Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures

FOOD-06457; No of Pages 6 International Journal of Food Microbiology xxx (2014) xxx–xxx

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures Antonio Valero a,⁎, Marta Hernandez b, Alessandra De Cesare c, Gerardo Manfreda c, Patricia González-García b, David Rodríguez-Lázaro b,d,⁎⁎ a

Department of Food Science and Technology, University of Cordoba, Campus de Rabanales, Edificio Darwin, 14014 Córdoba, Spain Subdirección de Investigación y Tecnología, Instituto Tecnológico Agrario de Castilla y León, Carretera de Burgos Km 119, Valladolid, Spain Department of Agricultural and Food Sciences, Alma Mater Studiorum-University of Bologna, Via del Florio 2, 40064 Ozzano dell'Emilia, BO, Italy d Microbiology Section, Faculty of Science, University of Burgos, Burgos, Spain b c

a r t i c l e

i n f o

Article history: Received 21 January 2014 Received in revised form 18 February 2014 Accepted 19 February 2014 Available online xxxx Keywords: Listeria monocytogenes Survival Raw sheep milk cured cheese Storage Modeling

a b s t r a c t Raw sheep milk cured cheese produced in the Castilla y Leon region (Spain) constitutes a traditional semi-hard aromatic cheese typically aged for three to six months. This product is catalogued as ready-to-eat since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens such as Listeria monocytogenes can represent a health concern for susceptible consumers. This study was aimed at evaluating the survival of L. monocytogenes on raw sheep milk cured cheese under different storage temperatures. Loglinear + shoulder and Weibull type models were fitted to data observed in order to estimate kinetic parameters. The Arrhenius relationship was further used to predict the impact of temperature on L. monocytogenes behavior during storage at 4, 12 and 22 °C. Additionally, growth of lactic acid bacteria (LAB) as a representative group of the indigenous microbiota was evaluated. Results obtained indicated that the time to eradication (time when absence of L. monocytogenes in the analyzed samples was observed) was 114, 104, and 77 days for cheese samples stored at 4, 12 and 22 °C, respectively. The LAB population showed an increase at 12 and 22 °C during storage. However, an increase of 1 log CFU/g was observed during the first 2 weeks irrespectively of the storage temperature. The log-linear + shoulder model indicated a good fit to observed data. Likewise, the Arrhenius relationship explained sufficiently the dependency of temperature on L. monocytogenes behavior. This study demonstrated that cheese storage at ambient temperatures could lead to the preservation of its quality properties as well as its safety against L. monocytogenes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Listeria monocytogenes is a Gram positive bacteria being the causative agent of listeriosis. Listeriosis is usually a severe disease with a mean mortality rate in humans of at least 20% (Chan and Wiedmann, 2009), which primarily causes infections in immunocompromised patients, pregnant women, and those at extremes of age (newborns and elderly) (Rodríguez-Lázaro and Hernández, 2013). L. monocytogenes has been isolated from a wide variety of environmental sources, including food-processing environments and a large variety of foods (Gandhi and Chikidas, 2007; Swaminathan and Gerner-Smidt, 2007). L. monocytogenes can survive and grow over a wide range of environmental conditions such as refrigeration temperatures, low pH and high salt concentration. This allows the pathogen to

⁎ Corresponding author. Tel.: +34 957218516. ⁎⁎ Correspondence to: D. Rodríguez-Lázaro, Microbiology Section, Faculty of Science, University of Burgos, Burgos, Spain. Tel.: +34 637451100. E-mail addresses: [email protected] (A. Valero), [email protected] (D. Rodríguez-Lázaro).

overcome food preservation and safety barriers, and pose a potential risk to human health (Gandhi and Chikidas, 2007). Epidemiologic data indicate that the foods involved in listeriosis outbreaks have a contamination significantly higher than 100 CFU/g (FAO/WHO, 2004). Due to these data, the Commission Regulation (EC) No. 2073/2005 established a maximum level of 100 CFU/g in those ready-to-eat (RTE) foods not supporting growth of the pathogen. For other RTE foods, absence in 25 g should be achieved. However, challenge test studies are recommended when pathogen behavior is not known in a particular RTE food (EC, 2008; Health Canada, 2012; Uyttendaele et al., 2004, 2009). Unlike many other bacterial food borne pathogens, L. monocytogenes can grow in milk at refrigeration temperatures and reach potentially infectious levels in certain high-moisture and surface-ripened cheeses (Bernini et al., 2013; Cogan, 2011). The survival and growth of L. monocytogenes in a dairy environment depends on the manufacturing, ripening and storage conditions used for the cheeses, even when the cheese is stored at refrigeration temperatures (Almeida et al., 2013). In addition, the strain-to-strain variability of survival in different storage conditions observed in some studies can be related to the different genotypic Listeria lineage to which the strains used in the study

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.017 0168-1605/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Valero, A., et al., Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.017

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A. Valero et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

belong (De Jesus and Whiting, 2003; Mataragas et al., 2008). In hard cheeses the bacterial presence could be due to recontamination during packaging, distribution and cheese preparation by the consumer (Schaffner et al., 2003 Raw sheep milk cured cheese produced in Castilla y Leon (Spain) is a traditional semi-hard aromatic cheese typically aged for three to six months. L. monocytogenes can be found in that type of cheese (Arrese and Arroyu-Izaga, 2012). Raw sheep milk cured cheeses are not submitted to any further culinary process in which a substantial reduction of L. monocytogenes contamination can be achieved, and as a result, it represents a health concern particularly for vulnerable consumers. Predictive microbiology is a multidisciplinary area of food microbiology, which is devoted to studying and predicting, by means of mathematical models, the effects of extrinsic factors (e.g. temperature, salt or preservatives) on microbial behavior in foods. Deviations of log linearity have been often used to describe pathogen survival kinetics. These models may reflect an initial “shoulder”, a linear reduction or sometimes the presence of a tail. Models aiming at describing nonlinear survival patterns, such as log-linear + shoulder or Weibull have been successfully applied for L. monocytogenes in various media or food matrices (Angelidis et al., 2010; Guentert et al., 2003; Mataragas et al., 2008; Peleg, 2006). The aim of the present work was to evaluate the fate of L. monocytogenes on raw sheep milk cured cheese under different storage temperatures. Nonlinear survival models were fitted to data observed in order to estimate kinetic parameters and to subsequently predict the impact of temperature on L. monocytogenes behavior during storage. Additionally, growth of lactic acid bacteria (LAB) as a representative group of the indigenous microbiota was evaluated. Based on the obtained results, recommendations to food operators, retailers and consumers were provided. 2. Material and methods 2.1. Cheese samples Raw sheep milk cured cheese was used in this study. Cheese samples were acquired in a local retailer. This type of cheese contains a high concentration of fat (approximately 37%) and more than 24% of proteins. The shelf life of the commercial portions of this type of products ranges between 1 and 3 months. The main ingredients are raw sheep milk, salt, rennet, starter cultures, and some additives: potassium nitrate and lysozyme. Ten 25-g cheese samples were tested to confirm the absence of L. monocytogenes using the ISO 11290-1/Amd 1:2004 detection method (ISO, 1996 and ISO, 2004a). The pH and aw values were measured on three non-inoculated samples every week along the frame time of the study. The aw was measured at 25 °C using an Aqualab water activity meter (Aqualab model Series 4, Decagon Devices, Inc., Pullman, WA), whereas the pH was measured after blending 10 g of cheese in 90 ml of distilled and deionized water using a Crison Basec 20+ pH meter equipped with a pH 0–14 electrode (Crison Instruments, S.A., Barcelona, Spain). 2.2. Bacterial strains and preparation of L. monocytogenes inoculum Three strains of L. monocytogenes were used in this study: LBMM334 and LBMM335 strains isolated from raw sheep milk cured cheeses at ITACyL, Spain and the C5 strain (Fox et al., 2011) isolated from a sheep farm environment in Ireland kindly provided by Dr. Kieran Jordan, TEAGASC, Dairy Products Research Centre, Ireland. The three strains were chosen either to represent the niche product raw sheep milk cured cheese (LBMM334 and LBMM335 strains), or due to previous evidence to survive the stressful environment of cheesemaking better than other strains (C5 strain) (Belessi et al., 2011a,b; Le Marc et al., 2010). All strains were maintained at − 80 °C in cryovial containing beads and cryopreservatives (Oxoid TP15731

Maintenance Freeze medium, Oxoid Ltd, Hampshire, UK). Prior to starting the experiment, a bead of each strain was surface plated onto a Petri dish with Brain Heart Infusion (BHI) agar (Beckton, Dickinson and Co.) and incubated at 37 °C for 24 h. Then, a loopful of one isolated cell was transferred aseptically into 10 ml of BHI Broth (Beckton, Dickinson and Co.) and incubated at 37 °C overnight. To determine the initial concentration of each working cocktail, an aliquot was serially diluted and surface plated onto BHI agar, incubated at 37 °C for 24 h and colonies were counted. Each working cocktail was diluted and mixed to create a final cocktail of the three L. monocytogenes strains (approx. 2.5 × 106 CFU/ml at stationary phase). 2.3. Artificial contamination of cheese samples and storage Cheese was aseptically removed from the commercial bags, cut in slides and transferred in sterile co-extruded polyamide/polyethylene packing bags (Industrias Pargón, Salamanca, Spain). Samples of 25 g of cheese slices were inoculated with 100 μl of PBS containing 104 CFU/g of L. monocytogenes onto 10 different surface areas, allowed to dry, and subsequently vacuum packaged, and stored at three different temperatures (4, 12 and 22 °C). 2.4. Microbiological analyses Microbiological studies on the presence and enumeration of L. monocytogenes at each sampling point were conducted in three independent inoculated cheese samples according to ISO 11290-1/Amd 1:2004 (ISO, 1996 and ISO, 2004a) and ISO 11290-2/Amd 1:2004 (ISO, 1998a and ISO, 2004b), respectively. The quantification limit was 10 CFU/g and the counts from the triplicate samples were expressed as log CFU/g. For each storage condition, the “time to eradication”, i.e. the time in which all of the replicate samples tested negative to the ISO 11290-1/Amd 1:2004 standard method was calculated (Angelidis et al., 2010). Cheese samples in each storage condition were not further analyzed after three successive negative results using the standard detection method. In parallel enumeration of lactic acid bacteria was conducted in each sampling time according to ISO 15214 (ISO, 1998b). 2.5. Data processing and modeling approach 2.5.1. Primary models A plot of log CFU/g versus time (in days) was created for each separated experiment in Microsoft Excel v2010 (Microsoft Corporation). The Baranyi primary model was fitted to observed growth data of lactic acid bacteria in cured sheep cheese (Baranyi and Roberts, 1994) by using the DMFit excel program (Institute of Food Research, Norwich, England). Kinetic growth parameters, lag phase (lag, d) and maximum growth rate (μmax, d− 1) were estimated from the observed data at each condition studied. Mean squared error (MSE) and coefficient of determination (R2) were determined to assess the goodness-of-fit of the model. For the sake of the evaluation of the fate of L. monocytogenes during storage, two different mathematical models were used. i) The “log-linear + shoulder” model (Geeraerd et al., 2000) which takes into account a shoulder phase before inactivation occurs. The model has the following form: −kmax t

N ¼ N0  e






ekmax  Sl  −1  e−kmax t

kmax Sl

1þ e

ð1Þ

where N is the cell concentration (CFU/ml) after a treatment time t (d), N0 is the initial cell concentration (CFU/g), kmax is the maximum inactivation rate (d− 1), and Sl is the shoulder length (d) (i.e., the length of the lag phase). To represent inactivation curves, the first

Please cite this article as: Valero, A., et al., Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.017

A. Valero et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

time point when the L. monocytogenes concentration fell below the LOQ (i.e. 10 CFU/g) was included in the model. ii) The Weibull model was as follows:  p t log10 Nðt Þ ¼ log10 N 0 − δ

f ¼

2.5.2. Secondary modeling To calculate the fate of L. monocytogenes at different storage temperatures, a relationship was performed between the scale parameter of the Weibull model (α) and temperature through the use of the Arrhenius model (Angelidis et al., 2013): Ea ðT ref −T Þ Rg T ref T

−

α T ¼ α T ref  e

ð3Þ

where αT is the a value at temperature T, αTref is the a value at a constant reference temperature, Tref, Ea is the activation energy (J/mol), and Rg is the universal gas constant, 8.314 J/(mol · K). Note that the temperatures T and Tref in Eq. (3) are representing absolute temperatures (i.e., expressed in °K). A reference temperature of 283.15 K (i.e., 10 °C) was employed. By considering as dependent variable the reduction ratio; N(t)/N0, the model allows estimate the potential cell reduction at other storage temperatures. Combining Eqs. (2) and (3) we obtain (Angelidis et al., 2013): 0

1β

N t B C log t ¼ −@ A E ðT −T Þ N0 − aR Tref T g ref α T ref  e

ð4Þ

A nonlinear regression procedure was followed in R v2.15.3 software (R Development Core Team, Vienna, Austria) to determine the kinetic parameters. Finally, a linear relationship was estimated between β parameter and temperature β ¼ −ða  T Þ þ b:

where l(θ) is the log likelihood, k the number of parameters and n the number of experimental data. The F test was used to decide whether the fitting performance of a model was statistically accepted. The value was calculated by the following equation:

ð2Þ

where δ is the first-decimal reduction time (d) and p is a shape parameter. Specifically, the Weibull-type model (Peleg and Cole, 1998; van Boekel, 2002) has been previously used to describe the nonloglinear nature of L. monocytogenes survival curves thanks to its flexibility and simplicity. The freeware add-in GInaFit v1.6 (Geeraerd et al., 2005) was used for the fitting procedure and statistical estimates.

3

MSEmodel MSEdata

ð7Þ

where MSEmodel is the mean square error of the model and MSEdata is the mean square error of the data indicating the measuring error of the data for replicate values. The lower the MSEdata is, the less variation is observed between the replicates for a given experimental condition. MSEdata value is calculated as follows: m X k  X

MSEdata ¼

average log10 Ni − logNij

2

i¼1 j¼1

ð8Þ

n−m

where n is the number of data points, m the number of time points, k is the number of replicates at each time point, average Ni is the mean value of the population at time point i (log CFU/g), and Nij is the population at time point i for specific replicate j (log CFU/g). The value was compared to F table value (95% confidence). If the calculated value is smaller than the F value from the table, the F test is accepted and indicates that the model fitting is statistically acceptable. 3. Results and discussion 3.1. Survival of L. monocytogenes on raw sheep milk cured cheese during storage at 4, 12 and 22 °C Fig. 1 shows the cell reduction of L. monocytogenes in inoculated cheeses at 4, 12 and 22 °C. A nonlinear survival pattern was observed deviating from the traditional linear kinetics. Survival curves were characterized by the presence of a shoulder region followed by a linear decay which was more pronounced at higher temperatures. Estimated kinetic parameters of both log-linear + shoulder and Weibull models are shown in Table 1. Average Sl values estimated by the loglinear + shoulder model ranged from 36.27 to 59.19 days and significant differences were obtained between 4/12 °C and 22 °C (p b 0.05). However, no significant differences were denoted for kmax (p N 0.05). Regarding Weibull model, significant differences were only obtained for δ values (p b 0.05). The adequacy of the primary models fitted was assessed through the F-test. The goodness-of-fit was satisfactory in the case of the Weibull model at the three storage temperatures tested, while the

ð5Þ

5

AIC C ¼ −1lðφÞ þ 2k þ

2kðk þ 1Þ n−k−1

ð6Þ

Log CFU/g

4

2.5.3. Statistical analyses The correlation coefficient (R2) and mean squared error (MSE) were provided by the GInaFit adding. An analysis of variance was used for all other analyses. These analyses were conducted with the Statistica for Windows v10 (Statsoft Iberica, Lisbon). Results were considered significant at p b 0.05. To allow for model comparison, the corrected Akaike Information Criterion (AICC) (Hurvich and Tsai, 1995) was calculated as it takes into account both the goodness of fit and the parsimony. The lower the AICC value the better the model is. AICC value is calculated as follows:

3

2

1

0 0

25

50

75

100

125

Time (d) Fig. 1. Estimation of the Weibull model for the fate of L. monocytogenes (mean log CFU/g ± SD) in cured sheep cheese estimated at 4 (■), 12 (□) and 22 °C ( ). Fitted models are also shown for 4 (⋯), 12 (- - -) and 22 °C (—).

Please cite this article as: Valero, A., et al., Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.017

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A. Valero et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

Table 1 Kinetic parameters ± SD of the log linear shoulder and Weibull models for the fate of L. monocytogenes in raw sheep milk cured cheese at 4, 12 and 22 °C: Sl (shoulder, d), kmax (maximum inactivation rate, d−1), N0 (initial level, log CFU/g), δ and p (Weibull parameters). T (°C)

4 12 22

Log linear shoulder

Weibull

Sl (d)

kmax (d−1)

N0 (log CFU/g)

δ

p

N0 (log CFU/g)

59.19 ± 2.36 50.47 ± 3.60 36.27 ± 3.86

0.16 ± 0.01 0.15 ± 0.01 0.20 ± 0.03

4.08 ± 0.06 3.95 ± 0.09 3.85 ± 0.13

73.07 ± 1.45 65.07 ± 2.36 45.25 ± 3.22

3.30 ± 0.17 3.03 ± 0.26 2.65 ± 0.39

4.15 ± 0.04 4.01 ± 0.07 3.97 ± 0.13

log-linear + shoulder model failed at 22 °C (Table 2). Besides, values of R2, MSEmodel and AICC were lower for the Weibull model thus showing its flexibility to be fitted to different environmental conditions (van Boekel, 2002). Although the Weibull model is of an empirical nature, a link can be made with physiological effects. As p parameter N 1 for the three tested temperatures (Table 1) it indicates that the remaining cells become increasingly damaged. The time to eradication (time when absence of L. monocytogenes in the analyzed samples was observed) was 114, 104, and 77 days for cheese samples stored at 4, 12 and 22 °C, respectively. The L. monocytogenes population remained relatively unchanged (less than 1 log CFU/g reduction) during at least 6 weeks irrespectively of the storage temperature: (0.99, 1 and 0.95 log CFU/g reductions after 42, 54 and 67 days at 22, 12 and 4 °C, respectively) (Fig. 1). After 6 weeks of storage, L. monocytogenes population started to behave differently depending on the temperature of storage. At 22 °C, approximately 1.76 log CFU/g reduction was observed at day 54, and afterwards the decrease of the L. monocytogenes population was faster: 2.45 log CFU/g reduction at day 67, 4.02 log CFU/g reduction at day 70, and only the presence of L. monocytogenes (below the limit of enumeration) was observed at day 74 (Fig. 1). However, the reduction was slower when the cheese samples were stored at 4 and 12 °C. At 12 °C, a 1.74 log CFU/g reduction was observed only after 77 days of storage, 2.90 log CFU/g reduction at day 91, and only the presence of L. monocytogenes was observed at day 98 (Fig. 1). Finally at 4 °C, the L. monocytogenes reduction was even slower: 2.10 log CFU/g reduction was only achieved after 91 days of storage, 3.19 log CFU/g reduction at day 101, and only the presence of L. monocytogenes was observed at day 111 (Fig. 1). Despite the absence of reported outbreaks in hard cheeses due to L. monocytogenes, due diligence is required with regard to potential contamination of cheeses during ripening or after production. These results corroborate what other studies have shown in hard cheeses such as Emmental (Schaffner et al., 2003), Gouda (Wemmenhove et al., 2013), Ricotta salata (Spanu et al., 2012), cheddar (Hystead et al., 2013) or Parmesan cheeses (Yousef and Marth, 1990) where L. monocytogenes was shown to produce decay after ripening and or storage periods. Lowsalt hard cheeses may present a potential risk for Listeria survival over longer time periods. However, Shrestha et al. (2011) did not find significant differences between cheddar cheeses formulated with low salt

(0.7%) or regular salt (1.8%) obtaining more than 1.5 log CFU/g reduction at 4, 10 and 21 °C. Traditional raw sheep milk cured cheeses are stored for long time periods thus Listeria if present, is normally at very low levels. However, although it presents a very low risk, it could be considered as significant especially for people with a deficient or diminished immune system. As a RTE product, the need for good sanitation practices to prevent post-manufacturing cross contamination remains.

3.2. Estimation of L. monocytogenes survival at different storage temperatures The kinetic parameters of the Arrhenius secondary model that illustrate the survival of L. monocytogenes in the studied cheese were calculated using a nonlinear regression procedure. Values of α10°C and Ea were 64.87 ± 2.29 and − 15,207.82 ± 3098.32 respectively for the studied L. monocytogenes strains. Subsequently, a linear regression was followed to estimate the relationship of the β parameter and temperature (°K). The resulting equation was: β = −(0.031 ⋅ T) + 11.82. As shown in previous studies since the Ea is negative the inactivation rate increases proportionally to temperature (Angelidis et al., 2013). This could be attributed to the loss of humidity and LAB growth which are able to produce metabolites that inhibit Listeria growth (Sip et al., 2012). Concentration levels of LAB at the time of the initial decay of L. monocytogenes were around 6.5–7.0 log CFU/g at the three temperatures. On top of that, the psychrotrophic nature of Listeria can also allow it to survive at low temperatures thus competing with the indigenous flora. The effect of all the kinetic parameters can be used to evaluate the effect of variations on storage temperature as shown in Fig. 2. It can be seen that a deviation of +1 °C and +2 °C from the initial storage temperature values can produce considerable variation to the bacterial reduction data, the effect propagating with storage time and being more pronounced at low storage temperatures. Temperature fluctuations from 4 to 6 °C produced an estimated 5 log reduction ranging between 0

-1

T (°C) 4

12

22

R2 MSEmodel AICC f R2 MSEmodel AICC f R2 MSEmodel AICC f

Log linear shoulder

Weibull

0.966 0.0458 50.34 1.453 0.930 0.098 70.24 0.820 0.912 0.140 61.21 2.130

0.985 0.0208 −28.19 0.660 0.965 0.052 1.45 0.435 0.931 0.110 18.57 1.666

-2 log N(t)/N0

Table 2 Statistical indices calculated for the primary inactivation models used to fit L. monocytogenes fate in cured sheep cheese. Bold values indicate that the F test was acceptable.

4ºC -3 12ºC -4

22ºC

-5 0

25

50

75

100

125

Time (d)

Fig. 2. Impact of increase of storage temperature (+1 °C [⋯] and +2 °C [- - -]) on the fate of L. monocytogenes in cured sheep cheese estimated at 4, 12 and 22 °C (—) represents fitted model for tested temperatures.

Please cite this article as: Valero, A., et al., Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.017

A. Valero et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

117 and 122 days. However, the effect was much lower at 22 °C (5 log reduction was estimated between 88 and 90 days). 3.3. LAB behavior on raw sheep milk cured cheese during storage at 4, 12 and 22 °C Lactic acid bacteria curves at the three storage temperatures are shown in Fig. 3. The LAB population showed an increase at 12 and 22 °C during storage. However, an increase of 1 log CFU/g was observed during the first 2 weeks irrespectively of the temperature of storage (increase of 1.05, 1.19 and 0.93 at day 13, 7 and 11when the samples were stored at 22, 12 and 4 °C, respectively) (Fig. 3). At 22 °C, LAB increased 2.28 log CFU after 35 days, and afterwards a gradual increase was observed until the eighth week of storage (2.68 log CFU increase), and subsequently the population reached a plateau (increase of 2.45 and 2 log at 101 and 122 days, respectively) (Fig. 3). At 12 °C, LAB increased 2 log after 39 days, and after that increase was reduced until the end (increase of 2.33 and 2.35 log at 101 and 122 days, respectively) (Fig. 3). At 4 °C, more unstable behavior was observed since, after an initial rapid increase, LAB population tend to be reduced up to the end of the storage period (Fig. 3). Growth results for LAB obtained at 4 °C showed an initial increase of 1 log CFU/g (approx) during the first 20 days, followed by a stationary phase up to 55 days and a decay up to the end of shelf life (122 days). This behavior could be explained by an initial development of psychrotrophic species at 4 °C on the cheese surface until reaching levels of approx. 6.8 log CFU/g. Subsequently, a loss of humidity and nutrient depletion could cause the inhibition of these predominant species thus producing product stabilization. It is shown in other studies that selection of different microbial communities throughout the shelf life can occur in hard cheeses at low temperatures (Dolci et al., 2008; Santarelli et al., 2013). This non-regular trend made it impossible to derive a primary model since results were not sufficiently consistent. Therefore the Baranyi model could not be fitted to these observed data. However, a satisfactory model fitting was obtained at 12 and 22 °C (R2 N 0.80) being the average maximum growth rate (μmax) equal to 0.035 and 0.059 d−1, respectively. No lag phase was determined since LAB growth was instantaneous from the initial inoculum level. 3.4. Physical–chemical characteristics of cured-sheep cheese during storage Two of the main extrinsic factors (pH and aw) affecting microbial growth and survival were analyzed during the storage of cheese samples. Values of pH and aw remained unchanged (5.46 ± 0.04 and 0.9450 ± 0.0006, respectively) without statistically significant differences (p N 0.05) irrespectively of time and temperature of storage.

Log CFU/g

8

7

6

5

4 0

25

50

75

100

125

Time (d) Fig. 3. Growth of lactic acid bacteria (mean log CFU/g ± SD) in cured sheep cheese estimated at 4 (■), 12 (□) and 22 °C ( ) storage. Fitted models are also shown for 12 (- - -) and 22 °C (—). At 4 °C, only observed values are presented since no growth was observed.

5

4. Conclusions In the present study, a more significant reduction in the numbers of L. monocytogenes occurred when the raw sheep milk cured cheese samples were stored at 22 °C. The use of a primary model such as loglinear + shoulder produced a good fit to observed survival data over storage time. Temperature dependency was appropriately described through the Arrhenius relationship. It can be concluded that storage at room temperature produced an accelerated decay rate of L. monocytogenes mainly due to loss of humidity and LAB growth. This fact leads to a negligible public health risk to consumers when the pathogen is present at low levels (b 100 CFU/g). However, according to the physical–chemical characteristics of pH and aw, this food could support L. monocytogenes survival and/or growth at certain storage conditions and whatever post-processing contamination or cross contamination during storage could represent the main transmission routes of L. monocytogenes to susceptible consumers when ingesting contaminated cheese cuts. It was demonstrated that storage at ambient temperatures can favor the maintenance of the quality as well as safety properties of this traditional product.

Acknowledgments This work was supported by the EU BASELINE project. DRL, PGG and MH acknowledge the support by the Project RTA2011-079-C02-01 of the Ministry of Economy and Competitiveness, Government of Spain.

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Please cite this article as: Valero, A., et al., Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.017