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Behaviour of Listeria monocytogenes and Staphylococcus aureus in sliced, vacuum-packaged raw milk cheese stored at two different temperatures and time periods
International Dairy Journal 57 (2016) 15e19
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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
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Behaviour of Listeria monocytogenes and Staphylococcus aureus in sliced, vacuum-packaged raw milk cheese stored at two different temperatures and time periods Alberto Bellio*, Sara Astegiano, Amaranta Traversa, Daniela Manila Bianchi, Silvia Gallina, Nicoletta Vitale, Fabio Zuccon, Lucia Decastelli Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta e IZSPLV, Via Bologna 148, 10154 Turin, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2015 Received in revised form 2 February 2016 Accepted 3 February 2016 Available online 12 February 2016
The behaviour of Listeria monocytogenes and Staphylococcus aureus in raw milk cheese slices packaged under vacuum was evaluated. Artificially contaminated 80-day ripened cheese was portioned, vacuum packaged, and then stored for 28 days at 4 C and for 56 days at 10 C. Bacterial counts were obtained before vacuum packaging and then weekly during storage. At the end of ripening, the initial L. monocytogenes count was 4.46 ± 0.89 log cfu g1; weekly bacterial counts remained substantially unchanged in the samples stored at 4 C but decreased to 3.54 ± 1.54 log cfu g1 in those stored at 10 C. The initial S. aureus count before vacuum packaging was 3.60 ± 0.78 log cfu g1; it then gradually decreased to 2.60 ± 1.32 log cfu g1 in the samples stored at 4 C and to about 1.9 log cfu g1 in those stored at 10 C. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Listeria monocytogenes is a common foodborne pathogen and a cause of severe illness. Older adults, pregnant women, neonates, and immune-compromised people are at higher risk for L. monocytogenes infection (listeriosis) (Kagkli, Iliopoulos, Stergiou, Lazaridou, & Nychas, 2009). Between 2012 and 2013, listeriosis incidence increased by 8.6%, with 1763 confirmed human cases reported in the European Union (EU). A total of 191 deaths were reported (case-fatality rate 15.6%) (EFSA, 2015). The ability of a food matrix to support, or not, the growth or survival of L. monocytogenes depends on the pH, water activity, and composition of the specific product and may vary even within the same food category (EFSA, 2013). L. monocytogenes is frequently detected in processed seafood and meat, ready-to-eat (RTE) food, and dairy products, among the last of which soft and semisoft cheeses have been identified as food vehicles in listeriosis outbreaks (Rosshaug, Detmer, Ingmer, & Larsen, 2012). Recognising this risk to consumer health, EU legislation, specifically Regulation 2073/2005/EC and later amendments (1441/2007/EC) (European Commission, 2005, 2007), set the L. monocytogenes limit of
* Corresponding author. Tel.: þ39 11 2686233. E-mail address: [email protected] (A. Bellio). http://dx.doi.org/10.1016/j.idairyj.2016.02.003 0958-6946/© 2016 Elsevier Ltd. All rights reserved.
100 colony-forming units (cfu) g1 during the shelf life of RTE foods. Among single samples of soft and semi-soft cheeses and other dairy products collected at retail during monitoring in 2012, the non-compliance rates were 0.3% and <0.1%, respectively. Staphylococcus aureus is one of the most pathogenic Grampositive aerobic organisms. Some strains elaborate toxins that cause gastroenteritis that typically manifests with nausea and vomiting, typically followed by abdominal cramps and diarrhoea. Staphylococcal food poisoning is caused by ingesting heat-stable enterotoxins produced by coagulase-positive staphylococci (CPS). S. aureus enterotoxins are a major causative agent of staphylococcal food poisoning (Le Loir, Baron, & Gautier, 2003). In 2013, 386 foodborne outbreaks caused by staphylococcal toxins were reported, accounting for 7.4% of all outbreaks reported in the EU, an increase compared with 2012. Ninety four (24.3%) of these were strong-evidence outbreaks; no case fatalities occurred. The largest proportion of strong-evidence outbreaks caused by staphylococcal toxins was attributed to the consumption of mixed food (19.1%), whereas cheese was implicated in 6.4% (EFSA, 2015). Dairy products are a recognised vehicle for staphylococcal enterotoxin food poisoning. A S. aureus count of approximately >5 log cfu g1 is necessary for enterotoxin production (Pelisser, Klein, Ascoli, Zotti, & Arisi, 2009). S. aureus contamination can occur in raw milk cheeses when made from raw milk obtained from cows suffering from mastitis. All cheeses can be contaminated by
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staphylococcal carriage in food handlers and substandard hygiene practices in general. For these reasons, a limit of <5 log cfu g1 CPS in cheese has been set for raw milk cheese. Because pasteurisation can virtually eliminate pathogens such as S. aureus and L. monocytogenes, the presence of these pathogenic microorganisms and their toxins is less likely to be a food safety issue with pasteurised milk cheese (Lancette & Bennett, 2001). More likely, the occurrence of L. monocytogenes in pasteurised milk is a result of postpasteurisation contamination or cross-contamination during cheese production due to the presence of the pathogen in the dairy plant (Samelis, Giannou, & Lianou, 2009), whereas human or animal contamination is implicated in the case of S. aureus. Whatever the origin of contamination, the increase or decrease in bacterial growth needs to be monitored during the shelf life of the cheese (Rosshaug et al., 2012). Cheese is a complex dynamic matrix in which several microbial, physical, and biochemical changes occur during maturation and storage. Vacuum packaging of cheese on retail premises before or at the moment of sale has become increasingly popular as a method of extending the product's shelf life. Therefore, the aim of this study was to evaluate the dynamic behaviour of L. monocytogenes and S. aureus under conditions simulating contamination during the processing and storage of sliced raw milk cheese packaged under vacuum, and then stored at two different temperatures and time periods (4 C for 28 days and 10 C for 56 days). 2. Materials and methods 2.1. Bacterial strains Two L. monocytogenes and two S. aureus strains were used separately. L. monocytogenes strains (IZSPLV 66163/3-2010, serotype 1/2b; IZSPLV 49640/1-2011, serotype 1/2a) and one S. aureus strain (IZSPLV 42875-2012) were isolated from 60 80-day ripened artisanal cheeses; the other S. aureus strain [American Type Culture Collection (ATCC) 33862] was obtained from a reference collection. Wild strains were confirmed using an API Listeria identification kit rieux, Marcy and an API Staph identification kit (both from: bioMe l'Etoile, France), respectively. Strains are available as frozen stock collection at 20 C on Cryobank™ (Copan Diagnostics, Murrieta, CA, USA).
prepared at different time periods (3 independent batches 2 foodborne pathogens 2 different strains ¼ 12 batches in total). The milk was homogenised, warmed to 37 C, and 1 mg 100 L1 of powdered rennet was added. After curdling, the curd was cut into pieces 0.5 cm in diameter and heated to 48 C for 1 h. The curd was transferred to moulds (50 cm by 7 cm) and mechanically pressed for 8 h to drain. The cheese was salted by immersion in 22% (w/v) brine at room temperature for 12 h, then transferred to a climatecontrolled ripening room (temperature 10 C, relative humidity 90%), where it was ripened for 80 days. The ripened cheese was sliced into portions (250 g each), vacuum packaged, and then stored under temperature controlled conditions at 4 C for 28 days and at 10 C for 56 days. 2.4. Sampling and analysis Before artificial contamination of the raw milk, L. monocytogenes and S. aureus cells were enumerated according to ISO 112902:1998/Amd1 2004 (ISO, 2004a) and ISO 6888-2:1999/Amd1:2003 (ISO, 2003). Samples were collected from each batch at days 0, 7, 14, 21, 28 (4 C condition) and at days 0, 7, 14, 21, 28, 35, 42, 49, 56 (10 C condition) after vacuum packaging. Ten g of each sample were added to 90 mL buffered peptone water (BPW) (Microbiol) and homogenised in a stomacher (Stomacher 400, PBI, Milan, Italy) for 1 min at 260 strokes min1. Analyses were performed on each batch. Mesophilic and thermophilic lactococci were counted using the pour plate technique on M17 agar plates (Microbiol) incubated at 30 C and 45 C for 48 h, respectively. Thermophilic lactobacilli were enumerated using the pour plate technique on de Man, Rogosa and Sharpe (MRS) agar plates (Microbiol) and incubated under anaerobic conditions at 45 C for 72 h. Enterococci were counted on kanamycin aesculin azide (KAA) agar plates (Microbiol) at 45 C for 48 h. L. monocytogenes and S. aureus were enumerated in triplicate on ALOA (Biolife, Milan, Italy) and Baird Parker agar added with rabbit plasma fibrinogen (RPF) (Liofilchem, Roseto degli Abruzzi, Italy) incubated at 37 C for 48 h, respectively; L. monocytogenes and CPS were enumerated according to ISO (2004a) and ISO (2003), respectively. Water activity (Aw) and pH were measured according to ISO 21807:2004 (ISO, 2004b) and MFHPB-03:2003 (MFHPB, 2003), respectively. 2.5. Statistical analysis
2.2. Preparation of inocula Each L. monocytogenes and S. aureus strain was grown on Agar Listeria according to Ottaviani Agosti (ALOA; Liofilchem, Roseto degli Abruzzi, Italy) and Columbia agar (Microbiol, Cagliari, Italy), respectively, incubated at 37 C for 24 h. A no. 3 McFarland standard (8.95 log cfu mL1) suspension of each strain was prepared using an rieux). Bacterial suspensions, 1 mL (L. ATB1550 densiometer (bioMe monocytogenes) and 100 mL (S. aureus), were gradually added to raw milk under stirring to achieve uniform distribution of the inoculum and a final concentration of approximately 3e4 log cfu mL1 and 5e6 log cfu mL1 respectively.
Data converted to log cfu are expressed as mean ± standard deviation. The effect of storage time (7e14e21e28 days), temperature (4 C, 10 C), batch (1, 2, 3) and strain (IZSPLV 66163/3-2010, IZSPLV 49640/1-2011 for L. monocytogenes and ATCC 33862, IZSPLV 42875-2012 for S. aureus) on microbial behaviour was evaluated using analysis of variance (ANOVA) factorial design. Data were analysed using a generalised linear model (GLM) procedure on SAS (version 9.2; SAS Institute, Cary, NC). Tukey's test was used to determine the significance differences among mean values at an a ¼ 0.05 over all comparisons. 3. Results
2.3. Cheese production 3.1. Behaviour of Listeria monocytogenes and LAB flora Each batch was prepared from 90 L of raw milk. The milk was delivered at the experimental dairy plant at 35 C and put into a stainless steel container. The batches were artificially contaminated with an inoculum of L. monocytogenes or S. aureus; 1.5 g 100 L1 of a freeze-dried starter culture containing a lactic acid bacteria (LAB) mixture of different species (Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii ssp. lactis) was then added. For each strain of the two pathogens, three different batches were
All raw milk samples tested negative for L. monocytogenes. At the end of 80-day ripening (pH 5.4 ± 0.2; Aw 0.92 ± 0.01), the L. monocytogenes count was 4.46 ± 0.89 log cfu g1 and the LAB concentration was high (8.28 ± 0.47 log cfu g1 mesophilic lactococci; 8.45 ± 0.37 log cfu g1 thermophilic lactococci; 8.05 ± 0.43 log cfu g1 thermophilic lactobacilli). The indigenous enterococci population was 6.08 ± 0.64 log cfu g1. The L.
A. Bellio et al. / International Dairy Journal 57 (2016) 15e19
monocytogenes count remained fairly stable (4.30 ± 0.89 log cfu g1) during storage for 28 days in the samples kept at 4 C, whereas in those refrigerated at 10 C it decreased slowly during the first 28 days (4.08 ± 1.42 log cfu g1), then rapidly during the fifth week (3.70 ± 1.35 log cfu g1), after which it remained stable (3.54 ± 1.54 log cfu g1) through to the end of the storage period (Fig. 1). Mesophilic and thermophilic lactococci counts were high during storage at 4 C and 10 C. Thermophilic lactobacilli counts remained stable (7.82 ± 0.60 log cfu g1) in the samples stored at 4 C, but decreased slightly from 8.05 ± 0.43 log cfu g1 to 7.52 ± 0.53 log cfu g1 in those stored at 10 C. The enterococci counts remained stable during storage at both temperature conditions. No differences in Aw or pH in the samples stored at 4 C were observed, whereas the pH decreased slightly from 5.4 ± 0.2 to pH 5.2 ± 0.1 in the samples stored at 10 C at the end of either storage period. 3.2. Behaviour of Staphylococcus aureus and LAB flora The raw milk delivered at the experimental dairy plant contained 1.50 ± 0.70 log cfu mL1 CPS. Before vacuum packaging, the S. aureus count was 3.60 ± 0.78 log cfu g1. The LAB population was composed of mesophilic lactococci (8.44 ± 0.39 log cfu g1), thermophilic lactococci (8.45 ± 0.37 log cfu g1), and thermophilic lactobacilli (8.06 ± 0.52 log cfu g1). The indigenous enterococci population was 6.70 ± 0.29 log cfu g1. The S. aureus count decreased to 2.60 ± 1.32 log cfu g1 after 28 days in the samples stored at 4 C and it decreased by about 1.5 log cfu g1 during the first 28 days in the samples stored at 10 C, before further decreasing to about 0.4 log cfu g1 during the last 28 days (Fig. 2). The mesophilic and thermophilic lactococci counts remained fairly stable at approximately >8.0 log cfu g1 in the samples stored at 4 C and at 10 C. At the end of the storage periods, the thermophilic lactobacilli count was 7.75 ± 0.42 log cfu g1 (4 C) and 7.42 ± 0.35 log cfu g1 (10 C). The enterococci counts, pH, and Aw remained substantially unchanged during the storage periods. 3.3. Statistical analysis L. monocytogenes survival was related to storage time (test F: 3.49; p ¼ 0.01) and temperature (test F: 12.1, p ¼ 0.001). The final model is shown in Table 1. Because L. monocytogenes behaviour was
17
related to batch (test F 216.6, p ¼ 0.0001), the mean estimated parameters were corrected for batch effect. The interaction between storage time and batch was statistically significant (test F ¼ 2.75, p ¼ 0.0003). Between day 0 (4.46 ± 0.89 log cfu g1) and day 21 (4.20 ± 0.10 log cfu g1), the L. monocytogenes count decreased in all batches, particularly in batch number 3 (3.1 ± 0.1 log cfu g1). A different effect of storage temperature on L. monocytogenes survival was observed between the samples kept at 4 C (4.30 ± 0.89 log cfu g1) and at 10 C (4.08 ± 1.42 log cfu g1) after 28 days, with a greater reduction in microbial count in the samples refrigerated at 10 C (Fig. 1). The samples stored at 10 C were monitored weekly until day 56. The L. monocytogenes count was lower at day 56 than at day 28 (3.54 ± 1.54 log cfu g1 versus 4.08 ± 1.42 log cfu g1). Similarly, S. aureus survival was also related to storage time (test F: 45.8; p ¼ 0.0001) and temperature (test F: 16.7, p ¼ 0.0001). The final model is shown in Table 2. Because S. aureus behaviour was related to batch (test F 152.2, p ¼ 0.0001), the mean estimated parameters were corrected for batch effect. The interaction between storage time and batch was statistically significant (test F ¼ 2.66, p ¼ 0.0005). An analogous difference in the effect of storage temperature on S. aureus survival between the samples stored at 4 C (2.60 ± 1.32 log cfu g1) and those kept at 10 C (2.09 ± 1.07 log cfu g1) after 28 days was seen in the greater reduction in those stored at 10 C (Fig. 2). The S. aureus count was 1.75 ± 0.90 log cfu g1 in the samples stored at 10 C at day 56. 4. Discussion Our study findings provide further data on L. monocytogenes and S. aureus behaviour under process simulation of contamination in sliced, raw milk cheese vacuum packaged and stored at 4 C and 10 C for 28 and 56 days, respectively. The Aw (90.92e0.93) and pH (5.2e5.5) of raw milk cheese allow S. aureus and L. monocytogenes to survive and grow under both aerobic and anaerobic conditions. The limits for S. aureus and L. monocytogenes pathogen growth are Aw 0.90 and 0.83, respectively, and pH 4.0 for both bacteria (Gallina et al., 2013; Lado & Yousef, 2007; Lungu, Ricke, & Johnson, 2009). While vacuum environment and modified atmosphere can inhibit the growth of spoilage microorganisms in foods and extend product shelf-life (Stanbridge & Davies, 1998), they can also allow the growth and/or survival of microaerophilic psychrotrofic pathogens,
Fig. 1. Changes in lactic acid bacteria, enterococci and L. monocytogenes mean counts in slices stored at 10 C: -, L. monocytogenes; , mesophilic lactococci; △, thermophilic lactococci; ,, thermophilic lactobacilli; :, enterococci.
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Fig. 2. Changes in lactic acid bacteria, enterococci and S. aureus mean counts in slices stored at 10 C: -, L. monocytogenes; , mesophilic lactococci; △, thermophilic lactococci; ,, thermophilic lactobacilli; :, enterococci.
including L. monocytogenes in particular, in various different foods n ~ ez, 1995). (García de Fernando, Nychas, Peck, & Ordo At the end of 80-day ripening, L. monocytogenes count was 4.46 ± 0.89 log cfu g1 and remained fairly stable for 28 d in the samples stored at ideal refrigeration conditions (4 C), whereas it decreased to 4.08 ± 1.42 log cfu g1 in the samples kept at 10 C, a temperature similar to that found in most home refrigerators. During vacuum storage, the microorganism did not multiply and it decreased slower at 4 C than at 10 C. The high concentration of L. monocytogenes used in this study was very unusual in real conditions. However it was necessary to contaminate with very high concentration to deeply study the behaviour of the pathogens. Previous studies on the effect of a higher storage temperature on dairy product shelf life (Genigeorgis, Carniciu, Dutulescu, & Farver Genigeorgis, 1991; Ryser & Marth, 1987), and on storage under vacuum conditions specifically (Giannou, Kakouri, Bogovic Matijasic, Rogelj, & Samelis, 2009), indicated that the lower the storage temperature, the higher the bacterial count and the longer the survival of L. monocytogenes. In contrast, other studies reported a decrease and/or no growth of L. monocytogenes in cheeses stored at refrigerated conditions (Finazzi et al., 2011). Furthermore, pathogen survival during storage can also vary among different L. monocytogenes strains (Kagkli et al., 2009). Some studies have highlighted the importance of competitive LAB flora on the growth or lack of growth of L. monocytogenes and demonstrated that Listeria survival is both strain-dependent and related to LAB starter culture (Gnanou Besse et al., 2006; Mellefont, McMeekin, & Ross, 2008). In the present study, the LAB flora count remained high (>8 log cfu g1) during storage at both temperatures, especially in the samples kept at 10 C. L. monocytogenes metabolism increased with higher temperature, probably leading to its earlier inactivation due to autolysis. Moreover, the greater competition from LAB
populations in cheese stored at higher temperatures may inhibit L. monocytogenes growth and/or survival (Kagkli et al., 2009; Shrestha, Grieder, McMahon, & Nummer, 2011). Both pH and Aw remained stable during storage at 4 C, whereas pH decreased slightly in the samples stored at 10 C. This may be another reason for the major reduction in L. monocytogenes in the samples stored at 10 C, as noted elsewhere (Murdock, Cleveland, Matthews, & Chikindas, 2007; Paramithiotis, Kagkli, Blana, Nychas, & Drosinos, 2008). Before vacuum packaging, the initial S. aureus count was 3.60 ± 0.78 log cfu g1; it decreased slightly to 2.60 ± 1.32 log cfu g1 in the samples stored at 4 C for 28 days. It decreased by about 1.5 log cfu g1 in the samples stored at 10 C during the first 28 days and then by about 0.4 log cfu g1 during the last 28 days. S. aureus did not grow during vacuum storage and the bacterial count decreased more quickly at 10 C than at 4 C. This difference could be explained by the fact that, similarly to L. monocytogenes, its metabolism increased with higher temperature (Shrestha et al., 2011), and that the LAB population may inhibit this microorganism (Gilliland & Speck, 1972). The L. monocytogenes count decreased slightly less than the S. aureus count due to differences in bacterial adaptability and resistance to the environment. Our experimental data show that the factors are related to bacterial behaviour: batch number, storage time and temperature resulted statistically significant for both L. monocytogenes and S. aureus. The highest variability in both pathogens was observed between batches. These results were expected and underline the importance of preparing at least three different batches for experimental challenge testing, as recommended by the French Agency for Food Safety ([AFSSA] Agence francaise de curite sanitaire des produits de sante ). se
Table 1 Output of generalised linear model for L. monocytogenes.a
Table 2 Output of generalised linear model for S. aureus.a
Factors
DF
SS type III
Root mean square
F value
Pr > F
Factors
DF
SS type III
Root mean square
F value
Pr > F
Batch Days Temperature Days*Batch
5 4 1 20
135.8657905 1.7494409 1.5184062 6.9062288
27.1731581 0.4373602 1.5184062 0.3453114
216.57 3.49 12.10 2.75
<0.0001 0.0094 0.0007 0.0003
Batch Days Temperature Days*Batch
1 5 4 19
2.9969072 136.6304396 32.9138210 9.0879313
2.9969072 27.3260879 8.2284552 0.4783122
16.70 152.24 45.84 2.66
<0.0001 <0.0001 <0.0001 0.0005
a
Abbreviations are: DF, degrees of freedom; SS, sum of square; Pr, probability.
a
Abbreviations are: DF, degrees of freedom; SS, sum of square; Pr, probability.
A. Bellio et al. / International Dairy Journal 57 (2016) 15e19
5. Conclusions The present study shows that storage temperature and packaging conditions may influence the survival of L. monocytogenes and S. aureus. Vacuum packaging may inhibit bacterial growth; indeed, neither pathogen grew under these conditions at ideal refrigeration (4 C) or at moderate temperature (10 C). However, if pathogens, especially L. monocytogenes, are present in ripened and aged cheese, they can survive during storage. In addition, because lower storage temperatures and vacuum conditions probably limit LAB flora activity, the decrease in L. monocytogenes and S. aureus counts is less at 4 C than at 10 C. Further investigations should be conducted with lower concentration of pathogens in order to evaluate the behaviour of L. monocytogenes in real conditions. References EFSA. (2013). Scientific report of EFSA and ECDC: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA Journal, 11, 3129. EFSA. (2015). Scientific report of EFSA and ECDC: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSA Journal, 13, 3991. European Commission. (2005). Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, 338, 1e26. European Commission. (2007). Regulation (EC) No 1441/2007 of 5 December 2007 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, 322, 12e29. Finazzi, G., Daminelli, P., Serraino, A., Pizzamiglio, V., Riu, R., Giacometti, F., et al. (2011). Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese. Letters in Applied Microbiology, 53, 364e370. Gallina, S., Bianchi, D. M., Bellio, A., Nogarol, C., Macori, G., Zaccaria, T., et al. (2013). Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping. Journal of Food Protection, 76, 2093e2096. n ~ ez, J. A. (1995). Growth/ García de Fernando, G. D., Nychas, G. J., Peck, M. W., & Ordo survival of psychrotrophic pathogens on meat packaged under modified atmospheres. International Journal of Food Microbiology, 28, 221e231. Genigeorgis, C., Carniciu, M., Dutulescu, D., & Farver, T. B. (1991). Growth and survival of Listeria monocytogenes in market cheeses stored at 4 to 30 C. Journal of Food Protection, 54, 662e668. Giannou, E., Kakouri, A., Bogovic Matijasic, B., Rogelj, I., & Samelis, J. (2009). Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25 C in air or vacuum packages: in situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition. Journal of Food Protection, 72, 531e538. Gilliland, S. E., & Speck, M. L. (1972). Interactions of food starter cultures and foodborne pathogens: lactic streptococci versus staphylococci and salmonellae. Journal of Milk and Food Technology, 35, 307e310. Gnanou Besse, N., Audinet, N., Barre, L., Cauquil, A., Cornu, M., & Colin, P. (2006). Effect of the inoculum size on Listeria monocytogenes growth in structured media. International Journal of Food Microbiology, 110, 43e51. ISO. (2003). ISO 6888-2:1999/Amd 1:2003. Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci
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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
Behaviour of Listeria monocytogenes and Staphylococcus aureus in sliced, vacuum-packaged raw milk cheese stored at two different temperatures and time periods Alberto Bellio*, Sara Astegiano, Amaranta Traversa, Daniela Manila Bianchi, Silvia Gallina, Nicoletta Vitale, Fabio Zuccon, Lucia Decastelli Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta e IZSPLV, Via Bologna 148, 10154 Turin, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2015 Received in revised form 2 February 2016 Accepted 3 February 2016 Available online 12 February 2016
The behaviour of Listeria monocytogenes and Staphylococcus aureus in raw milk cheese slices packaged under vacuum was evaluated. Artificially contaminated 80-day ripened cheese was portioned, vacuum packaged, and then stored for 28 days at 4 C and for 56 days at 10 C. Bacterial counts were obtained before vacuum packaging and then weekly during storage. At the end of ripening, the initial L. monocytogenes count was 4.46 ± 0.89 log cfu g1; weekly bacterial counts remained substantially unchanged in the samples stored at 4 C but decreased to 3.54 ± 1.54 log cfu g1 in those stored at 10 C. The initial S. aureus count before vacuum packaging was 3.60 ± 0.78 log cfu g1; it then gradually decreased to 2.60 ± 1.32 log cfu g1 in the samples stored at 4 C and to about 1.9 log cfu g1 in those stored at 10 C. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Listeria monocytogenes is a common foodborne pathogen and a cause of severe illness. Older adults, pregnant women, neonates, and immune-compromised people are at higher risk for L. monocytogenes infection (listeriosis) (Kagkli, Iliopoulos, Stergiou, Lazaridou, & Nychas, 2009). Between 2012 and 2013, listeriosis incidence increased by 8.6%, with 1763 confirmed human cases reported in the European Union (EU). A total of 191 deaths were reported (case-fatality rate 15.6%) (EFSA, 2015). The ability of a food matrix to support, or not, the growth or survival of L. monocytogenes depends on the pH, water activity, and composition of the specific product and may vary even within the same food category (EFSA, 2013). L. monocytogenes is frequently detected in processed seafood and meat, ready-to-eat (RTE) food, and dairy products, among the last of which soft and semisoft cheeses have been identified as food vehicles in listeriosis outbreaks (Rosshaug, Detmer, Ingmer, & Larsen, 2012). Recognising this risk to consumer health, EU legislation, specifically Regulation 2073/2005/EC and later amendments (1441/2007/EC) (European Commission, 2005, 2007), set the L. monocytogenes limit of
* Corresponding author. Tel.: þ39 11 2686233. E-mail address: [email protected] (A. Bellio). http://dx.doi.org/10.1016/j.idairyj.2016.02.003 0958-6946/© 2016 Elsevier Ltd. All rights reserved.
100 colony-forming units (cfu) g1 during the shelf life of RTE foods. Among single samples of soft and semi-soft cheeses and other dairy products collected at retail during monitoring in 2012, the non-compliance rates were 0.3% and <0.1%, respectively. Staphylococcus aureus is one of the most pathogenic Grampositive aerobic organisms. Some strains elaborate toxins that cause gastroenteritis that typically manifests with nausea and vomiting, typically followed by abdominal cramps and diarrhoea. Staphylococcal food poisoning is caused by ingesting heat-stable enterotoxins produced by coagulase-positive staphylococci (CPS). S. aureus enterotoxins are a major causative agent of staphylococcal food poisoning (Le Loir, Baron, & Gautier, 2003). In 2013, 386 foodborne outbreaks caused by staphylococcal toxins were reported, accounting for 7.4% of all outbreaks reported in the EU, an increase compared with 2012. Ninety four (24.3%) of these were strong-evidence outbreaks; no case fatalities occurred. The largest proportion of strong-evidence outbreaks caused by staphylococcal toxins was attributed to the consumption of mixed food (19.1%), whereas cheese was implicated in 6.4% (EFSA, 2015). Dairy products are a recognised vehicle for staphylococcal enterotoxin food poisoning. A S. aureus count of approximately >5 log cfu g1 is necessary for enterotoxin production (Pelisser, Klein, Ascoli, Zotti, & Arisi, 2009). S. aureus contamination can occur in raw milk cheeses when made from raw milk obtained from cows suffering from mastitis. All cheeses can be contaminated by
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staphylococcal carriage in food handlers and substandard hygiene practices in general. For these reasons, a limit of <5 log cfu g1 CPS in cheese has been set for raw milk cheese. Because pasteurisation can virtually eliminate pathogens such as S. aureus and L. monocytogenes, the presence of these pathogenic microorganisms and their toxins is less likely to be a food safety issue with pasteurised milk cheese (Lancette & Bennett, 2001). More likely, the occurrence of L. monocytogenes in pasteurised milk is a result of postpasteurisation contamination or cross-contamination during cheese production due to the presence of the pathogen in the dairy plant (Samelis, Giannou, & Lianou, 2009), whereas human or animal contamination is implicated in the case of S. aureus. Whatever the origin of contamination, the increase or decrease in bacterial growth needs to be monitored during the shelf life of the cheese (Rosshaug et al., 2012). Cheese is a complex dynamic matrix in which several microbial, physical, and biochemical changes occur during maturation and storage. Vacuum packaging of cheese on retail premises before or at the moment of sale has become increasingly popular as a method of extending the product's shelf life. Therefore, the aim of this study was to evaluate the dynamic behaviour of L. monocytogenes and S. aureus under conditions simulating contamination during the processing and storage of sliced raw milk cheese packaged under vacuum, and then stored at two different temperatures and time periods (4 C for 28 days and 10 C for 56 days). 2. Materials and methods 2.1. Bacterial strains Two L. monocytogenes and two S. aureus strains were used separately. L. monocytogenes strains (IZSPLV 66163/3-2010, serotype 1/2b; IZSPLV 49640/1-2011, serotype 1/2a) and one S. aureus strain (IZSPLV 42875-2012) were isolated from 60 80-day ripened artisanal cheeses; the other S. aureus strain [American Type Culture Collection (ATCC) 33862] was obtained from a reference collection. Wild strains were confirmed using an API Listeria identification kit rieux, Marcy and an API Staph identification kit (both from: bioMe l'Etoile, France), respectively. Strains are available as frozen stock collection at 20 C on Cryobank™ (Copan Diagnostics, Murrieta, CA, USA).
prepared at different time periods (3 independent batches 2 foodborne pathogens 2 different strains ¼ 12 batches in total). The milk was homogenised, warmed to 37 C, and 1 mg 100 L1 of powdered rennet was added. After curdling, the curd was cut into pieces 0.5 cm in diameter and heated to 48 C for 1 h. The curd was transferred to moulds (50 cm by 7 cm) and mechanically pressed for 8 h to drain. The cheese was salted by immersion in 22% (w/v) brine at room temperature for 12 h, then transferred to a climatecontrolled ripening room (temperature 10 C, relative humidity 90%), where it was ripened for 80 days. The ripened cheese was sliced into portions (250 g each), vacuum packaged, and then stored under temperature controlled conditions at 4 C for 28 days and at 10 C for 56 days. 2.4. Sampling and analysis Before artificial contamination of the raw milk, L. monocytogenes and S. aureus cells were enumerated according to ISO 112902:1998/Amd1 2004 (ISO, 2004a) and ISO 6888-2:1999/Amd1:2003 (ISO, 2003). Samples were collected from each batch at days 0, 7, 14, 21, 28 (4 C condition) and at days 0, 7, 14, 21, 28, 35, 42, 49, 56 (10 C condition) after vacuum packaging. Ten g of each sample were added to 90 mL buffered peptone water (BPW) (Microbiol) and homogenised in a stomacher (Stomacher 400, PBI, Milan, Italy) for 1 min at 260 strokes min1. Analyses were performed on each batch. Mesophilic and thermophilic lactococci were counted using the pour plate technique on M17 agar plates (Microbiol) incubated at 30 C and 45 C for 48 h, respectively. Thermophilic lactobacilli were enumerated using the pour plate technique on de Man, Rogosa and Sharpe (MRS) agar plates (Microbiol) and incubated under anaerobic conditions at 45 C for 72 h. Enterococci were counted on kanamycin aesculin azide (KAA) agar plates (Microbiol) at 45 C for 48 h. L. monocytogenes and S. aureus were enumerated in triplicate on ALOA (Biolife, Milan, Italy) and Baird Parker agar added with rabbit plasma fibrinogen (RPF) (Liofilchem, Roseto degli Abruzzi, Italy) incubated at 37 C for 48 h, respectively; L. monocytogenes and CPS were enumerated according to ISO (2004a) and ISO (2003), respectively. Water activity (Aw) and pH were measured according to ISO 21807:2004 (ISO, 2004b) and MFHPB-03:2003 (MFHPB, 2003), respectively. 2.5. Statistical analysis
2.2. Preparation of inocula Each L. monocytogenes and S. aureus strain was grown on Agar Listeria according to Ottaviani Agosti (ALOA; Liofilchem, Roseto degli Abruzzi, Italy) and Columbia agar (Microbiol, Cagliari, Italy), respectively, incubated at 37 C for 24 h. A no. 3 McFarland standard (8.95 log cfu mL1) suspension of each strain was prepared using an rieux). Bacterial suspensions, 1 mL (L. ATB1550 densiometer (bioMe monocytogenes) and 100 mL (S. aureus), were gradually added to raw milk under stirring to achieve uniform distribution of the inoculum and a final concentration of approximately 3e4 log cfu mL1 and 5e6 log cfu mL1 respectively.
Data converted to log cfu are expressed as mean ± standard deviation. The effect of storage time (7e14e21e28 days), temperature (4 C, 10 C), batch (1, 2, 3) and strain (IZSPLV 66163/3-2010, IZSPLV 49640/1-2011 for L. monocytogenes and ATCC 33862, IZSPLV 42875-2012 for S. aureus) on microbial behaviour was evaluated using analysis of variance (ANOVA) factorial design. Data were analysed using a generalised linear model (GLM) procedure on SAS (version 9.2; SAS Institute, Cary, NC). Tukey's test was used to determine the significance differences among mean values at an a ¼ 0.05 over all comparisons. 3. Results
2.3. Cheese production 3.1. Behaviour of Listeria monocytogenes and LAB flora Each batch was prepared from 90 L of raw milk. The milk was delivered at the experimental dairy plant at 35 C and put into a stainless steel container. The batches were artificially contaminated with an inoculum of L. monocytogenes or S. aureus; 1.5 g 100 L1 of a freeze-dried starter culture containing a lactic acid bacteria (LAB) mixture of different species (Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii ssp. lactis) was then added. For each strain of the two pathogens, three different batches were
All raw milk samples tested negative for L. monocytogenes. At the end of 80-day ripening (pH 5.4 ± 0.2; Aw 0.92 ± 0.01), the L. monocytogenes count was 4.46 ± 0.89 log cfu g1 and the LAB concentration was high (8.28 ± 0.47 log cfu g1 mesophilic lactococci; 8.45 ± 0.37 log cfu g1 thermophilic lactococci; 8.05 ± 0.43 log cfu g1 thermophilic lactobacilli). The indigenous enterococci population was 6.08 ± 0.64 log cfu g1. The L.
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monocytogenes count remained fairly stable (4.30 ± 0.89 log cfu g1) during storage for 28 days in the samples kept at 4 C, whereas in those refrigerated at 10 C it decreased slowly during the first 28 days (4.08 ± 1.42 log cfu g1), then rapidly during the fifth week (3.70 ± 1.35 log cfu g1), after which it remained stable (3.54 ± 1.54 log cfu g1) through to the end of the storage period (Fig. 1). Mesophilic and thermophilic lactococci counts were high during storage at 4 C and 10 C. Thermophilic lactobacilli counts remained stable (7.82 ± 0.60 log cfu g1) in the samples stored at 4 C, but decreased slightly from 8.05 ± 0.43 log cfu g1 to 7.52 ± 0.53 log cfu g1 in those stored at 10 C. The enterococci counts remained stable during storage at both temperature conditions. No differences in Aw or pH in the samples stored at 4 C were observed, whereas the pH decreased slightly from 5.4 ± 0.2 to pH 5.2 ± 0.1 in the samples stored at 10 C at the end of either storage period. 3.2. Behaviour of Staphylococcus aureus and LAB flora The raw milk delivered at the experimental dairy plant contained 1.50 ± 0.70 log cfu mL1 CPS. Before vacuum packaging, the S. aureus count was 3.60 ± 0.78 log cfu g1. The LAB population was composed of mesophilic lactococci (8.44 ± 0.39 log cfu g1), thermophilic lactococci (8.45 ± 0.37 log cfu g1), and thermophilic lactobacilli (8.06 ± 0.52 log cfu g1). The indigenous enterococci population was 6.70 ± 0.29 log cfu g1. The S. aureus count decreased to 2.60 ± 1.32 log cfu g1 after 28 days in the samples stored at 4 C and it decreased by about 1.5 log cfu g1 during the first 28 days in the samples stored at 10 C, before further decreasing to about 0.4 log cfu g1 during the last 28 days (Fig. 2). The mesophilic and thermophilic lactococci counts remained fairly stable at approximately >8.0 log cfu g1 in the samples stored at 4 C and at 10 C. At the end of the storage periods, the thermophilic lactobacilli count was 7.75 ± 0.42 log cfu g1 (4 C) and 7.42 ± 0.35 log cfu g1 (10 C). The enterococci counts, pH, and Aw remained substantially unchanged during the storage periods. 3.3. Statistical analysis L. monocytogenes survival was related to storage time (test F: 3.49; p ¼ 0.01) and temperature (test F: 12.1, p ¼ 0.001). The final model is shown in Table 1. Because L. monocytogenes behaviour was
17
related to batch (test F 216.6, p ¼ 0.0001), the mean estimated parameters were corrected for batch effect. The interaction between storage time and batch was statistically significant (test F ¼ 2.75, p ¼ 0.0003). Between day 0 (4.46 ± 0.89 log cfu g1) and day 21 (4.20 ± 0.10 log cfu g1), the L. monocytogenes count decreased in all batches, particularly in batch number 3 (3.1 ± 0.1 log cfu g1). A different effect of storage temperature on L. monocytogenes survival was observed between the samples kept at 4 C (4.30 ± 0.89 log cfu g1) and at 10 C (4.08 ± 1.42 log cfu g1) after 28 days, with a greater reduction in microbial count in the samples refrigerated at 10 C (Fig. 1). The samples stored at 10 C were monitored weekly until day 56. The L. monocytogenes count was lower at day 56 than at day 28 (3.54 ± 1.54 log cfu g1 versus 4.08 ± 1.42 log cfu g1). Similarly, S. aureus survival was also related to storage time (test F: 45.8; p ¼ 0.0001) and temperature (test F: 16.7, p ¼ 0.0001). The final model is shown in Table 2. Because S. aureus behaviour was related to batch (test F 152.2, p ¼ 0.0001), the mean estimated parameters were corrected for batch effect. The interaction between storage time and batch was statistically significant (test F ¼ 2.66, p ¼ 0.0005). An analogous difference in the effect of storage temperature on S. aureus survival between the samples stored at 4 C (2.60 ± 1.32 log cfu g1) and those kept at 10 C (2.09 ± 1.07 log cfu g1) after 28 days was seen in the greater reduction in those stored at 10 C (Fig. 2). The S. aureus count was 1.75 ± 0.90 log cfu g1 in the samples stored at 10 C at day 56. 4. Discussion Our study findings provide further data on L. monocytogenes and S. aureus behaviour under process simulation of contamination in sliced, raw milk cheese vacuum packaged and stored at 4 C and 10 C for 28 and 56 days, respectively. The Aw (90.92e0.93) and pH (5.2e5.5) of raw milk cheese allow S. aureus and L. monocytogenes to survive and grow under both aerobic and anaerobic conditions. The limits for S. aureus and L. monocytogenes pathogen growth are Aw 0.90 and 0.83, respectively, and pH 4.0 for both bacteria (Gallina et al., 2013; Lado & Yousef, 2007; Lungu, Ricke, & Johnson, 2009). While vacuum environment and modified atmosphere can inhibit the growth of spoilage microorganisms in foods and extend product shelf-life (Stanbridge & Davies, 1998), they can also allow the growth and/or survival of microaerophilic psychrotrofic pathogens,
Fig. 1. Changes in lactic acid bacteria, enterococci and L. monocytogenes mean counts in slices stored at 10 C: -, L. monocytogenes; , mesophilic lactococci; △, thermophilic lactococci; ,, thermophilic lactobacilli; :, enterococci.
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Fig. 2. Changes in lactic acid bacteria, enterococci and S. aureus mean counts in slices stored at 10 C: -, L. monocytogenes; , mesophilic lactococci; △, thermophilic lactococci; ,, thermophilic lactobacilli; :, enterococci.
including L. monocytogenes in particular, in various different foods n ~ ez, 1995). (García de Fernando, Nychas, Peck, & Ordo At the end of 80-day ripening, L. monocytogenes count was 4.46 ± 0.89 log cfu g1 and remained fairly stable for 28 d in the samples stored at ideal refrigeration conditions (4 C), whereas it decreased to 4.08 ± 1.42 log cfu g1 in the samples kept at 10 C, a temperature similar to that found in most home refrigerators. During vacuum storage, the microorganism did not multiply and it decreased slower at 4 C than at 10 C. The high concentration of L. monocytogenes used in this study was very unusual in real conditions. However it was necessary to contaminate with very high concentration to deeply study the behaviour of the pathogens. Previous studies on the effect of a higher storage temperature on dairy product shelf life (Genigeorgis, Carniciu, Dutulescu, & Farver Genigeorgis, 1991; Ryser & Marth, 1987), and on storage under vacuum conditions specifically (Giannou, Kakouri, Bogovic Matijasic, Rogelj, & Samelis, 2009), indicated that the lower the storage temperature, the higher the bacterial count and the longer the survival of L. monocytogenes. In contrast, other studies reported a decrease and/or no growth of L. monocytogenes in cheeses stored at refrigerated conditions (Finazzi et al., 2011). Furthermore, pathogen survival during storage can also vary among different L. monocytogenes strains (Kagkli et al., 2009). Some studies have highlighted the importance of competitive LAB flora on the growth or lack of growth of L. monocytogenes and demonstrated that Listeria survival is both strain-dependent and related to LAB starter culture (Gnanou Besse et al., 2006; Mellefont, McMeekin, & Ross, 2008). In the present study, the LAB flora count remained high (>8 log cfu g1) during storage at both temperatures, especially in the samples kept at 10 C. L. monocytogenes metabolism increased with higher temperature, probably leading to its earlier inactivation due to autolysis. Moreover, the greater competition from LAB
populations in cheese stored at higher temperatures may inhibit L. monocytogenes growth and/or survival (Kagkli et al., 2009; Shrestha, Grieder, McMahon, & Nummer, 2011). Both pH and Aw remained stable during storage at 4 C, whereas pH decreased slightly in the samples stored at 10 C. This may be another reason for the major reduction in L. monocytogenes in the samples stored at 10 C, as noted elsewhere (Murdock, Cleveland, Matthews, & Chikindas, 2007; Paramithiotis, Kagkli, Blana, Nychas, & Drosinos, 2008). Before vacuum packaging, the initial S. aureus count was 3.60 ± 0.78 log cfu g1; it decreased slightly to 2.60 ± 1.32 log cfu g1 in the samples stored at 4 C for 28 days. It decreased by about 1.5 log cfu g1 in the samples stored at 10 C during the first 28 days and then by about 0.4 log cfu g1 during the last 28 days. S. aureus did not grow during vacuum storage and the bacterial count decreased more quickly at 10 C than at 4 C. This difference could be explained by the fact that, similarly to L. monocytogenes, its metabolism increased with higher temperature (Shrestha et al., 2011), and that the LAB population may inhibit this microorganism (Gilliland & Speck, 1972). The L. monocytogenes count decreased slightly less than the S. aureus count due to differences in bacterial adaptability and resistance to the environment. Our experimental data show that the factors are related to bacterial behaviour: batch number, storage time and temperature resulted statistically significant for both L. monocytogenes and S. aureus. The highest variability in both pathogens was observed between batches. These results were expected and underline the importance of preparing at least three different batches for experimental challenge testing, as recommended by the French Agency for Food Safety ([AFSSA] Agence francaise de curite sanitaire des produits de sante ). se
Table 1 Output of generalised linear model for L. monocytogenes.a
Table 2 Output of generalised linear model for S. aureus.a
Factors
DF
SS type III
Root mean square
F value
Pr > F
Factors
DF
SS type III
Root mean square
F value
Pr > F
Batch Days Temperature Days*Batch
5 4 1 20
135.8657905 1.7494409 1.5184062 6.9062288
27.1731581 0.4373602 1.5184062 0.3453114
216.57 3.49 12.10 2.75
<0.0001 0.0094 0.0007 0.0003
Batch Days Temperature Days*Batch
1 5 4 19
2.9969072 136.6304396 32.9138210 9.0879313
2.9969072 27.3260879 8.2284552 0.4783122
16.70 152.24 45.84 2.66
<0.0001 <0.0001 <0.0001 0.0005
a
Abbreviations are: DF, degrees of freedom; SS, sum of square; Pr, probability.
a
Abbreviations are: DF, degrees of freedom; SS, sum of square; Pr, probability.
A. Bellio et al. / International Dairy Journal 57 (2016) 15e19
5. Conclusions The present study shows that storage temperature and packaging conditions may influence the survival of L. monocytogenes and S. aureus. Vacuum packaging may inhibit bacterial growth; indeed, neither pathogen grew under these conditions at ideal refrigeration (4 C) or at moderate temperature (10 C). However, if pathogens, especially L. monocytogenes, are present in ripened and aged cheese, they can survive during storage. In addition, because lower storage temperatures and vacuum conditions probably limit LAB flora activity, the decrease in L. monocytogenes and S. aureus counts is less at 4 C than at 10 C. Further investigations should be conducted with lower concentration of pathogens in order to evaluate the behaviour of L. monocytogenes in real conditions. References EFSA. (2013). Scientific report of EFSA and ECDC: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA Journal, 11, 3129. EFSA. (2015). Scientific report of EFSA and ECDC: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSA Journal, 13, 3991. European Commission. (2005). Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, 338, 1e26. European Commission. (2007). Regulation (EC) No 1441/2007 of 5 December 2007 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, 322, 12e29. Finazzi, G., Daminelli, P., Serraino, A., Pizzamiglio, V., Riu, R., Giacometti, F., et al. (2011). Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese. Letters in Applied Microbiology, 53, 364e370. Gallina, S., Bianchi, D. M., Bellio, A., Nogarol, C., Macori, G., Zaccaria, T., et al. (2013). Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping. Journal of Food Protection, 76, 2093e2096. n ~ ez, J. A. (1995). Growth/ García de Fernando, G. D., Nychas, G. J., Peck, M. W., & Ordo survival of psychrotrophic pathogens on meat packaged under modified atmospheres. International Journal of Food Microbiology, 28, 221e231. Genigeorgis, C., Carniciu, M., Dutulescu, D., & Farver, T. B. (1991). Growth and survival of Listeria monocytogenes in market cheeses stored at 4 to 30 C. Journal of Food Protection, 54, 662e668. Giannou, E., Kakouri, A., Bogovic Matijasic, B., Rogelj, I., & Samelis, J. (2009). Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25 C in air or vacuum packages: in situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition. Journal of Food Protection, 72, 531e538. Gilliland, S. E., & Speck, M. L. (1972). Interactions of food starter cultures and foodborne pathogens: lactic streptococci versus staphylococci and salmonellae. Journal of Milk and Food Technology, 35, 307e310. Gnanou Besse, N., Audinet, N., Barre, L., Cauquil, A., Cornu, M., & Colin, P. (2006). Effect of the inoculum size on Listeria monocytogenes growth in structured media. International Journal of Food Microbiology, 110, 43e51. ISO. (2003). ISO 6888-2:1999/Amd 1:2003. Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci
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