Three slices (5 mm thick) were taken from centre and intermediate parts from each 2.6. Statistical Analysis. TBARs values recorded for different salami.Thesesamplesweremincedfor10secondswitha categories (wax or not coating) and periods (0, 1, and 2 Moulinette5 and 2.5 g of the homogenate was analysed for months of storage) were summarized graphically as box and 2-Thiobarbituric Acid Reactive Substances (TBARS) using whiskers plots. Statistical analyses were performed using the method developed by Wang et al. [7] for meat and meat the R package “stats” (version 2.15.3) [8]. Data relative to products. The entire protocol (sampling and analyses) was samples from different categories and period were analysed repeated two times (i.e., after 2-3 days) using other portions with Bartlett’s test to assess homoscedasticity (homogene- of each salami. A total of eight measurements (2 salamis, 2 ity of variance). When departures from normality of data replicates, and 2 repetitions) for each treatment group and were observed nonparametric alternatives to the analysis of storagetimewereobtained.Samplehomogenateswereputin variance (ANOVA) were used. In particular, the Wilcoxon vials in an ice bath, mixed with 20 mL of chilled extracting signed-rank test was used for comparing the MDA equivalent solution, and homogenised at 16,000 rpm for 2 minutes with TBARS values observed in salami packaged with or without Ultra-turrax5 (model T25 basic IKA, Labortechnic, Italy). wax. Differences among samples taken at 5, 6, and 7 months Additional 5 mL of extracting solution was used to wash were analysed with the Kruskal-Wallis rank sum test. When the blades of the Ultra-turrax (final dilution 1 : 10); then differences were considered to be significant at 𝑝 ≤ 0.01 the the extracted samples were filtered (Whatman5 paper filter significance of individual pair differences (aging periods) was n.4). Samples were continuously maintained in a chilled bath tested for inequality using the multiple comparison test after until the filtration. Two mL of the filtrates was mixed with Kruskal-Wallis using the R package “pgirmess.” ∘ 2 mL of the TBA reagent and incubated at 40 Cinawater ∘ bath for 90 minutes and then chilled in fridge at 6–8 Cfor 3. Results and Discussion 30 minutes. Therefore the absorbance at 532 nm was read (5 replicates) on a spectrophotometer (Perkin-Elmer, model 3.1. Effect of the Beeswax Coating on the TBARS Concen- Lambda 1). A calibration curve was designed using standards tration. Slower drying and ripening at low temperatures of −1 at concentration in the range of 0.025 to 0.7mg mL of TMP the beeswax-coated salami resulted in less lipid oxidative −1 (5 mg kg ) working solution. By using the above-mentioned changes. The concentration of TBARS (MDA equivalents) −1 TMP solutions, malondialdehyde (MDA) standards in the is reported in Table 1. Values were below 0.8 mg kg in Journal of Food Quality 3

Table 1: TBARS concentrations in salami according to aging period and beeswax coating.

−1 Ageing TBARS (MDA mg kg ) 1 Coating 2 Wilcoxon signed-rank test (months) Mean Median Range Beeswax 0.693a (±0.017) 0.696 0.653–0.713 5 𝑝 = 0.0039 Control 1.016b (±0.045) 1.029 0.938–1.059 Beeswax 0.678a (±0.075) 0.697 0.561–0.795 6 𝑝 = 0.0039 Control 1.166c (±0.045) 1.176 1.104–1.227 Beeswax 1.106b (±0.039) 1.098 1.048–1.173 7 𝑝 = 0.0039 Control 1.869d (±0.023) 1.872 1.819–1.897 Note: mean, median, and range calculated from 𝑛=8measurements (i.e., 2 samples, 2 replicates, and 2 repetitions for each treatment group). Samples with 1 significant differences in their malondialdehyde level are indicated by different letters; significant differences detected between samples at the same aging period (Wilcoxon test). 2 Significant differences detected between samples at different aging period (Kruskal-Wallis test) chi-square = 15.3934; 𝑝 value = 0.0004543.

the beeswax-coated salami until 6 months of aging (median Table 2: Changes in the water activity values (Aw) in salamis with 0.697, max 0.795) and significantly higher in the uncoated or without beeswax coating. salami (median 1.176, max 1.227). At 7 months the MDA −1 Ageing period Control Beeswax coating median values were equal to 1.098 and 1.872 mg kg in wax 0.821 ± 0.001a 0.903 ± 0.002c coated and uncoated salami, respectively. Limits for TBARS 5months −1 0.812 ± 0.002a 0.899 ± 0.002d (MDA equivalents) have been suggested at 0.5 mg kg of 6months a e meat for threshold of consumer detection of rancidity [9–11] 7months 0.820 ± 0.011 0.888 ± 0.004 −1 and 1.0 mg kg for sausage products [12]. However, detection Aging period at the beginning of the shelf life = 5 months. Differencesrelatedtocoating:Fcalculated=1192.81;Fcritical=4.75;𝑝 value limits have not been set for salami. The lipid peroxidation −13 = 2.21 ∗ 10 . in raw ripened sausages (salami) involves transformation Differences related to aging (in salami with beeswax coating): F calculated = of primary products of lipid degradation (alkyl free radical 23.03; F critical = 5.14; 𝑝 value = 0.0015. with a group of conjugated bonds) into secondary products, Means with different letters are significantly different. including MDA [13–15], and this is correlated to the increase of TBARS concentration. The presence of high TBARS values in sausages after ripening can be explained by availability of treatment groups, whereas texture was softer in the beeswax- oxygen consequently to the mechanical process [16]. How- coated salami (Figure 1). The softer texture was related to ever, the use of vacuum stuffing, antioxidants, the protective the lower loss of water. There is a statistically significant effect of some starters [17], and also the storage of ripening relationship between TBARS and Aw values, but only for the sausage in vacuum or modified atmosphere without oxygen beeswax-coated salami (Spearman’s rank correlation rho = [18]cansignificantlycontributetoanincreasedoxidative −0.623; 𝑝 value = 0.0011). Within this group the concentration stability. The latter condition can occur with the use of of TBARS slightly increased in consequence of the water beeswax coating. In a study of Novelli et al. [19] concerning loss, but this effect was masked in controls by the large “Milano” salami with similar fat content (approximately Aw variability observed at 7 months (Aw = 0.820 ± 0.011; 30%), the TBARS values were equal to 1.39 ± 1.08 mg MDA −1 range 0.814–0.832) (Table 2 and Figure 2). Any difference kg at >3 months of aging, which is a value higher than those was perceived during the sensory test with regard to sharp of the beeswax-coated salami at 7 months of aging. burning taste (Wilcoxon paired sign test 𝑝 value > 0.05). Even if the consumers do not perceive any flavour deterio- Also the presence of rancid off-flavour was not perceived ration, lipids oxidation involves loss of unsaturated fatty acids and only two panel members (out of eight) reported value (nutrient loss) and the end products of lipid oxidation may be above the limit of perception in the uncoated salami at 7 mutagenic and carcinogenic. Malondialdehyde (MDA) can months of aging. The panel test probably did not give rise react with DNA and form MDA adducts [20, 21]. Storage toanegativescore(rancidoff-flavour)becausetheTBARS −1 of ripening sausage in vacuum or modified atmosphere has concentration was relatively low (1.819–1.897 mg MDA kg ) resulted in increased oxidative stability of raw sausage after 2 also in these samples. An unpleasant fruity flavour was and 5 months of storage [18]. Beeswax coating appears to give perceived by some panel members in the salami coated with similar protection. beeswax at 7 months of aging. This flavour deterioration might be related to spoilage by heterofermentative lactic acid 3.2. Correlation between TBARS Concentration, Aw, and Sen- bacteria in consequence of the higher Aw [22, 23]. There sory Evaluations. The beeswax-coated salami had signifi- are no published studies available that define TBARS values cantly higher mean Aw values, from 0.903 to 0.888, between that are associated with the presence of rancid off-flavours in 5 and 7 months of aging. The uncoated salami had Aw values “Felino” salami. in a range of 0.812 to 0.821 (Table 2). The sensory test did not Sojiˇ c´ et al. [24] reported that vacuum and MAP packaging reveal relevant differences in the taste and flavour of different can contribute to better oxidative and sensory stability of 4 Journal of Food Quality

Taste Rancid off-flavour (1 = sharp burning; 10 = mildly acidic) Texture (1 = hard; 10 = soft) (1 = none; 3 = sligthly perceived; 10 = strong) 10.0 10.0 10.0 9.0 9.0 9.0 8.0 8.0 8.0 7.0 7.0 7.0 6.0 6.0 6.0 5.0 5.0 5.0 4.0 4.0 4.0 3.0 3.0 3.0 2.0 2.0 2.0 1.0 1.0 1.0 0.0 0.0 0.0 m wax m wax m wax m wax m wax m wax 6 7 6 6 7 7 mcontrol mcontrol mcontrol mcontrol mcontrol mcontrol 7 6 7 7 6 6

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 line the median. The lines (“whiskers”) show the largest or the smallest observation. The symbol Q indicates average values.

1.8 1.1

1.6 1.0

0.9 1.4

MDA, control MDA, 0.8 MDA, beeswax MDA,

1.2 0.7

1.0 0.6

0.810 0.820 0.830 0.885 0.895 0.905 Aw, control Aw, beeswax Figure 2: Relationship between TBARS and Aw values. dry fermented sausage and that TBARS values are negatively reduces the development of lipid peroxidation products in correlatedwithodourandtasteofsalamiagedfor7months. these salamis. Ban˜on´ et al. [25] observed that the main causes of loss of sensory quality of salami limiting their shelf life under the Additional Points tested retail (aerobic) conditions include flavour deterioration associated with rancidity, bitterness, and mouldy off- Practical Applications. Beeswax coating can be used to pre- flavour, together with hardening and loss of juiciness. serve and improve the sensorial quality of salami during aging. It can be a natural packaging material that does not 4. Conclusions harm the environment and has good oxygen moisture barrier properties. Theresultsofthispreliminarystudyindicatethatbeeswax coating can be a useful alternative to the plastic packaging. Competing Interests Thisnaturalcoatingmaterialcanbeusedtoincreasetheaging period without compromising the texture, which remains soft The authors declare that there is no conflict of interests and is appreciated for its better flavour. Beeswax effectively regarding the publication of this paper. Journal of Food Quality 5

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