Survival of Salmonella spp. in minced meat packaged under vacuum and modified atmosphere

Jasna Djordjević Marija Bošković Marija Starčević Jelena Ivanović Nedjeljko Karabasil Mirjana Dimitrijević Ivana Branković Lazić Milan Ž. Baltić About the authors

Abstract

The effect of different modified atmosphere packaging regimes on the behavior of Salmonella spp. on minced meat was studied. Minced meat was experimentally contaminated with a Salmonella spp. cocktail (S. Enteritidis, S. Typhimurium, S. Infantis and S. Arizonae), packaged under vacuum or modified atmosphere with initial headspaces containing 20%O2/50%CO2/30%N2 and 20%O2/30%CO2/50%N2) and stored at 3 ± 1 °C for 12 days. Samples were analyzed for Salmonella spp., viable and lactic acid bacteria count every third day. Salmonella spp. counts decreased during storage in all packaging types, with reductions of about 1.5 log CFU/g. A significant difference (p < 0.01) was noted between Salmonella spp. counts in meat packaged in vacuum and modified atmospheres, although there was no significant difference in Salmonella spp. count between meat packaged in 50%CO2, and meat packaged in 30%CO2. At the end of the study, there were significant differences (p < 0.01; p < 0.05) in total viable and lactic acid bacterial counts between meat packaged in vacuum and modified atmosphere, and the lowest counts were noted in meat packaged in modified atmosphere with 50%CO2.

Keywords:
Minced meat; Modified atmosphere packaging; Vacuum; Salmonella; pH

Introduction

Pork and beef minced meat are widely consumed in Europe.11 Koppel R, Eugster A, Ruf J, Rentsch J. Quantification of meat proportions by measuring DNA contents in raw and boiled sausages using matrix-adapted calibrators and multiplex real-time PCR. J AOAC Int. 2012;95(2):494-499. In Serbia as well as in other Balkan and some Mediterranean countries, minced meat is an inseparable part of traditional dishes (e.g. moussaka, sarma), and meat products (e.g. sausages, ćevapčići, hamburger). The mincing process disrupts the meat cellular structure, releasing tissue fluids and making the minced meat a highly nutritious medium supporting bacterial growth; mincing also allows migration of surface bacteria throughout the product.22 Motjaremi Y, Moy G, Todd E, eds. Encyclopedia of Food Safety. vol. 1. San Diego, CA, USA: Academic Press Elsevier; 2014. Therefore, it presents a highly perishable product that need to be wrapped or packaged and chilled immediately to an internal temperature of not more than 2 °C or frozen to -18 °C during storage and transport (Regulation (EC) 853/2004).33 Efsa Biohaz. Panel (EFSA Panel on Biological Hazards) Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 2 (minced meat from all species). EFSA J. 2014;12(7):3783.

Despite measures to control foodborne pathogens from farm to fork the burden of diseases caused by foodborne pathogens remains important health and economic issue.44 Bacon RT, Sofos JN. Characteristics of biological hazards in foods. In: Schmidt HR, Rodrick G, eds. Food Safety Handbook. Hoboken, New Jersey: Willey Interscience, John Wiley & Sons, Inc.; 2003:712–1641.

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This highlights the need for improved prevention and control of Salmonella spp. in food.

The food industry has developed numerous preservation techniques in order to prevent and control Salmonella and other food-borne pathogens and spoilage microorganisms in fresh meat products, making the meat safer for consumption and extending its shelf life. Vacuum packaging (VP) and modified atmosphere packaging (MAP) are the most commonly used packaging methods for meat and meat products. MAP is considered to be an effective technique for raw meat preservation.1919 Narasimha RD, Sachindra NM. Modified atmosphere and vacuum packaging of meat and poultry products. Food Rev Int. 2002;18(4):263-293.

20 Skandamis PN, Nychas GJE. Preservation of fresh meat with active and modified atmosphere packaging conditions. Int J Food Microbiol. 2002;79(1):35-45.

21 Chouliara E, Karatapanis A, Savvaidis IN, Kontominas MG. Combined effect of oregano essential oil and modified atmosphere packaging on shelf-life extension of fresh chicken breast meat, stored at 4 °C. Food Microbiol. 2007;24(6):607-617.

22 McMillin KW. Where is MAP going? A review and future potential of modified atmosphere packaging for meat. Meat Sci. 2008;80(1):43-65.
-2323 Zhou GH, Xu XL, Liu Y. Preservation technologies for fresh meat-a review. Meat Sci. 2010;86(1):119-128. These methods are based on removal of the surrounding atmosphere (VP) or flushing it out and replacing it with a gas mixture (MAP) before sealing in gas barrier materials.2020 Skandamis PN, Nychas GJE. Preservation of fresh meat with active and modified atmosphere packaging conditions. Int J Food Microbiol. 2002;79(1):35-45.

21 Chouliara E, Karatapanis A, Savvaidis IN, Kontominas MG. Combined effect of oregano essential oil and modified atmosphere packaging on shelf-life extension of fresh chicken breast meat, stored at 4 °C. Food Microbiol. 2007;24(6):607-617.

22 McMillin KW. Where is MAP going? A review and future potential of modified atmosphere packaging for meat. Meat Sci. 2008;80(1):43-65.
-2323 Zhou GH, Xu XL, Liu Y. Preservation technologies for fresh meat-a review. Meat Sci. 2010;86(1):119-128. Gases most often used in MAP are carbon dioxide, which inhibits bacterial growth, oxygen, which prevents anaerobic growth and retains meat color, and nitrogen, which avoids oxidation of fats and pack collapse. Depending on the type of food or effect desired, these gases can be used separately or in combination in various concentrations.1919 Narasimha RD, Sachindra NM. Modified atmosphere and vacuum packaging of meat and poultry products. Food Rev Int. 2002;18(4):263-293.,2121 Chouliara E, Karatapanis A, Savvaidis IN, Kontominas MG. Combined effect of oregano essential oil and modified atmosphere packaging on shelf-life extension of fresh chicken breast meat, stored at 4 °C. Food Microbiol. 2007;24(6):607-617.

Considering the prevalence of Salmonella in minced meat and the frequency of its consumption via many traditional products and, taking into account that packaging of meat is the most common method of food preservation, there is a need to explore the effect of packaging methods on Salmonella spp. survival, especially in mixed minced meat (pork and beef). Therefore, the aim of this study was to compare the effects of vacuum and two initial headspace-modified atmosphere conditions (20%O2/50%CO2/30%N2 and 20%O2/30%CO2/50%N2) on the survival of Salmonella spp., total viable bacteria and lactic acid bacteria in minced meat stored at 3 ± 1 °C.

Materials and methods

Pork and beef muscles from leg of different carcasses used in the study were provided 48 h post-slaughter by a local slaughterhouse (Pećinci-Subotište, Serbia). Connective tissues and visible fat were trimmed after which the pieces of meat were minced separately in a sterile grinder (4 mm perforation diameter in the meat grinder plate), mixed in a 50:50 ratio of pork:beef and transported under refrigeration to the laboratory within an hour.

Four serovars of S. enterica (S. Enteritidis ATCC 13076, S. Typhimurium ATCC 14028, S. Arizonae ATCC 13314 and S. Infantis ATCC 51741) (www.atcc.org) were used in this study. The serovars were stored in Brain Heart Infusion (BHI; Merck, Germany) with 20% glycerol at -80 °C until needed. One ml of each frozen Salmonella serovar was added to 10 ml of BHI (Merck, Germany), incubated at 37 °C for 24 h, then were streaked on Xylose Lysine Tergitol-4 Agar (XLT4) (Merck, Germany) to verify their characteristics. In order to get a second subculture isolated, black colonies were picked from the XLT4 plates and inoculated into BHI tubes (1 colony per tube) and further incubated for another 24 h at 37 °C. After incubation, the cultures were centrifuged at 5000 × g (Eppendorf, Hamburg, Germany) for 10 min and suitable dilutions were prepared in BHI. A Salmonella cocktail was prepared by combining equal portions of standardized cell suspensions to yield approximately 8 log CFU/ml of each serovar in the mixture. Salmonella counts were determined by serial dilution and subsequent enumeration on XLT4. This Salmonella cocktail (40 ml of the cocktail) was used to inoculate 9 kg of minced meat in the sterile mixer in the experimental laboratory of the Faculty of Veterinary Medicine, University of Belgrade. According to legal requirement for the absence of Salmonella in 25 g of raw meat, meat used in the present study was not naturally contaminated with Salmonella. Minced meat was divided in portions of 100 g, and packaged in three different conditions: VP, modified atmosphere package 1 (MAP1, containing 20%O2/50%CO2/30%N2) and modified atmosphere package 2 (MAP2, containing 20%O2/30%CO2/50%N2). MAP treatments were conducted considering ratio of 1:3 (v/w) between the volume of gas and weight of the minced meat (G/P ratio). A Variovac packaging machine (Variovac Primus, Zarrentin, Germany) was used for VP and MAP. Minced meat was packaged in a OPA/EVOH/PE foil (oriented polyamide/ethylene vinyl alcohol/polyethylene Dynopack, POLIMOON, Kristiansand, Norway), with low gas permeability (O2 - 3.2 cm3/m2/day at 23 °C, N2 - 1 cm3/m2/day at 23 °C, CO2 - 14 cm3/m2/day at 23 °C, water vapor - 15 g/m2/day at 38 °C). All minced meat samples weighed 100 ± 5 g and were refrigerated at 3 ± 1 °C.

Minced meat was analyzed for Salmonella spp., total viable count (TVC-mesophiles, 30 °C), and lactic acid bacteria (LAB) count immediately and on days 3, 6, 9 and 12 of storage. For bacterial enumeration, approximately 10 g of meat were weighed aseptically after package opening, transferred into sterile Stomacher bags and 90 ml of Buffered Peptone Water (BPW) (Merck, Germany) was added to each sample. Meat samples were homogenized in a Stomacher blender (Stomacher 400 Circulator, Seward, UK) for 2 min. Serial decimal dilutions were prepared in buffered peptone water (Merck, Germany) and 1 ml or 0.1 ml of appropriately diluted homogenized meat was inoculated directly on the surface of XLT4 (Merck, Germany) for Salmonella spp. enumeration2424 Pathania A, McKee SR, Bilgili SF, Singh M. 2010 inhibition of nalidixic acid-resistant Salmonella on marinated chicken skin. J Food Protect. 2010;73(11):2072-2078. and incubated for 24 h at 37 °C, Plate Count Agar (PCA; Merck, Germany) for TVC-mesophiles enumeration according to ISO 4833:2003,2525 ISO 4833:2003 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of microorganisms - Colony-count technique at 30 degrees C. and incubated at 30 °C for 72 h and MRS Agar (Merck, Germany) for LAB enumeration according to ISO 15214:1998,2626 ISO 15214:1998 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of mesophilic lactic acid bacteria - Colony-count technique at 30 degrees C. and incubated at 30 °C for 72 h. After incubation, plates were examined visually for typical colonies and morphological characteristics associated with each growth medium, the number of colonies was counted and results were recorded as colony forming units per g (CFU/g). Suspect colonies of Salmonella spp. were tested using API 20e (BioMerieux Italia-Bagno a Ripoli, Florence), while suspect colonies of lactic acid bacteria were stained by Gram and catalase test was done.

The meat pH was measured after 10 min at room temperature using a hand-held pH meter, Testo 205 (Testo AG, Lenzkirch, Germany), equipped with a penetrating glass electrode.

Measurement of headspace gas composition in the minced meat packaging was conducted using the gas composition tester, Oxybaby (WITT Gasetechnik GmbH & Co. KG, Witten, Germany). The measurement range of the instrument is 0-100% by volume (vol) for oxygen (O2) and carbon dioxide (CO2). The nitrogen content is calculated as the difference from 100% after the measured values of oxygen and carbon dioxide are deducted. The accuracy of the device is 0.1% for oxygen and carbon dioxide.

Statistical analysis

The study was conducted in a completely randomized design, six repetitions were carried out for each treatment and the treatments were arranged in a 3 × 5 factorial design (3 treatments, 5 sampling days). Numbers of bacteria (CFU/g) were transformed into logarithms (log) before statistical analysis. Statistical analyses of the results were conducted using the software GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The results were expressed as mean ± standard error of the mean and are reported in tables. The effects of different treatments during the storage period were appraised by one-factor analysis of variance- ANOVA and Tukey's multiple comparison test (p < 0.05).

Results

Microbiological status of the minced meat

The initial Salmonella spp. count in the inoculated minced meat was 8.8 ± 0.04 log CFU/g (Table 1). The Salmonella spp. count decreased until the day 6 in all groups, with significantly higher (p < 0.05) counts in VP than in MAP1 minced meat. A significant reduction of Salmonella spp. count was found on day 6 (average reduction of 1.9 log CFU/g). From day 9, slightly increasing Salmonella spp. counts were observed in all packaging types, except in packages with 50% CO2 where it decrease again from day 9 to day 12. Number of Salmonella spp. in VP meat was significantly higher (p < 0.05) than in both MAP meat on day 12 (Table 1). Significant difference (p < 0.05) also was noted between two MAP packaging, with lower count in MAP with 50% CO2.

Table 1
Change in Salmonella spp. count, LAB count and TVC (log CFU/g) in packaged minced meat samples during storage at 3 ± 1 ºC (mean ± SEM).

The LAB count in minced meat increased during storage in all packaging types (Table 1). On day 12, the maximum detected LAB count was in VP meat, while the lowest LAB count was in MAP1, i.e. in meat packaged with modified atmosphere with a higher concentration of carbon dioxide. Significant differences (p < 0.05) were detected between the LAB count in VP meat and MAP1, as well as between MAP1 and MAP2.

The maximum detected TVC was in the VP on day 12. The lowest TVC at the end of experiment was recorded in the MAP1, and it was significantly lower (p < 0.05) than it other groups.

Headspace gas

The headspace gas data for the two MAP types are shown in Table 2.

Table 2
Concentrations of CO2, O2 and N2 in headspace gas of packaged minced meat samples during storage at 3 ± 1 ºC (%).

Meat pH

In all meat sampled, the pH was 5.7 at the beginning of the study and then increased during storage (Table 3). A significant difference (p < 0.05) in pH was observed between VP and MAP1 on day 9.

Table 3
Change of pH in packaged minced meat samples during storage at 3 ± 1 ºC (mean ± SEM).

Discussion

The decrease of Salmonella spp. count until day 6 in the MAP packaging in the present study partly can be attributed to the inhibitory action of carbon dioxide, especially in the MAP1 with high CO2 content (50%), which was more effective than VP in the reducing Salmonella count (Table 1). Gram-negative bacteria, including Salmonella are highly sensitive to CO2 because its interact with membrane lipids, causing changes in ion membrane transport, penetrates the membrane and causes cytoplasmic acidification, disordered synthesis of specific enzymes, reduces cell metabolism extended the lag phase of microbial growth and reduced the growth rate during the logarithmic phase.2222 McMillin KW. Where is MAP going? A review and future potential of modified atmosphere packaging for meat. Meat Sci. 2008;80(1):43-65.,2727 Martinez L, Djenane D, Cilla I, Beltran JA, Roncales P. Effect of different concentrations of carbon dioxide and low concentration of carbon monoxide on the shelf-life of fresh pork sausages packaged in modified atmosphere. Meat Sci. 2005;71(3):563-570.

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Carbon dioxide is highly soluble in high moisture and fatty foods such as meat.3030 Cooksey K. Modified atmosphere packaging of meat poultry and fish. In: Han H, Jung, eds. Innovations in Food Packaging. 2nd ed. Elsevier; 2014:475–493. In addition to the level of solubility of carbon dioxide an important factor affecting bacterial growth is storage temperature of the packaged meat. Storage temperatures below 5 °C, as in the current study where meat samples were stored at 3 ± 1 °C, increase the solubility of carbon dioxide, whose antimicrobial activity increases, but also increase the sensitivity of bacterial cells to the effects of carbon dioxide.2727 Martinez L, Djenane D, Cilla I, Beltran JA, Roncales P. Effect of different concentrations of carbon dioxide and low concentration of carbon monoxide on the shelf-life of fresh pork sausages packaged in modified atmosphere. Meat Sci. 2005;71(3):563-570.,3030 Cooksey K. Modified atmosphere packaging of meat poultry and fish. In: Han H, Jung, eds. Innovations in Food Packaging. 2nd ed. Elsevier; 2014:475–493.,3333 Devlieghere F, Debevere J, Van Impe J. Concentration of carbon dioxide in the water-phase as a parameter to model the effect of a modified atmosphere on microorganisms. Int J Food Microbiol. 1998;43:105-113.,3434 Mullan WMA. Science and Technology of Modified Atmosphere Packaging; 2002. http://www.dairyscience.info/index.php/packaging-/117-modified-atmosphere-packaging.html.
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In the present study, the CO2 concentration was 50% (MAP1) and 30% (MAP2) at the beginning of the experiment. Decrease of CO2 during first tree days of storage is attributed to its absorption in meat and was fallowed by its increase until the end of the storage period. Increase of CO2 was caused by bacterial activity. Similar results were reported by Goulas.3535 Goulas AE. 2008 Combined effect of chill storage and modified atmosphere packaging on mussels (Mytilus galloprovincialis) preservation. Packag Technol Sci. 2008;21:247-255. The decrease of O2 concentration observed in both MAP can be attributed to the growth of aerobic bacteria and microbial respiration, which utilize O2 and produce CO2 that contributes to spoilage.

Competitive microbiota, as well as the initial concentration of bacterial cells, has an influence on the growth of Salmonella spp. Although Salmonella spp. are able to grow and compete with other microorganisms,3636 Mackey BM, Kerridge AL. The effect of incubation temperature and inoculum size on growth of Salmonellae in minced beef. Int J Food Microbiol. 1998;6:57-65. these bacteria are described in the literature as a relatively weak competitor.3030 Cooksey K. Modified atmosphere packaging of meat poultry and fish. In: Han H, Jung, eds. Innovations in Food Packaging. 2nd ed. Elsevier; 2014:475–493.,3737 Borch E, Kant-Muermans ML, Blixt Y. Bacterial spoilage of meat and cured meat product. Int J Food Microbiol. 1996;33:103-120.

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39 Blixt Y, Borch E. Comparison of shelf life of vacuum-packed pork and beef. Meat Sci. 2002;60:371-378.
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TVC is a parameter which also determines meat shelf-life. The initial microbial load is one of the most important parameters determining the shelf life of meat.3939 Blixt Y, Borch E. Comparison of shelf life of vacuum-packed pork and beef. Meat Sci. 2002;60:371-378. The biggest proportions of the initial microbiota on fresh meat are mesophilic and psychrotrophic bacteria, and this latter group of bacteria is mainly responsible for meat spoilage. For these reasons TVC is used as an important microbiological quantitative indicator of production process hygiene, and for safety evaluation, as well as a spoilage indicator of raw meat.22 Motjaremi Y, Moy G, Todd E, eds. Encyclopedia of Food Safety. vol. 1. San Diego, CA, USA: Academic Press Elsevier; 2014.,5454 Jay JM. Food preservation with modified atmosphere. In: Modern Food Microbiology. Gaithersburg, MD: Aspen Publisher; 2000:283–300.,5555 Tao F, Peng Y. A non-destructive method for prediction of total viable count in pork meat by hyperspectral scattering imaging. Food Bioprocess Technol. 2015;8(17):30. Based on numerous investigations, a TVC value of 107 CFU/g in meat is considered as a critical value for assessment of spoilage.5656 ICMSF. Microbiological specifications of food pathogens. Salmonellae. In: Microorganisms in Food 5. London: Blackie Academic and Professional; 1996:217–264.

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58 Berruga MI, Vergara H, Linares MB. Control of microbial growth and rancidity in rabbit carcasses by modified atmosphere packaging. J Sci Food Agric. 2005;85:1987-1991.
-5959 Djenane D, Martinez L, Blanco D, Yanguela J, Beltran JA, Roncales P. Effect of lactic acid bacteria on extension of shelf life and growth of Listeria monocytogenes in beef stored in CO2-rich atmosphere. Braz J Microbiol. 2005;36:405-412. The number of microorganisms including bacteria depends on the intrinsic and extrinsic factors including pH, meat surface morphology, O2 availability, temperature and the presence and development of other bacteria.6060 Ercolini D, Russo F, Nasi A, Ferranti P, Villani F. Mesophilic and psychrotrophic bacteria from meat and their spoilage potential in vitro and in beef. Appl Environ Microbiol. 2009;75(7):. Changes in these factors and bacterial competition could influence the changes in the TVC in the present study. At the end of experiment, lower TVC was present in meat samples packaged with modified atmosphere, especially MAP with 50% CO2. Results from the present study showed lower values of TVC in meat packaged in the higher concentration of carbon dioxide, which can be attributed to antibacterial effect of modified atmosphere, especially carbon dioxide are consistent with the results of other authors.2727 Martinez L, Djenane D, Cilla I, Beltran JA, Roncales P. Effect of different concentrations of carbon dioxide and low concentration of carbon monoxide on the shelf-life of fresh pork sausages packaged in modified atmosphere. Meat Sci. 2005;71(3):563-570.,6161 Labadie J. Consequences of packaging on bacterial growth. Meat is an ecological niche. Meat Sci. 1999;52(3):299-305.

Many factors can affect the pH of packaged meat, but it is considered that a major factor responsible for its decline is the LAB population.6262 Fernández-López J, Sendra E, Sayas-Barberá E, Navarro C, Pérez-Alvarez JA. Physico-chemical and microbiological profiles of "salchichón" (Spanish dry-fermented sausage) enriched with orange fiber. Meat Sci. 2008;80:410-417. Microorganisms' growth as well as chemical reactions occurring during proteolytic processes throughout storage cause increases in meat pH (Table 3). These processes create alkali compounds (ammonia, trimethylamine, dimethylamine) responsible for pH increases.6363 Baltić T, PhD Thesis Influence of Marinating on Salmonella spp. Growth in Broiler Meat. Belgrade, Serbia: Faculty of Veterinary Medicine, University of Belgrade; 2014. The increasing pH in all meat packaging regimes can be explained due to the high concentration of bacteria, resulting in production of alkali compounds. Furthermore, the pH increases could also be due to proteolysis, causing the production of free amino acids and leading to the formation of NH3 and amines.6464 Karabagias I, Badeka A, Kontominas MG. Shelf life extension of lamb meat using thyme or oregano essential oils and modified atmosphere packaging. Meat Sci. 2011;88(1):109-116. The results obtained in the present study are consistent with those obtained by Milijašević,6565 Milijašević M, Velebit B, Jovanović J. The effect of different gas mixtures on colour and microbiological compliance of beef packaged in protective atmosphere. In: International 54th meat Industry Conference. 2007:93. Bozec et al.,6666 Bozec A, Zuliani V, Le Roux A, Ellouze M. Shelf-life evaluation of pork meat stored under different packaging atmospheres. In: 57th International Congress of Meat Science and Technology. 2011:1–4. and Cachaldora et al.4545 Cachaldora A, García G, Lorenzo JM, García-Fontán MC. Effect of modified atmosphere and vacuum packaging on some quality characteristics and the shelf-life of "morcilla", a typical cooked blood sausage. Meat Sci. 2013;93:220-225. while in the results obtained by Schirmer and Langsrud4444 Schirmer BC, Langsrud S. A dissolving CO2 headspace combined with organic acids prolongs the shelf-life of fresh pork. Meat Sci. 2010;85:280-284. and Babić et al.,6767 Babić J, Matekalo-Sverak V, Borović B, et al. Uticaj pakovanja u modifikovanoj atmosferi na održivost ćevapčića. Tehnol Mesa. 2012;53(1):36-42. meat pH remained consistent during the storage period.

Conclusions

All types of packaging used in present study decreased the Salmonella spp. count during first days of storage. This pathogen was best inhibited by MAP containing higher CO2 concentration (50%), followed by MAP with 30% CO2 and VP. Furthermore, MAP with a higher CO2 level exhibited greater antibacterial activity against TVC and LAB.

Acknowledgments

This paper was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, through the funding of the project Selected biological hazards to the safety/quality of food of animal origin and the control measures from farm to consumer (No. 31034).

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Publication Dates

  • Publication in this collection
    Jul-Sep 2018

History

  • Received
    8 Nov 2016
  • Accepted
    23 Sept 2017
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
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