FOOD-06481; No of Pages 4 International Journal of Food Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg Džiuginta Jakočiūnė a, Magne Bisgaard a, Gaëlle Hervé b,c, Jocelyne Protais b,c, John Elmerdahl Olsen a,⁎, Marianne Chemaly b,c a b c
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark Anses — French Agency for Food, Environmental and Occupational Health and Safety, HQPAP — Unit of Hygiene and Quality of Poultry and Pork Products, BP 53, 22440 Ploufragan, France UEB — European University of Brittany, 5 Boulevard Laënnec, 35000 Rennes, France
a r t i c l e
i n f o
Available online xxxx Keywords: Salmonella Pasteurized whole eggs Prediction NaCl Survival Growth
a b s t r a c t This study investigated the influence of three parameters (time, temperature and NaCl concentration) on survival and four parameters (temperature, NaCl and lysozyme concentrations and pH) on growth of Salmonella enterica serovar Enteritidis (S. Enteritidis) in pasteurized whole egg (PWE). Doehlert uniform shell design was employed to choose conditions for trials and data was fitted to polynomial models and were presented as estimated response surfaces. A model for prediction of reduction of S. Enteritidis in PWE within temperatures between 50 and 58 °C, NaCl concentrations of 0–12%, and heating times between 30 and 210 s and a model for prediction of growth rate of S. Enteritidis in PWE in the temperature range of 1–25 °C, NaCl concentration of 0–12%, pH between 5 and 9, and lysozyme concentrations of 107–1007 U/mg proteins were developed. The maximum reduction condition was 58 °C, 0% of NaCl at a fixed heating time of 120 s, while maximum growth rate was estimated at 25 °C and 0% of NaCl. pH and lysozyme concentration were shown not to influence growth performance significantly in the range of values studied. Results inform industry of the optimal pasteurization and storage parameters for liquid whole egg. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Salmonella enterica serovar Enteritidis (S. Enteritidis) is an important food borne pathogenic bacterium. Most outbreaks caused by this bacterium have been associated to eggs and egg products (Anon., 2013). Pasteurization of liquid egg products is widely applied in the industry to reduce this risk, however several Salmonella outbreaks associated with consumption of pasteurized egg products indicate that Salmonella may still be present in the final product (HPA, 2007, 2012). Hurdle effects might be used to prevent regrowth of Salmonella in food products (Leistner, 2000). Addition of NaCl is often used as a preservative in food, however, already several decades ago Garibaldi et al. (1969) observed that addition of both salt and sucrose to egg yolk increased thermal resistance of Salmonella. Likewise, a number of studies have concluded that heat tolerance of Salmonella increases when aw decreases both in broth or buffer systems (Goepfert et al., 1970), and in liquid egg products (Garibaldi et al., 1969; Ng et al., 1979; Palumbo et al., 1995). This has however been contradicted in studies by Mattick et al. (2001) who observed the opposite effect when the temperature was lower than 65 °C, and Aljarallah and Adams (2007) reported a switch from protective to sensitizing effect of salt at even lower temperatures ⁎ Corresponding author. Tel.: +45 353 32784. E-mail address: [email protected] (J.E. Olsen).
(53 °C–55 °C). There is therefore a need to revisit this problem, if the Salmonella risk from liquid egg products shall be reduced by the use of hurdle effects. Predictive models have been used to estimate the influence of multiple parameters on growth of Salmonella in growth media (Park et al., 2007; Thayer et al., 1987), but so far nobody have used such multiple parameter models to describe Salmonella growth in liquid egg. In the current investigation we used predictive modeling to determine individual and combined effects of three environmental conditions: time, temperature and NaCl concentration on survival of S. Enteritidis in pasteurized whole egg (PWE) and to investigate growth of S. Enteritidis as a function of four parameters: NaCl and lysozyme concentrations, temperature and pH in the same product. 2. Materials and methods 2.1. Bacterial strains, media and egg products S. Enteritidis strain E2187 (Olsen et al., 1994) was used for survival experiments. It was plated on Xylose-Lysine-Deoxycholate Agar (XLD, Oxoid, Basingstoke, United Kingdom) and incubated overnight at 37 °C and colonies were harvested in physiological saline (0.9% NaCl, Merck, Darmstadt, Germany) to an OD-value corresponding to a concentration of approximately 109 CFU/ml. PWE (Danæg, Roskilde,
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015 0168-1605/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
2
D. Jakočiūnė et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx
Denmark) was spiked to a final concentration of approximately 107 CFU/ml. Actual concentrations were determined by plating appropriate decimal dilutions in saline on Luria-Bertani (LB) agar (Becton, Dickinson and Company, Sparks, MD, USA) plates. S. Enteritidis strain MJG01 (previously isolated from egg product) was used for growth experiments. It was plated on a Plate Count Agar (PCA, AES, Bruz, France) and incubated at 37 °C for 24 h. One colony was then transferred to a Brain Heart Infusion broth (AES, Bruz, France) and incubated at 37 °C for 24 h. This stock solution was used to inoculate the PWE (ADRO Ouest, France) after appropriate dilutions in a Tryptone Salt (Biomérieux, Craponne, France) broth to reach the final concentration as shown below in the description of the growth experiment. PWE used for both survival and growth experiments was prepared by industry by pasteurization to ensure sterility.
Table 2 Range of parameters for growth experiments according to Doehlert shell design. Factors
No. of levels
Values
pH Temperature (°C) Lysozyme (U/mg proteins) NaCl (%, w/w)
3 5 7 7
5–7–9 1–7–13–19–25 107–257–407–557–707–857–1007 0–2–4–6–8–10–12
Before the experiments the defined pH values were obtained by adding acid (HCl) or base (NaOH) at a suitable titer in order to avoid introducing an excessive dilution factor into the PWE. Solid NaCl was added at the defined concentrations after a moderate heat treatment and ionization to ensure the sterility before adding it to the PWE. Numbers of bacteria were determined by CFU counts on PCA.
2.2. Doehlert uniform shell design 2.5. Statistical analysis Both growth and survival experiments were carried out according to a Doehlert experimental design (Doehlert, 1970). For survival experiments, two experimental matrices 1 and 2 were designed with low (50, 52, 54 °C) and high (54, 56, 58 °C) temperatures, respectively (Table 1). Each experimental matrix contained 13 trials with variation in three variables (NaCl concentration (%, w/w), time (s) and temperature (°C)). Three repeats were carried out of experiment with conditions at the center to ensure reproducibility (total number of trials 16). Results were presented as log10 reduction of Salmonella in PWE. For growth experiments 21 trials were performed with variation in four variables (pH, temperature (°C), lysozyme concentration (U/mg proteins) and NaCl concentration (%, w/w)) (Table 2). To ensure reproducibility, two repeats of experiment were carried out with conditions at the center (total number of trials 23). For each trial a growth curve was determined based on CFU and the growth rate was calculated according to the primary model of Rosso (1995) based on 10 to 15 enumerations for each growth curve.
For survival experiments Grubb's test of outliers with P b 0.05 (http://graphpad.com/quickcalcs/Grubbs1.cfm) identified one CFU count out of four in three samples as outliers. These were eliminated before further analysis. Data from matrices 1 and 2 were merged and used to build one model for survival experiment. Statistical analysis of data was performed using Statgraphics© software. Results explained the individual effect of each parameter and their interactions. Polynomial models were built for prediction of S. Enteritidis behavior using the same software. Moreover, the results were illustrated as estimated response surfaces. Lack-of-fit and R2 were used to determine whether the selected model is adequate to describe the observed data, or whether a more complicated model should be used. Lack-of-fit test was performed by comparing the variability of the current model residuals to the variability between observations at replicate settings of the factors. 3. Results and discussion
2.3. Survival experiments
3.1. Effects of environmental conditions on survival of Salmonella in PWE
Solid NaCl was added at the defined concentrations to PWE maximum 24 h before the experiment and samples of 90 μl of salted PWE in 1.5 ml Eppendorf tubes were pre-heated to different temperatures (+/−1 °C) listed in Table 1 with an Eppendorf thermomixer with shaking at 1000 rpm. Temperatures were measured with a penetration thermometer Ebro TDC 200. 10 μl of spiked PWE without NaCl was added to reach final concentrations of NaCl and a final concentration of approximately 106 CFU/ml. Samples were incubated for different times as indicated in Table 1 followed by placing samples on ice and addition of 900 μl ice cold saline to stop the heating. Further decimal dilutions were prepared in saline and plated on LB agar plates. Two independent experiments were performed for each matrix with two replicates for each trial. Numbers of bacteria were converted to log10 CFU/ml and subtracted from initial concentration to obtain reductions.
Three environmental conditions (NaCl concentration (%, w/w), time (s) and temperature (°C)) were investigated in relation to survival of S. Enteritidis in PWE. Data presented in Table 3 show the estimated individual and combined effects of all parameters on S. Enteritidis log10 reduction and their significance in both matrices. Time and temperature had significant positive effect on log10 reduction, while increasing NaCl concentration protected against killing. Fig. 1 shows estimated response surface of log10 reduction as a function of temperature and NaCl concentration with time fixed at the central value of 120 s. In both matrices the effect of NaCl on heat killing of S. Enteritidis was protective. This corresponds well with results of Garibaldi et al. (1969) and a protective effect of NaCl was also observed by others (Ng et al., 1979; Palumbo et al., 1995), however the challenge temperatures in their Table 3 Estimated effects of factors individually and their interactions for reduction of S. Enteritidis in PWE in the temperature range of 50° to 58 °C.
2.4. Growth experiments PWE samples were inoculated with 103 CFU/ml of S. Enteritidis, where after growth kinetics within the given range of temperature, pH, salt and lysozyme concentrations (Table 2) were determined.
Table 1 Range of parameters for survival experiments according to Doehlert uniform shell design. Factors
No. of levels
Values
Temperature (°C)
3
NaCl (%, w/w) Time (s)
5 7
50–52–54 (matrix 1) 54–56–58 (matrix 2) 0–3–6–9–12 30–60–90–120–150–180–210
Factors
Estimated effects on log10 reduction
SD
P valuea
A: NaCl conc. B: Time C: Temperature AA AB AC BB BC CC Lack-of-fit R2
−1.14 2.095 2.56636 2.28772 −0.779082 −0.106122 0.12839 0.947347 0.587489
0.165366 0.143211 0.141024 0.273981 0.316945 0.298819 0.205485 0.258784 0.252847
0.0005 0.0000 0.0000 0.0002 0.0492 0.7346 0.5551 0.0106 0.0592 0.0742 0.921892
a
Significant values are indicated in bold.
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
D. Jakočiūnė et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx
Estimated Response Surface Time=120,0
Table 4 Estimated effects of factors individually and their interactions for growth rate of S. Enteritidis in PWE.
Log10 reduction
5 4 3 2 1 0 0
2
4
6
Salt
8
10
12
50
52
54
56
3
58
Temperature
Fig. 1. Estimated surface response of log10 reduction of S. Enteritidis in PWE as a function of NaCl concentration and temperature with a fixed value of time (120 s).
experiments were higher than in this study (over 60 °C). Blackburn et al. (1997) also showed a protective effect, however the experiments were performed using growth media and the model was related to publish data on Salmonella growth in egg products. Based on their results, a maximal protective concentration of 5–7% of salt within the range of 54.5–64.5 °C was predicted (Blackburn et al., 1997). Importantly, results obtained in our study are contradictory to those of Aljarallah and Adams (2007), who showed that D-values increased for Salmonella at 55 °C and 60 °C at low aw values in broth media whereas at 48 °C, 50 °C and 53 °C the D-values were lower in low aw media. They concluded that heat must have different targets in the cell at different temperatures. At low heating temperatures (below 53 °C–55 °C) the cytoplasmic membrane is injured and the additional osmotic stress due to low aw leads to cell death. At higher temperatures ribosomes are targeted and then NaCl has a protective effect (Aljarallah and Adams, 2007). In our study with PWE, NaCl had a protective effect both at low and high temperatures. The reason for this is currently unknown and further studies are indicated to investigate this. The results of the current study shows that data obtained from experiments with growth media cannot be used to conclude on similar situations in PWE. A significant negative combined effect of NaCl concentration and time and significant positive combined effect of time and temperature were observed (Table 3). Lack-of-fit was not significant and R2 was very high (Table 3) meaning that the model gave a good prediction of the experimental data. The equation of the model derived from the survival data was as follows:
Factors
Estimated effect on growth rate
SD
P valuea
A: Temperature B: NaCl conc. C: Lysozyme conc. D: pH AA AB AC AD BB BC BD CC CD DD Lack-of-fit R2
0.24 −0.25 0.009 −0.024 0.18 −0.48 −0.068 0.029 0.19 −0.06 −0.013 0.032 −0.06 0.0030
0.012 0.01 0.009 0.009 0.025 0.027 0.028 0.029 0.018 0.023 0.024 0.015 0.022 0.013
0.0025 0.0016 0.4388 0.1212 0.0184 0.0031 0.1364 0.4155 0.0077 0.1244 0.6396 0.1648 0.1137 0.8411 0.0512 0.94
a
Significant values are indicated in bold.
significantly affected the growth rate of S. Enteritidis, i.e. growth declined with decreasing temperature and increasing NaCl concentration. An interaction between these two parameters was observed, i.e. combined they constituted a hurdle effect (Fig. 2). These results are similar to results of previous investigation of S. Enteritidis growth in culture medium, showing that high storage temperature (19 °C), low NaCl concentration and neutral pH were optimal for Salmonella growth (Thayer et al., 1987). Studies performed with egg products have also shown inhibition of growth by decreased temperature (Sakha and Fujikawa, 2013; Singh et al., 2011) and increased NaCl concentration (McQuestin et al., 2010; Musgrove et al., 2009). Park et al. (2007) showed a decreased growth rate of Salmonella in broth culture outside neutral pH, and this was confirmed for high pH (pH = 9) valued during growth in egg white (Kang et al., 2006). However, this effect of pH was not evident in PWE, probably reflecting differences between the egg products used for experiments. We also saw no influence of lysozyme concentration, which confirms observation by Kang et al. (2006) that the antimicrobial activity of egg white, with lysozyme as an important active compound, is not active at the low and ambient temperature investigated in the present investigation. A model was fitted to the growth curves in order to be able to predict the behavior of S. Enteritidis in PWE. An insignificant lack-of-fit and high R2 values (Table 4) indicated that the model reflected the experimental data well.
log10 reduction ¼ 45:2031−0:270334 Salt−0:056986 Time
∧
−1:8066 Temperature þ 0:0317739 Salt 2 −0:000721372 Salt Time−0:00221088 Salt ∧ ∗ Temperature þ 0:00000792531 Time 2 þ 0:00131576 ∧ ∗ Time Temperature þ 0:018359 Temperature 2: 0,74 0,54
µ
This equation can be used to estimate log10 reduction of Salmonella for different concentrations of NaCl, time and temperatures in the model range. Based on results from both matrices we concluded that best reduction was obtained using maximal temperature and the lowest NaCl concentration. Special attention should be drawn to the fact that NaCl had protective effect to heat tolerance also in the low temperature range.
Lysozyme=557.0; pH=7
0,34 0,14 -0,06 0
3.2. Effects of environmental conditions on growth of S. Enteritidis in PWE The second part of this study was to investigate influence of four environmental conditions on growth of S. Enteritidis in PWE. Results presented in Table 4 show that temperature and NaCl concentration
5
10
15
Temperature
20
25
0
2
4
6
8
10
12
Salt
Fig. 2. Estimated surface response of growth rate of S. Enteritidis in PWE as a function of NaCl concentration and temperature with fixed values of lysozyme (557.0 U/mg protein) and pH (7.0).
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
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D. Jakočiūnė et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx
The following equation describes the growth rate model: μ ¼ −0:134604 þ 0:0134802 Temperature−0:00367957 Salt þ 0:000295239 Lysozyme þ 0:00288486 pH þ 0:000592593 ∧
∗ Temperature 2−0:00321528 Temperature Salt−0:00000608025 ∗ Temperature Lysozyme þ 0:000587963 Temperature pH ∧ þ 0:00277083 Salt 2−0:000011142 Salt Lysozyme−0:000546296 ∧ ∗ Salt pH þ 7:8738E–8 Lysozyme 2−0:0000328601 Lysozyme ∧
∗ pH þ 0:000382716 pH 2:
Based on this equation, S. Enteritidis growth rate in PWE with different concentrations of NaCl and lysozyme, time and temperatures in the model range can be predicted. In conclusion the current study has produced two models for prediction of log10 reduction and growth of S. Enteritidis in PWE in ranges of NaCl concentration, heating time and temperature with regard to survival and NaCl concentration, pH, lysozyme concentrations and temperatures with regard to growth. NaCl concentration had negative effect on reduction of S. Enteritidis in PWE while time and temperature had positive effects and temperature, NaCl concentration and an interaction between these two parameters significantly affected the growth rate for S. Enteritidis in PWE. The results presented can be used by industry to choose optimal pasteurization and storage parameters for liquid whole egg products. Acknowledgments This study was supported by the European Commission through the project BASELINE, grant agreement 222738, and the areas of Brittany and Pays de la Loire in France through the federation of PAO and Valorial. Pasteurized whole egg products were kindly provided by Danæg and by egg product producers in France (ADRO Ouest). References Aljarallah, K.M., Adams, M.R., 2007. Mechanisms of heat inactivation in Salmonella serotype Typhimurium as affected by low water activity at different temperatures. J. Appl. Microbiol. 102, 153–160. Anon., 2013. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA J. 11 (4), 250. Blackburn, C.W., Curtis, L.M., Humpheson, L., Billon, C., McClure, P.J., 1997. Development of thermal inactivation models for Salmonella enteritidis and Escherichia coli O157:
H7 with temperature, pH and NaCl as controlling factors. Int. J. Food Microbiol. 38, 31–44. Doehlert, D.H., 1970. Uniform shell designs. Appl. Stat. 19, 231–239. Garibaldi, J.A., Straka, R.P., Ijichi, K., 1969. Heat resistance of Salmonella in various egg products. Appl. Microbiol. 17, 491–496. Goepfert, J.M., Iskander, I.K., Amundson, C.H., 1970. Relation of the heat resistance of salmonellae to the water activity of the environment. Appl. Microbiol. 19, 429–433. HPA, 2007. Two outbreaks of salmonellosis in the United Kingdom and Channel Islands putatively linked to imported pasteurised egg products. Health Protection Report 1. Health Protection Agency, London, United Kingdom (http://www.hpa.org.uk/hpr/archives/2007/hpr4507.pdf). HPA, 2012. Salmonella Enteritidis PT1 associated with consumption of liquid egg product. Health Protection Report 6. Health Protection Agency, London, United Kingdom (http://www.hpa.org.uk/hpr/archives/2012/hpr4212.pdf). Kang, H., Loui, C., Clavijo, R.I., Riley, L.W., Lu, S., 2006. Survival characteristics of Salmonella enterica serovar Enteritidis in chicken egg albumen. Epidemiol. Infect. 134 (5), 967–976. Leistner, L., 2000. Basic aspects of food preservation by hurdle technology. Int. J. Food Microbiol. 55, 181–186. Mattick, K.L., Jorgensen, F., Wang, P., Pound, J., Vandeven, M.H., Ward, L.R., Legan, J.D., Lappin-Scott, H.M., Humphrey, T.J., 2001. Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity. Appl. Environ. Microbiol. 67, 4128–4136. McQuestin, O.J., Musgrove, M.T., Tamplin, M.L., 2010. Kinetics of growth and inactivation of Salmonella enterica serotype Typhimurium DT104 in pasteurised liquid egg products. Food Microbiol. 27 (3), 396–402. Musgrove, M.T., Mcquestin, O.J., Tamplin, M., Kelley, L.C., 2009. Growth and survival of antibiotic-resistant Salmonella typhimurium DT104 in liquid egg products. J. Food Prot. 72 (9), 1992–1996. Ng, H., Garibaldi, J.A., Ijichi, K., Mihara, K.L., 1979. Pasteurization of salted whole egg inoculated with Arizona or Salmonella. Appl. Environ. Microbiol. 37, 1091–1095. Olsen, J.E., Skov, M.N., Threlfall, E.J., Brown, D.J., 1994. Clonal lines of Salmonella enterica serotype Enteritidis documented by IS200-, ribo-, pulsed-field gel electrophoresis and RFLP typing. J. Med. Microbiol. 40, 15–22. Palumbo, M.S., Beers, S.M., Bhaduri, S., Palumbo, S.A., 1995. Thermal resistance of Salmonella spp. and Listeria monocytogenes in liquid egg yolk and egg yolk products. J. Food Prot. 58, 960–966. Park, S.Y., Seo, K.Y., Ha, S.D., 2007. A response surface model based on absorbance data for the growth rates of Salmonella enterica serovar Typhimurium as a function of temperature, NaCl, and pH. J. Microbiol. Biotechnol. 15, 1323–1329. Rosso, L., 1995. Modélisation et microbiologie prévisionnelle: élaboration d'un nouvel outil pour l'agro-alimentaire. (PhD thesis in Biometry) Université Claude Bernard Lyon-I. Sakha, M.Z., Fujikawa, H., 2013. Prediction of Salmonella Enteritidis growth in pasteurized and unpasteurized liquid egg products with a growth model. Biocontrol Sci. 18, 89–93. Singh, A., Korasapati, N.R., Juneja, V.K., Subbiah, J., Froning, G., Thippareddi, H., 2011. Dynamic predictive model for the growth of Salmonella spp. in liquid whole egg. J. Food Sci. 76, M225–M232. Thayer, D.W., Muller, W.S., Buchanan, R.L., Phillips, J.G., 1987. Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium. Appl. Environ. Microbiol. 53, 1311–1315.
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg Džiuginta Jakočiūnė a, Magne Bisgaard a, Gaëlle Hervé b,c, Jocelyne Protais b,c, John Elmerdahl Olsen a,⁎, Marianne Chemaly b,c a b c
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark Anses — French Agency for Food, Environmental and Occupational Health and Safety, HQPAP — Unit of Hygiene and Quality of Poultry and Pork Products, BP 53, 22440 Ploufragan, France UEB — European University of Brittany, 5 Boulevard Laënnec, 35000 Rennes, France
a r t i c l e
i n f o
Available online xxxx Keywords: Salmonella Pasteurized whole eggs Prediction NaCl Survival Growth
a b s t r a c t This study investigated the influence of three parameters (time, temperature and NaCl concentration) on survival and four parameters (temperature, NaCl and lysozyme concentrations and pH) on growth of Salmonella enterica serovar Enteritidis (S. Enteritidis) in pasteurized whole egg (PWE). Doehlert uniform shell design was employed to choose conditions for trials and data was fitted to polynomial models and were presented as estimated response surfaces. A model for prediction of reduction of S. Enteritidis in PWE within temperatures between 50 and 58 °C, NaCl concentrations of 0–12%, and heating times between 30 and 210 s and a model for prediction of growth rate of S. Enteritidis in PWE in the temperature range of 1–25 °C, NaCl concentration of 0–12%, pH between 5 and 9, and lysozyme concentrations of 107–1007 U/mg proteins were developed. The maximum reduction condition was 58 °C, 0% of NaCl at a fixed heating time of 120 s, while maximum growth rate was estimated at 25 °C and 0% of NaCl. pH and lysozyme concentration were shown not to influence growth performance significantly in the range of values studied. Results inform industry of the optimal pasteurization and storage parameters for liquid whole egg. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Salmonella enterica serovar Enteritidis (S. Enteritidis) is an important food borne pathogenic bacterium. Most outbreaks caused by this bacterium have been associated to eggs and egg products (Anon., 2013). Pasteurization of liquid egg products is widely applied in the industry to reduce this risk, however several Salmonella outbreaks associated with consumption of pasteurized egg products indicate that Salmonella may still be present in the final product (HPA, 2007, 2012). Hurdle effects might be used to prevent regrowth of Salmonella in food products (Leistner, 2000). Addition of NaCl is often used as a preservative in food, however, already several decades ago Garibaldi et al. (1969) observed that addition of both salt and sucrose to egg yolk increased thermal resistance of Salmonella. Likewise, a number of studies have concluded that heat tolerance of Salmonella increases when aw decreases both in broth or buffer systems (Goepfert et al., 1970), and in liquid egg products (Garibaldi et al., 1969; Ng et al., 1979; Palumbo et al., 1995). This has however been contradicted in studies by Mattick et al. (2001) who observed the opposite effect when the temperature was lower than 65 °C, and Aljarallah and Adams (2007) reported a switch from protective to sensitizing effect of salt at even lower temperatures ⁎ Corresponding author. Tel.: +45 353 32784. E-mail address: [email protected] (J.E. Olsen).
(53 °C–55 °C). There is therefore a need to revisit this problem, if the Salmonella risk from liquid egg products shall be reduced by the use of hurdle effects. Predictive models have been used to estimate the influence of multiple parameters on growth of Salmonella in growth media (Park et al., 2007; Thayer et al., 1987), but so far nobody have used such multiple parameter models to describe Salmonella growth in liquid egg. In the current investigation we used predictive modeling to determine individual and combined effects of three environmental conditions: time, temperature and NaCl concentration on survival of S. Enteritidis in pasteurized whole egg (PWE) and to investigate growth of S. Enteritidis as a function of four parameters: NaCl and lysozyme concentrations, temperature and pH in the same product. 2. Materials and methods 2.1. Bacterial strains, media and egg products S. Enteritidis strain E2187 (Olsen et al., 1994) was used for survival experiments. It was plated on Xylose-Lysine-Deoxycholate Agar (XLD, Oxoid, Basingstoke, United Kingdom) and incubated overnight at 37 °C and colonies were harvested in physiological saline (0.9% NaCl, Merck, Darmstadt, Germany) to an OD-value corresponding to a concentration of approximately 109 CFU/ml. PWE (Danæg, Roskilde,
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015 0168-1605/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
2
D. Jakočiūnė et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx
Denmark) was spiked to a final concentration of approximately 107 CFU/ml. Actual concentrations were determined by plating appropriate decimal dilutions in saline on Luria-Bertani (LB) agar (Becton, Dickinson and Company, Sparks, MD, USA) plates. S. Enteritidis strain MJG01 (previously isolated from egg product) was used for growth experiments. It was plated on a Plate Count Agar (PCA, AES, Bruz, France) and incubated at 37 °C for 24 h. One colony was then transferred to a Brain Heart Infusion broth (AES, Bruz, France) and incubated at 37 °C for 24 h. This stock solution was used to inoculate the PWE (ADRO Ouest, France) after appropriate dilutions in a Tryptone Salt (Biomérieux, Craponne, France) broth to reach the final concentration as shown below in the description of the growth experiment. PWE used for both survival and growth experiments was prepared by industry by pasteurization to ensure sterility.
Table 2 Range of parameters for growth experiments according to Doehlert shell design. Factors
No. of levels
Values
pH Temperature (°C) Lysozyme (U/mg proteins) NaCl (%, w/w)
3 5 7 7
5–7–9 1–7–13–19–25 107–257–407–557–707–857–1007 0–2–4–6–8–10–12
Before the experiments the defined pH values were obtained by adding acid (HCl) or base (NaOH) at a suitable titer in order to avoid introducing an excessive dilution factor into the PWE. Solid NaCl was added at the defined concentrations after a moderate heat treatment and ionization to ensure the sterility before adding it to the PWE. Numbers of bacteria were determined by CFU counts on PCA.
2.2. Doehlert uniform shell design 2.5. Statistical analysis Both growth and survival experiments were carried out according to a Doehlert experimental design (Doehlert, 1970). For survival experiments, two experimental matrices 1 and 2 were designed with low (50, 52, 54 °C) and high (54, 56, 58 °C) temperatures, respectively (Table 1). Each experimental matrix contained 13 trials with variation in three variables (NaCl concentration (%, w/w), time (s) and temperature (°C)). Three repeats were carried out of experiment with conditions at the center to ensure reproducibility (total number of trials 16). Results were presented as log10 reduction of Salmonella in PWE. For growth experiments 21 trials were performed with variation in four variables (pH, temperature (°C), lysozyme concentration (U/mg proteins) and NaCl concentration (%, w/w)) (Table 2). To ensure reproducibility, two repeats of experiment were carried out with conditions at the center (total number of trials 23). For each trial a growth curve was determined based on CFU and the growth rate was calculated according to the primary model of Rosso (1995) based on 10 to 15 enumerations for each growth curve.
For survival experiments Grubb's test of outliers with P b 0.05 (http://graphpad.com/quickcalcs/Grubbs1.cfm) identified one CFU count out of four in three samples as outliers. These were eliminated before further analysis. Data from matrices 1 and 2 were merged and used to build one model for survival experiment. Statistical analysis of data was performed using Statgraphics© software. Results explained the individual effect of each parameter and their interactions. Polynomial models were built for prediction of S. Enteritidis behavior using the same software. Moreover, the results were illustrated as estimated response surfaces. Lack-of-fit and R2 were used to determine whether the selected model is adequate to describe the observed data, or whether a more complicated model should be used. Lack-of-fit test was performed by comparing the variability of the current model residuals to the variability between observations at replicate settings of the factors. 3. Results and discussion
2.3. Survival experiments
3.1. Effects of environmental conditions on survival of Salmonella in PWE
Solid NaCl was added at the defined concentrations to PWE maximum 24 h before the experiment and samples of 90 μl of salted PWE in 1.5 ml Eppendorf tubes were pre-heated to different temperatures (+/−1 °C) listed in Table 1 with an Eppendorf thermomixer with shaking at 1000 rpm. Temperatures were measured with a penetration thermometer Ebro TDC 200. 10 μl of spiked PWE without NaCl was added to reach final concentrations of NaCl and a final concentration of approximately 106 CFU/ml. Samples were incubated for different times as indicated in Table 1 followed by placing samples on ice and addition of 900 μl ice cold saline to stop the heating. Further decimal dilutions were prepared in saline and plated on LB agar plates. Two independent experiments were performed for each matrix with two replicates for each trial. Numbers of bacteria were converted to log10 CFU/ml and subtracted from initial concentration to obtain reductions.
Three environmental conditions (NaCl concentration (%, w/w), time (s) and temperature (°C)) were investigated in relation to survival of S. Enteritidis in PWE. Data presented in Table 3 show the estimated individual and combined effects of all parameters on S. Enteritidis log10 reduction and their significance in both matrices. Time and temperature had significant positive effect on log10 reduction, while increasing NaCl concentration protected against killing. Fig. 1 shows estimated response surface of log10 reduction as a function of temperature and NaCl concentration with time fixed at the central value of 120 s. In both matrices the effect of NaCl on heat killing of S. Enteritidis was protective. This corresponds well with results of Garibaldi et al. (1969) and a protective effect of NaCl was also observed by others (Ng et al., 1979; Palumbo et al., 1995), however the challenge temperatures in their Table 3 Estimated effects of factors individually and their interactions for reduction of S. Enteritidis in PWE in the temperature range of 50° to 58 °C.
2.4. Growth experiments PWE samples were inoculated with 103 CFU/ml of S. Enteritidis, where after growth kinetics within the given range of temperature, pH, salt and lysozyme concentrations (Table 2) were determined.
Table 1 Range of parameters for survival experiments according to Doehlert uniform shell design. Factors
No. of levels
Values
Temperature (°C)
3
NaCl (%, w/w) Time (s)
5 7
50–52–54 (matrix 1) 54–56–58 (matrix 2) 0–3–6–9–12 30–60–90–120–150–180–210
Factors
Estimated effects on log10 reduction
SD
P valuea
A: NaCl conc. B: Time C: Temperature AA AB AC BB BC CC Lack-of-fit R2
−1.14 2.095 2.56636 2.28772 −0.779082 −0.106122 0.12839 0.947347 0.587489
0.165366 0.143211 0.141024 0.273981 0.316945 0.298819 0.205485 0.258784 0.252847
0.0005 0.0000 0.0000 0.0002 0.0492 0.7346 0.5551 0.0106 0.0592 0.0742 0.921892
a
Significant values are indicated in bold.
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
D. Jakočiūnė et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx
Estimated Response Surface Time=120,0
Table 4 Estimated effects of factors individually and their interactions for growth rate of S. Enteritidis in PWE.
Log10 reduction
5 4 3 2 1 0 0
2
4
6
Salt
8
10
12
50
52
54
56
3
58
Temperature
Fig. 1. Estimated surface response of log10 reduction of S. Enteritidis in PWE as a function of NaCl concentration and temperature with a fixed value of time (120 s).
experiments were higher than in this study (over 60 °C). Blackburn et al. (1997) also showed a protective effect, however the experiments were performed using growth media and the model was related to publish data on Salmonella growth in egg products. Based on their results, a maximal protective concentration of 5–7% of salt within the range of 54.5–64.5 °C was predicted (Blackburn et al., 1997). Importantly, results obtained in our study are contradictory to those of Aljarallah and Adams (2007), who showed that D-values increased for Salmonella at 55 °C and 60 °C at low aw values in broth media whereas at 48 °C, 50 °C and 53 °C the D-values were lower in low aw media. They concluded that heat must have different targets in the cell at different temperatures. At low heating temperatures (below 53 °C–55 °C) the cytoplasmic membrane is injured and the additional osmotic stress due to low aw leads to cell death. At higher temperatures ribosomes are targeted and then NaCl has a protective effect (Aljarallah and Adams, 2007). In our study with PWE, NaCl had a protective effect both at low and high temperatures. The reason for this is currently unknown and further studies are indicated to investigate this. The results of the current study shows that data obtained from experiments with growth media cannot be used to conclude on similar situations in PWE. A significant negative combined effect of NaCl concentration and time and significant positive combined effect of time and temperature were observed (Table 3). Lack-of-fit was not significant and R2 was very high (Table 3) meaning that the model gave a good prediction of the experimental data. The equation of the model derived from the survival data was as follows:
Factors
Estimated effect on growth rate
SD
P valuea
A: Temperature B: NaCl conc. C: Lysozyme conc. D: pH AA AB AC AD BB BC BD CC CD DD Lack-of-fit R2
0.24 −0.25 0.009 −0.024 0.18 −0.48 −0.068 0.029 0.19 −0.06 −0.013 0.032 −0.06 0.0030
0.012 0.01 0.009 0.009 0.025 0.027 0.028 0.029 0.018 0.023 0.024 0.015 0.022 0.013
0.0025 0.0016 0.4388 0.1212 0.0184 0.0031 0.1364 0.4155 0.0077 0.1244 0.6396 0.1648 0.1137 0.8411 0.0512 0.94
a
Significant values are indicated in bold.
significantly affected the growth rate of S. Enteritidis, i.e. growth declined with decreasing temperature and increasing NaCl concentration. An interaction between these two parameters was observed, i.e. combined they constituted a hurdle effect (Fig. 2). These results are similar to results of previous investigation of S. Enteritidis growth in culture medium, showing that high storage temperature (19 °C), low NaCl concentration and neutral pH were optimal for Salmonella growth (Thayer et al., 1987). Studies performed with egg products have also shown inhibition of growth by decreased temperature (Sakha and Fujikawa, 2013; Singh et al., 2011) and increased NaCl concentration (McQuestin et al., 2010; Musgrove et al., 2009). Park et al. (2007) showed a decreased growth rate of Salmonella in broth culture outside neutral pH, and this was confirmed for high pH (pH = 9) valued during growth in egg white (Kang et al., 2006). However, this effect of pH was not evident in PWE, probably reflecting differences between the egg products used for experiments. We also saw no influence of lysozyme concentration, which confirms observation by Kang et al. (2006) that the antimicrobial activity of egg white, with lysozyme as an important active compound, is not active at the low and ambient temperature investigated in the present investigation. A model was fitted to the growth curves in order to be able to predict the behavior of S. Enteritidis in PWE. An insignificant lack-of-fit and high R2 values (Table 4) indicated that the model reflected the experimental data well.
log10 reduction ¼ 45:2031−0:270334 Salt−0:056986 Time
∧
−1:8066 Temperature þ 0:0317739 Salt 2 −0:000721372 Salt Time−0:00221088 Salt ∧ ∗ Temperature þ 0:00000792531 Time 2 þ 0:00131576 ∧ ∗ Time Temperature þ 0:018359 Temperature 2: 0,74 0,54
µ
This equation can be used to estimate log10 reduction of Salmonella for different concentrations of NaCl, time and temperatures in the model range. Based on results from both matrices we concluded that best reduction was obtained using maximal temperature and the lowest NaCl concentration. Special attention should be drawn to the fact that NaCl had protective effect to heat tolerance also in the low temperature range.
Lysozyme=557.0; pH=7
0,34 0,14 -0,06 0
3.2. Effects of environmental conditions on growth of S. Enteritidis in PWE The second part of this study was to investigate influence of four environmental conditions on growth of S. Enteritidis in PWE. Results presented in Table 4 show that temperature and NaCl concentration
5
10
15
Temperature
20
25
0
2
4
6
8
10
12
Salt
Fig. 2. Estimated surface response of growth rate of S. Enteritidis in PWE as a function of NaCl concentration and temperature with fixed values of lysozyme (557.0 U/mg protein) and pH (7.0).
Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
4
D. Jakočiūnė et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx
The following equation describes the growth rate model: μ ¼ −0:134604 þ 0:0134802 Temperature−0:00367957 Salt þ 0:000295239 Lysozyme þ 0:00288486 pH þ 0:000592593 ∧
∗ Temperature 2−0:00321528 Temperature Salt−0:00000608025 ∗ Temperature Lysozyme þ 0:000587963 Temperature pH ∧ þ 0:00277083 Salt 2−0:000011142 Salt Lysozyme−0:000546296 ∧ ∗ Salt pH þ 7:8738E–8 Lysozyme 2−0:0000328601 Lysozyme ∧
∗ pH þ 0:000382716 pH 2:
Based on this equation, S. Enteritidis growth rate in PWE with different concentrations of NaCl and lysozyme, time and temperatures in the model range can be predicted. In conclusion the current study has produced two models for prediction of log10 reduction and growth of S. Enteritidis in PWE in ranges of NaCl concentration, heating time and temperature with regard to survival and NaCl concentration, pH, lysozyme concentrations and temperatures with regard to growth. NaCl concentration had negative effect on reduction of S. Enteritidis in PWE while time and temperature had positive effects and temperature, NaCl concentration and an interaction between these two parameters significantly affected the growth rate for S. Enteritidis in PWE. The results presented can be used by industry to choose optimal pasteurization and storage parameters for liquid whole egg products. Acknowledgments This study was supported by the European Commission through the project BASELINE, grant agreement 222738, and the areas of Brittany and Pays de la Loire in France through the federation of PAO and Valorial. Pasteurized whole egg products were kindly provided by Danæg and by egg product producers in France (ADRO Ouest). References Aljarallah, K.M., Adams, M.R., 2007. Mechanisms of heat inactivation in Salmonella serotype Typhimurium as affected by low water activity at different temperatures. J. Appl. Microbiol. 102, 153–160. Anon., 2013. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA J. 11 (4), 250. Blackburn, C.W., Curtis, L.M., Humpheson, L., Billon, C., McClure, P.J., 1997. Development of thermal inactivation models for Salmonella enteritidis and Escherichia coli O157:
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Please cite this article as: Jakočiūnė, D., et al., Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.015
