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Survival of Salmonella enteric in skim milk powder with different water activity and water mobility
Food Control 47 (2015) 1e6
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Survival of Salmonella enteric in skim milk powder with different water activity and water mobility Feng Lian a, Wei Zhao a, *, Rui-jin Yang a, Yali Tang b, Wendy Katiyo a a b
State Key Laboratory of Food Science & Technology, School of Food Science and Technology, Jiangnan University, No. 1800 Lihu Road, Wuxi 214122, China School of Mechanical Engineering, Department of Packaging Engineering, Jiangnan University, No. 1800 Lihu Road, Wuxi 214122, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 March 2014 Received in revised form 10 June 2014 Accepted 17 June 2014 Available online 25 June 2014
Recently, the safety of low water activity (aw) foods started to become a major concern. It has been observed that microorganisms may not grow in low-aw foods but can survive for rather long periods of time, representing significant risk. Most researchers have been concentrating on the role of water activity. However, little is known about the effect of water mobility on influencing the survival of Salmonella in low-aw foods. In this study, water mobility of skim milk powder was manipulated using ultrahigh pressure to change tertiary structure, thus changing watereprotein interactions. Skim milk powder with different levels of water mobility at similar aw level were obtained to evaluate how aw and water mobility influenced the survival of Salmonella enteric in low-aw foods. Results showed that aw could influence the survival of S. enteric and the lower the aw, the higher the survival. Water mobility had little influence on the survival of S. enteric when aw was 0.33 and 0.53. However, it influenced its survival when aw was increased to 0.81. The survival population of S. enteric was higher in the sample with low water mobility. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Low-water activity foods Water activity Water mobility
1. Introduction Low-water activity foods are those with water activity (aw) levels lower than 0.85 (Beuchat et al., 2013). Low-aw foods include milk powder, egg powder, chocolate, peanut butter and so on. They are usually believed to have the advantages of controlling the growth of pathogenic and spoilage microorganisms. Hence, their microbial safety is usually ignored by the public. Recently, a number of outbreaks associated with low-aw foods and pathogens occurred, resulting in the safety of these foods becoming a major concern (Awuah, Ramaswamy, Economides, & Mallikarjunan, 2005; Beuchat et al., 2013; CDC, 2007; Farakos, Frank, & Schaffner, 2013; Finstad, O'Bryan, Marcy, Crandall, & Ricke, 2012; Koch et al., 2005). The contamination of low-aw foods with Salmonella is not an unknown problem. It has been observed that foodborne pathogens may not grow in low-aw foods but can survive for rather long periods of time once the food is contaminated, thus representing significant risk even at low levels. However, current practices appear inadequate to address this challenge, as
* Corresponding author. Tel./fax: þ86 510 85919150. E-mail addresses: [email protected] (W. Zhao), [email protected] (R.-j. Yang). http://dx.doi.org/10.1016/j.foodcont.2014.06.036 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
contamination continues, even in large multi-national companies that employ experienced food microbiologists and risk managers. It is known that aw is one of the basic properties of a food that exerts major influence on microbial survival and growth (Beuchat et al., 2013; Farakos et al., 2013; Laroche, Fine, & Gervais, 2005). Some research has been conducted in this aspect, but the conclusions about the influence of aw on microbial survival in low-aw foods by different groups are inconsistent. Jung and Beuchat (1999) concluded that Salmonella died more rapidly in higher aw egg white powder than in lower aw powder. However, Mattick et al., (2001) observed that the relationship between microbial survival and aw may change at different temperatures (Mattick et al., 2001). Their study demonstrated that Salmonella at low aw (0.65) was more heat tolerant at temperatures above 70 C than when at higher aw (0.90), but the reverse was true at lower temperatures. Laroche et al., (2005) used Saccharomyces cerevisiae and Lactobacillus plantarum as test organisms. The microorganisms exhibited different survival ability in the low-aw foods (wheat flour and skim milk) when they were adjusted to the same water activity. There was a range of aw for greatest heat resistance for each microorganism and low-aw food across all temperatures. Moreover, some researchers (Hills, Manning, Ridge, & Brocklehurst, 1997) indicated that the molecular mobility of water in low-aw foods is an important factor in microbial survival. Microbial survival in low-aw foods did not
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correlate to water activity, but did correlate to molecular mobility of the water. Vittadini, Chinachoti, Lavoie, and Pham (2005) provided evidence that microbial behavior was more closely associated with molecular mobility in low-aw foods and aw provided good predictions of microbial behavior only in high-aw foods. A recent study (Farakos et al., 2013) determined and discussed how the physical state of water in low-aw foods influences the survival of Salmonella, employing whey protein powder with different water mobility at similar aw levels by pH adjustment and heat denaturation. The results demonstrated that aw significantly influenced the survival of Salmonella at all temperatures, survival increasing with decreasing aw. Water mobility did not significantly influence survival independent of aw. In their study, the water mobility of whey protein powder was manipulated using pH adjustments to change the tertiary structure, thus changing watereprotein interactions. In this way, the solutes for the low-aw foods with different water mobility at similar aw levels are difficult to keep the same. It has been evidenced that use of different solutes to achieve similar water activities produces markedly different microbial survival kinetics (Mattick et al., 2001). In low-aw foods, most of the water is in glassy and rubbery states so the water has limited mobility. Since water molecules must contact the bacterial cell for interaction, the ease of transition between physical states, i.e. molecular mobility, should indicate the potential for interaction between bacterial cells and water. In the present work, water mobility of skim milk powder was manipulated using ultra-high pressure to change the tertiary structure of protein, thus changing watereprotein interactions. Skim milk powder, with different water mobility at similar aw levels and other conditions (such as solutes), was obtained to evaluate how aw and water mobility influence the survival of Salmonella in low-aw foods. 2. Materials and methods 2.1. Modification of skim milk powder The water mobility of skim milk powder (Bright dairy corporation, China) was manipulated using ultra-high pressure adjustments to change the secondary and tertiary structure of protein, thus changing watereprotein interactions. Skim milk powder (500 g) and water (500 mL) were made into a solution. The solution was then transferred into polyethylene bags and a vacuum sealer (A300/16, Multiuac, Germany) was used to remove most of the air. The sealed samples were stored overnight at 4 C. Ultra-high pressure treatments were performed by ultra-high pressure equipment (UHPF-800MPa-5L, Baotou Kefa Co., China) for 10 min at approximately 25 C. The temperature of the vessel was kept at room temperature. The compression and decompression rate was 25 MPa/s. The samples were treated at 100, 200, 300, 400 or 500 MPa. The control sample was hydrated overnight at 4 C but was not treated by ultra-high pressure. After treatment, samples were then poured into sterile Petri dishes and frozen to 80 C overnight in a freezer (HFU 586 Basic, Thermal Scientific, Germany). The samples were then placed in a vacuum freeze drier (7948030, Labconco, US) for 3 days to obtain aw levels below 0.10. Once freeze dried, the samples were broken down to homogenous particles with a mortar and pestle. 2.2. Adjustment of the aw of re-dried samples Re-dried samples were adjusted to the targeted aw values in vacuum desiccators containing various saturated salt solutions of known relative vapor pressures. The targeted aw levels were: 0.33 (Magnesium Chloride), 0.53 (Magnesium Nitrate) and 0.81 (ammonium sulfate). Equilibrium was assumed when there was no
further change in weight. The aw of samples was measured using a water activity meter (Hygrolab2, Rotronic, Switzerland). 2.3. Water mobility determination A low-field 1H Nuclear Magnetic Resonance analyzer (MicroMRCL, Niumag Corporation, Shanghai, China) with a resonance frequency of 21.798 MHz and 0.55 T magnetic field was used for measurements of proton transversal relaxation time (T2). The transversal relaxation time was measured with a CarrePurcelleMeiboomeGill (CPMG) pulse sequence. The experimental parameters were as follows: the 90 pulse width was 3 ms, the 180 pulse width was 7 ms, the Number of Scans (NS) was 16, the Receiver Gain (RG) was set to 20 dB, the echo time (TE) was 80 ms and the echo number was set to 1000. The values of T21 relaxation time were recorded. 2.4. Procedure for inoculating milk powder for storage experiments Salmonella enteric (JFM-125) isolated from powdered infant formula was a gift from Food Biotechnology Center of Jiangnan University. The cultures were maintained at 4 C on LuriaeBertani (LB) ager and activated by transferring a loop of inocula into 150 mL nutrient broth and cultured for 14 h at 37 C. Cultures were harvested by centrifugation (9000 rpm, 5 min), and pellets were washed twice in 150 mL of sterile 0.1% peptone water. Finally, the pellets were re-suspended in 3 mL sterile 0.1% peptone water. The suspension was poured into a sterile Petri dish and put in a desiccator for 3 days to ensure aw of below 0.10. The dried cells were ground with a mortar and pestle. The dried inoculum (0.01 g) was mixed with 0.99 g of equilibrated skim milk powder to provide a 1.00 g sample in a polypropylene vial. It was assumed that the addition of inoculum did not influence aw of the milk powder because the dose was too small to be significant. The samples were placed in retort pouches, then vacuum packaged. The vacuum packaged samples were stored in desiccators at their corresponding relative humidity in an incubator at 37 C. The samples were taken at: 0, 3, 6, 12, 30 and 60 days for survival experiments. 2.5. Enumeration of cultivable Salmonella The plate count method was adopted to enumerate Salmonella populations. Each inoculated sample (1.00 g) was assayed for population of Salmonella at selected time intervals. All samples were respectively dissolved in 9 mL sterile saline and homogenized for 1 min with a vortex (Lab Dancer, IKA, Germany). After homogenization, 1 mL dissolved sample was serially diluted using sterile test tubes containing 9 mL sterile saline to obtain the appropriate degree of dilution. Then 1 mL appropriate dilution was transferred to a Petri dish, and soon afterwards approximately 15 mL nutrient ager, with the temperature about 46 C, was added. When the nutrient agar (Sinopharm Chemical Reagent, China) solidified, the petri dishes were inverted and placed in an incubator. The plates were enumerated after incubation at 37 C for 24 h. 2.6. Statistical analysis and curve fitting Cultivability of S. enteric for each combination of water activity and water mobility was performed. Each experiment was carried out at least in triplicate. Data was analyzed in Microsoft Excel 2010 (Microsoft Corporation, US). Initial data (CFU/g) was converted into log of the survival of S. enteric at a given time. Comparison was made by SPSS software. Curves were fitted using the Curve Expert software (Version 1.3). This software contained 60 models. The most appropriate model was chosen to describe the curves.
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3. Results and discussion 3.1. The survival of S. enteric influenced by water activity The viability results for S. enteric in skim milk powder at three levels of aw at 37 C are shown in Fig. 1. After adjusting to the targeted aw and inoculating with S. enteric, the initial populations were 9.90, 10.01 and 10.19 logs, in samples with aw of 0.33, 0.53 and 0.81, respectively. A reduction of about 3.96 logs occurred within the first month of storage, and about 0.27 logs in the second month at aw 0.33. At aw 0.53, the reduction population during the first month was 3.89 logs, and it was similar to that at aw 0.33. Reduction during the second month was 0.8 logs, and this was significantly higher than at aw 0.33 (p < 0.05). Reduction populations at aw of 0.81 were 4.69 and 1.16 logs, respectively. They were both significantly higher than that at aw 0.33 and 0.53 (p < 0.05). This indicates that the death rate during the first month was higher than the second, at the three aw of skim milk powder. This phenomenon is in agreement with observations by Licari and Potter (1970). These researchers reported that Salmonella in skim milk powder died in a two-stage process: rapid death during the first several weeks followed by slow death during the remaining shelf life. After storage for two months, the reduction populations were 4.23, 4.69 and 5.85 logs at aw levels of 0.33, 0.53 and 0.81, respectively. This is similar with the results of Jung and Beuchat (1999), who reported that after storage of two months at 37 C, log populations in all the samples were less than 1.00 log with an initial population of about 5.00 logs. It also indicates that survival is enhanced as the aw of skim milk powder decreases. Vesterlund, Salminen, and Salminen (2012) studied the effect of aw on the storage stability of probiotics included in crushed flaxseed to identify the right aw level for storage at room temperature. They observed that at the highest aw tested (0.43), the probiotic product was unstable. Within 4 months of storage, the reduction of viable cells was 3.70, 0.81 and 0.29 logs at aw of 0.43, 0.22 and 0.11, respectively, demonstrating that lower aw is more suitable for the survival of microorganisms in dried foods. 3.2. Preparation of samples with different water mobility by ultrahigh pressure Ultra-high pressure is classified as non-thermal processing technique, using the fluid medium (water or oil) for transmitting pressure. Proteins or complex biopolymers subjected to ultra-high
Fig. 2. Continuous relaxation time spectra of samples at three aw and six water mobility (water mobility 1: control, water mobility 2: protein denatured at 100 MPa, water mobility 3: protein denatured at 200 MPa, water mobility 4: protein denatured at 300 MPa, water mobility 5: protein denatured at 400 MPa, water mobility 5: protein denatured at 500 MPa). Fig. 1. Viability of S. enteric at 37 C and 3 water activities (aw) during 2 months of storage in skim milk powder.
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pressure may have their tertiary and secondary structures modified by disrupting hydrophobic and electrostatic interactions (Molina, Papadopoulou, & Ledward, 2001). It has been widely used in the modification of proteins. In this study, water mobility of skim milk powder was manipulated using ultra-high pressure to change the tertiary structure of proteins, thus changing watereprotein interactions, that is water mobility. Skim milk powder with different water mobility at similar aw levels and other conditions (such as solutes) were obtained to evaluate the influence of aw and water mobility on the survival of Salmonella in low-aw foods. LF 1H NMR has been used to investigate water mobility in materials and foods (Choi & Kerr, 2003; Fan et al., 2013). LF 1H NMR can measure proton relaxation, thus can be used to study changes in water mobility (Carneiro et al., 2013). Continuously distributed exponential curve fitting was performed on the obtained T2 relaxation time from samples. Dependence of the nature of skim milk powder, the faster relaxation component which ranges from 0 to 10 ms in the following is called T21. In the present study, T21 represents the water which is closely associated with macromolecules and microbial cells (Li et al., 2012). Fig. 2 shows the distributed T2 relaxation time spectra of skim milk powder submitted to different treatments with ultra-high pressure. Table 1 shows the values of T21 detected by LF 1H NMR according to the different treatments of skim milk powder. Comparison of the continuous distributed curves, at three aw levels of 0.33, 0.53 and 0.81, reveals visible differences between the treated skim milk powder and the control. The treated samples mostly tended to exhibit a broader T21 distribution than the control, which is likely due to the differences in water distribution caused by ultra-high pressure. Fig. 2 shows significant differences in the distribution of water mobility between different treated samples. As shown in Fig. 2(a), a significant increase (p < 0.05) in relaxation time (T21) was observed in the samples treated with pressure of 100, 200, 300 and 400 MPa. The T21 distribution of the sample treated with 500 MPa pressure was broader than the control. However, it was narrower than the rest of the samples. This phenomena indicates that pressure ranging from 100 to 500 MPa affects the T21 distribution at aw 0.33. As shown in Fig. 2(b), the curve of the sample at aw 0.53 is similar to that at aw
Table 1 Range of the LF 1H NMR obtained in skim milk powder, according to the different treatments. aw
Water mobility a
0.33 ± 0.05
1 2b 3c 4d 5e 6f 1 2 3 4 5 6 1 2 3 4 5 6
0.53 ± 0.04
0.81 ± 0.06
a b c d e f
Control. Protein denatured Protein denatured Protein denatured Protein denatured Protein denatured
at at at at at
100 200 300 400 500
MPa. MPa. MPa. MPa. MPa.
T21 (ms) 0.187 0.756 0.498 0.572 0.498 0.248 0.327 0.658 0.572 0.658 0.756 0.572 0.756 1.000 1.000 1.000 1.000 0.376
Fig. 3. Survival of S. enteric in samples at three aw and six water mobility (water mobility 1: control, water mobility 2: protein denatured at 100 MPa, water mobility 3: protein denatured at 200 MPa, water mobility 4: protein denatured at 300 MPa, water mobility 5: protein denatured at 400 MPa, water mobility 6: protein denatured at 500 MPa).
F. Lian et al. / Food Control 47 (2015) 1e6
0.33. The samples treated with pressure ranging from 100 to 400 MPa had similar T21 distributions, indicating that they had similar water mobility. Fig. 2(c) shows the T2 distribution of skim milk powder at aw 0.81. It was also discovered that the samples treated with pressure ranging from 100 to 400 MPa had similar T21 distributions, and as shown in Table 1, the T21 values of these samples were the same. The other significant difference is that the T21 value of the sample treated with 500 MPa is lower than the control, which suggests that its water mobility is lower. 3.3. The cultivability of S. enteric influenced by water mobility Fig. 3 shows the cultivability of S. enteric in skim milk powder treated at different pressure at three aw. It was observed that about 5.00 logs of S. enteric were inactive after two months storage in all the samples from Fig. 3(a). This indicates that water mobility has little influence on the cultivability of S. enteric compared with the control at aw 0.33 (p > 0.05). This was also demonstrated by Farakos et al., (2013). They revealed that water activity significantly influenced the survival of Salmonella in low-aw foods (aw < 0.60), while water mobility had no effect independent of aw. Vittadini et al. (2005) investigated the correlation of microbial response in model food systems with physico-chemical and “mobility” descriptors of the media. They studied the role of aw, “macromolecular” mobility and water molecular dynamics in multicomponent model food (ranging from solid to liquid) systems in their correlation with microbial activity. It was found that a correlation between cell death and increased 1H and 2H NMR mobility in low-aw foods (solid and semisolid media), indicating NMR molecular mobility is a possible tool to describe water availability in solid and semisolid systems (dry and intermediate moisture foods), while in a high moisture system, the use of water activity remains a valid indicator for microbial activity. However, it is difficult to differentiate the action of water mobility from that of aw because different water mobility with different aw were employed to correlate with microbial response. To further discriminate the influence of molecular mobility on microbial survival behavior in low-aw foods, the present study prepared skim milk powder with different water mobility at similar aw levels to evaluate how aw and water mobility influence the survival of Salmonella in low-aw foods. As shown in Fig. 3(b), all samples had similar reduction during the two months storage period, which was similar at aw 0.33. It also indicates that
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water mobility has little influence on the cultivability of S. enteric at this level of aw (p > 0.05). The cultivability of S. enteric at aw 0.81 after storage at 37 C was assayed in the treated and control skim milk powder for up to two months (Fig. 3(c)). During the first month, the curve for the sample with water mobility 6 was similar to others'. However, in the second month, the S. enteric in the sample with water mobility 6 had a reduction of 0.39 logs which was significantly lower (p < 0.05) than the reduction of approximately 1.28 logs for the other samples. The spectra of LF 1H NMR (Fig. 2(c)) has indicates that water mobility of the sample treated with 500 MPa is weaker (p < 0.05) than the rest. These results suggest that water mobility has little influence on the survival of S. enteric at low aw (aw < 0.53). However, it could influence the cultivability of S. enteric in skim milk powder at aw 0.81 (Fig. 3(c)). After two months storage, the decrease of microorganisms in the sample with water mobility 6 was about 5.00 logs. The rest of the samples had about 6.00 logs decrease. This indicates that water mobility may exert its effect at higher aw in low moisture content foods and the cultivability population of S. enteric is higher in the sample with lower water mobility. This level of aw is similar with the aw (0.75e0.97) used by Pham, Vittadini, Levin, and Chinachoti (1999), who reported that the germination time of the L-sorbose resistant strain of Aspergillus nidulans was highly dependent on solid composition and aw (0.75e0.95) but correlation was best with water mobility as indicated by 2H NMR T2 relaxation time. Vittadini, Dickinson, and Chinachoti (2002) reported that a strong mannitolewater interaction, leading to a decreased mobility, played a role in protecting cells from death. The published literature to date indicates little attempt to reveal molecular mechanisms involved in cell survival to discriminate water mobility from aw. It was speculated that a higher water molecular mobility might have allowed initiation but not completion of metabolic reactions leading to cells death (Vittadini et al., 2005). Further studies are needed to understand the metabolic response of the cell to water mobility and aw. 3.4. Fitting of inactivation models of S. enteric in samples with different water mobility and aw Farakos et al. (2013) developed models for non-fat food systems. They observed that the survival data at 36 C could be described by all models with the exception of the log-linear model. In this study,
Table 2 Statistical parameter, standard error (S) and relation index (r) of the model of Logistic model and Exponential association. Water activity
0.33 ± 0.05
0.53 ± 0.04
0.81 ± 0.06
a, b, c
Configuration
0.187 0.756 0.498 0.572 0.498 0.248 0.327 0.658 0.572 0.658 0.756 0.572 0.756 1.000 1.000 1.000 1.000 0.376
Logistic model
Exponential association
a
b
c
r
S
a
b
r
S
4.060 4.354 4.376 4.392 4.226 4.198 4.151 4.644 4.395 4.451 4.370 4.623 5.267 5.135 5.088 5.376 5.521 4.458
11.972 14.567 44.382 41.470 107.747 17.982 13.728 14.282 21.277 6.285 14.973 7.286 6.749 5.626 8.560 10.328 7.675 23.223
0.282 0.284 0.352 0.345 0.448 0.300 0.293 0.318 0.343 0.244 0.300 0.165 0.216 0.188 0.223 0.240 0.117 0.272
0.986 0.995 0.997 0.997 0.999 0.994 0.990 0.984 0.987 0.968 0.987 0.985 0.966 0.950 0.961 0.979 0.968 0.992
0.377 0.253 0.200 0.188 0.106 0.259 0.329 0.470 0.413 0.592 0.408 0.410 0.722 0.843 0.776 0.610 0.723 0.323
4.333 4.703 5.012 5.025 4.739 4.573 4.479 5.046 4.784 4.597 4.716 4.988 5.634 5.553 5.676 5.802 6.204 5.173
0.076 0.068 0.05 0.050 0.054 0.0646 0.072 0.075 0.0696 0.100 0.071 0.057 0.076 0.071 0.062 0.067 0.039 0.047
0.992 0.993 0.983 0.982 0.964 0.988 0.990 0.988 0.979 0.991 0.997 0.997 0.991 0.980 0.987 0.991 0.992 0.989
0.254 0.256 0.399 0.430 0.592 0.323 0.289 0.356 0.463 0.274 0.229 0.157 0.325 0.468 0.388 0.341 0.316 0.332
the parameters in equation, r relation index,
S
standard error.
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F. Lian et al. / Food Control 47 (2015) 1e6
the Curve Expert software was used to find a suitable model with time as X-axis and log (N0/Nt) as Y-axis. The Logistic model (Equation (1)) and Exponential association (Equation (2)) were more suitable for the survival of S. enteric in comparison with other models. (1) Logistic model
y¼
a 1 þ becx
(1)
(2) Exponential association
y ¼ a 1 ebx
(2)
Table 2 shows the parameter details for these two models for all samples. It was observed that Exponential association is more suitable for the inactivation of S. enteric for all samples compared with Logistic model. Exponential association was therefore selected as the model to describe the inactivation of S. enteric. It was also observed that the parameters for the sample with aw 0.81 and T21 0.376 are different from other samples with aw 0.81. This may indicate that water mobility may influence the survival of S. enteric at aw 0.81. 4. Conclusions S. enteric could survive for a long period of time in skim milk powder. The survival could be influenced by water activity and water mobility. The higher the aw of skim milk powder, the lower the S. enteric survival. When aw was at 0.33 and 0.53, water mobility had little effect. However, water mobility also influenced the survival of S. enteric when aw was increased to 0.81. The survival population of S. enteric was higher in the sample with low water mobility. Acknowledgments The authors gratefully acknowledge the financial support provided by Project 31101376 of the National Natural Science Foundation of China, the Program for New Century Excellent Talents in University of Ministry of Education of China (Grant No. NCET-130834) and the Fundamental Research Funds for the Central Universities (JUSRP51406A). References Awuah, G. B., Ramaswamy, H. S., Economides, A., & Mallikarjunan, K. (2005). Inactivation of Escherichia coli K-12 and Listeria innocua in milk using radio
frequency (RF) heating. Innovative Food Science and Emerging Technologies, 6(4), 396e402. Beuchat, L. R., Komitopoulou, E., Beckers, H., Betts, R. P., Bourdichon, F., Fanning, S., et al. (2013). Low-water activity foods: increased concern as vehicles of foodborne pathogens. Journal of Food Protection, 76(1), 150e172. Carneiro, C. S., M arsico, E. T., Ribeiro, R. O. R., Júnior, C. A. C., Alvares, T. S., & Jesus, E. F. O. (2013). Studies of the effect of sodium tripolyphosphate on frozen shrimp by physicochemical analytical methods and Low Field Nuclear Magnetic Resonance (LF 1H NMR). LWT e Food Science and Technology, 50(2), 401e407. CDC (Centers for Disease Control and Prevention). (2007). Multistate outbreak of Salmonella serotype Tennessee infections associated with peanut butter e United States, 2006e2007. CDC e Morbidity and Mortality Weekly Report, 42, 516e517, 56:521e524. Choi, S. G., & Kerr, W. L. (2003). 1H NMR studies of molecular mobility in wheat starch. Food Research International, 36(4), 341e348. Fan, D., Ma, S., Wang, L., Zhao, H., Zhao, J., Zhang, H., et al. (2013). 1H NMR studies of starchewater interactions during microwave heating. Carbohydrate Polymers, 97(2), 406e412. Farakos, S. M., Frank, J. F., & Schaffner, D. W. (2013). Modeling the influence of temperature, water activity and water mobility on the persistence of Salmonella in low-moisture foods. International Journal of Food Microbiology, 166(2), 280e293. Finstad, S., O'Bryan, C. A., Marcy, J. A., Crandall, P. G., & Ricke, S. C. (2012). Salmonella and broiler processing in the United States: relationship to foodborne salmonellosis. Food Research International, 45(2), 789e794. Hills, B. P., Manning, C. E., Ridge, Y., & Brocklehurst, T. (1997). Water availability and the survival of Salmonella typhimurium in porous systems. International Journal of Food Microbiology, 36(2), 187e198. Jung, Y. S., & Beuchat, L. R. (1999). Survival of multidrug-resistant Salmonella typhimurium DT104 in egg powders as affected by water activity and temperature. International Journal of Food Microbiology, 49(1), 1e8. Koch, J., Schrauder, A., Alpers, K., Werber, D., Frank, C., Prager, R., et al. (2005). Salmonella Agona outbreak from contaminated aniseed, Germany. Emerging Infectious Diseases, 11(7), 1124e1128. Laroche, C., Fine, F., & Gervais, P. (2005). Water activity affects heat resistance of microorganisms in food powders. International Journal of Food Microbiology, 97(3), 307e315. Li, C., Liu, D., Zhou, G., Xu, X., Qi, J., Shi, P., et al. (2012). Meat quality and cooking attributes of thawed pork with different low field NMR T21. Meat Science, 92(2), 79e83. Licari, J. J., & Potter, N. N. (1970). Salmonella survival during spray drying and subsequent handling of skimmilk powder. III. Effects of storage temperature on salmonella and dried milk properties. Journal of Dairy Science, 53(7), 877e882. Mattick, K. L., Jørgensen, F., Wang, P., Pound, J., Vandeven, M. H., Ward, L. R., et al. (2001). Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity. Applied and Environmental Microbiology, 67(9), 4128e4136. Molina, E., Papadopoulou, A., & Ledward, D. A. (2001). Emulsifying properties of high pressure treated soy protein isolate and 7S and 11S globulins. Food Hydrocolloids, 15, 263e269. Pham, X., Vittadini, E., Levin, R. E., & Chinachoti, P. (1999). Role of water mobility on mold spore germination. Journal of Agricultural and Food Chemistry, 47(12), 4976e4983. Vesterlund, S., Salminen, K., & Salminen, S. (2012). Water activity in dry foods containing live probiotic bacteria should be carefully considered: a case study with Lactobacillus rhamnosus GG in flaxseed. International Journal of Food Microbiology, 157(2), 319e321. Vittadini, E., Chinachoti, P., Lavoie, J. P., & Pham, X. (2005). Correlation of microbial response in model food systems with physicochemical and “mobility” descriptors of the media. Innovative Food Science and Emerging Technologies, 6(1), 21e28. Vittadini, E., Dickinson, L. C., & Chinachoti, P. (2002). A study of 1H and 2H NMR molecular mobility and structural relaxation in xanthan and locust bean gum mixtures: effect on microbial response. Carbohydrate Polymers, 49(3), 261e269.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Survival of Salmonella enteric in skim milk powder with different water activity and water mobility Feng Lian a, Wei Zhao a, *, Rui-jin Yang a, Yali Tang b, Wendy Katiyo a a b
State Key Laboratory of Food Science & Technology, School of Food Science and Technology, Jiangnan University, No. 1800 Lihu Road, Wuxi 214122, China School of Mechanical Engineering, Department of Packaging Engineering, Jiangnan University, No. 1800 Lihu Road, Wuxi 214122, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 March 2014 Received in revised form 10 June 2014 Accepted 17 June 2014 Available online 25 June 2014
Recently, the safety of low water activity (aw) foods started to become a major concern. It has been observed that microorganisms may not grow in low-aw foods but can survive for rather long periods of time, representing significant risk. Most researchers have been concentrating on the role of water activity. However, little is known about the effect of water mobility on influencing the survival of Salmonella in low-aw foods. In this study, water mobility of skim milk powder was manipulated using ultrahigh pressure to change tertiary structure, thus changing watereprotein interactions. Skim milk powder with different levels of water mobility at similar aw level were obtained to evaluate how aw and water mobility influenced the survival of Salmonella enteric in low-aw foods. Results showed that aw could influence the survival of S. enteric and the lower the aw, the higher the survival. Water mobility had little influence on the survival of S. enteric when aw was 0.33 and 0.53. However, it influenced its survival when aw was increased to 0.81. The survival population of S. enteric was higher in the sample with low water mobility. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Low-water activity foods Water activity Water mobility
1. Introduction Low-water activity foods are those with water activity (aw) levels lower than 0.85 (Beuchat et al., 2013). Low-aw foods include milk powder, egg powder, chocolate, peanut butter and so on. They are usually believed to have the advantages of controlling the growth of pathogenic and spoilage microorganisms. Hence, their microbial safety is usually ignored by the public. Recently, a number of outbreaks associated with low-aw foods and pathogens occurred, resulting in the safety of these foods becoming a major concern (Awuah, Ramaswamy, Economides, & Mallikarjunan, 2005; Beuchat et al., 2013; CDC, 2007; Farakos, Frank, & Schaffner, 2013; Finstad, O'Bryan, Marcy, Crandall, & Ricke, 2012; Koch et al., 2005). The contamination of low-aw foods with Salmonella is not an unknown problem. It has been observed that foodborne pathogens may not grow in low-aw foods but can survive for rather long periods of time once the food is contaminated, thus representing significant risk even at low levels. However, current practices appear inadequate to address this challenge, as
* Corresponding author. Tel./fax: þ86 510 85919150. E-mail addresses: [email protected] (W. Zhao), [email protected] (R.-j. Yang). http://dx.doi.org/10.1016/j.foodcont.2014.06.036 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
contamination continues, even in large multi-national companies that employ experienced food microbiologists and risk managers. It is known that aw is one of the basic properties of a food that exerts major influence on microbial survival and growth (Beuchat et al., 2013; Farakos et al., 2013; Laroche, Fine, & Gervais, 2005). Some research has been conducted in this aspect, but the conclusions about the influence of aw on microbial survival in low-aw foods by different groups are inconsistent. Jung and Beuchat (1999) concluded that Salmonella died more rapidly in higher aw egg white powder than in lower aw powder. However, Mattick et al., (2001) observed that the relationship between microbial survival and aw may change at different temperatures (Mattick et al., 2001). Their study demonstrated that Salmonella at low aw (0.65) was more heat tolerant at temperatures above 70 C than when at higher aw (0.90), but the reverse was true at lower temperatures. Laroche et al., (2005) used Saccharomyces cerevisiae and Lactobacillus plantarum as test organisms. The microorganisms exhibited different survival ability in the low-aw foods (wheat flour and skim milk) when they were adjusted to the same water activity. There was a range of aw for greatest heat resistance for each microorganism and low-aw food across all temperatures. Moreover, some researchers (Hills, Manning, Ridge, & Brocklehurst, 1997) indicated that the molecular mobility of water in low-aw foods is an important factor in microbial survival. Microbial survival in low-aw foods did not
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correlate to water activity, but did correlate to molecular mobility of the water. Vittadini, Chinachoti, Lavoie, and Pham (2005) provided evidence that microbial behavior was more closely associated with molecular mobility in low-aw foods and aw provided good predictions of microbial behavior only in high-aw foods. A recent study (Farakos et al., 2013) determined and discussed how the physical state of water in low-aw foods influences the survival of Salmonella, employing whey protein powder with different water mobility at similar aw levels by pH adjustment and heat denaturation. The results demonstrated that aw significantly influenced the survival of Salmonella at all temperatures, survival increasing with decreasing aw. Water mobility did not significantly influence survival independent of aw. In their study, the water mobility of whey protein powder was manipulated using pH adjustments to change the tertiary structure, thus changing watereprotein interactions. In this way, the solutes for the low-aw foods with different water mobility at similar aw levels are difficult to keep the same. It has been evidenced that use of different solutes to achieve similar water activities produces markedly different microbial survival kinetics (Mattick et al., 2001). In low-aw foods, most of the water is in glassy and rubbery states so the water has limited mobility. Since water molecules must contact the bacterial cell for interaction, the ease of transition between physical states, i.e. molecular mobility, should indicate the potential for interaction between bacterial cells and water. In the present work, water mobility of skim milk powder was manipulated using ultra-high pressure to change the tertiary structure of protein, thus changing watereprotein interactions. Skim milk powder, with different water mobility at similar aw levels and other conditions (such as solutes), was obtained to evaluate how aw and water mobility influence the survival of Salmonella in low-aw foods. 2. Materials and methods 2.1. Modification of skim milk powder The water mobility of skim milk powder (Bright dairy corporation, China) was manipulated using ultra-high pressure adjustments to change the secondary and tertiary structure of protein, thus changing watereprotein interactions. Skim milk powder (500 g) and water (500 mL) were made into a solution. The solution was then transferred into polyethylene bags and a vacuum sealer (A300/16, Multiuac, Germany) was used to remove most of the air. The sealed samples were stored overnight at 4 C. Ultra-high pressure treatments were performed by ultra-high pressure equipment (UHPF-800MPa-5L, Baotou Kefa Co., China) for 10 min at approximately 25 C. The temperature of the vessel was kept at room temperature. The compression and decompression rate was 25 MPa/s. The samples were treated at 100, 200, 300, 400 or 500 MPa. The control sample was hydrated overnight at 4 C but was not treated by ultra-high pressure. After treatment, samples were then poured into sterile Petri dishes and frozen to 80 C overnight in a freezer (HFU 586 Basic, Thermal Scientific, Germany). The samples were then placed in a vacuum freeze drier (7948030, Labconco, US) for 3 days to obtain aw levels below 0.10. Once freeze dried, the samples were broken down to homogenous particles with a mortar and pestle. 2.2. Adjustment of the aw of re-dried samples Re-dried samples were adjusted to the targeted aw values in vacuum desiccators containing various saturated salt solutions of known relative vapor pressures. The targeted aw levels were: 0.33 (Magnesium Chloride), 0.53 (Magnesium Nitrate) and 0.81 (ammonium sulfate). Equilibrium was assumed when there was no
further change in weight. The aw of samples was measured using a water activity meter (Hygrolab2, Rotronic, Switzerland). 2.3. Water mobility determination A low-field 1H Nuclear Magnetic Resonance analyzer (MicroMRCL, Niumag Corporation, Shanghai, China) with a resonance frequency of 21.798 MHz and 0.55 T magnetic field was used for measurements of proton transversal relaxation time (T2). The transversal relaxation time was measured with a CarrePurcelleMeiboomeGill (CPMG) pulse sequence. The experimental parameters were as follows: the 90 pulse width was 3 ms, the 180 pulse width was 7 ms, the Number of Scans (NS) was 16, the Receiver Gain (RG) was set to 20 dB, the echo time (TE) was 80 ms and the echo number was set to 1000. The values of T21 relaxation time were recorded. 2.4. Procedure for inoculating milk powder for storage experiments Salmonella enteric (JFM-125) isolated from powdered infant formula was a gift from Food Biotechnology Center of Jiangnan University. The cultures were maintained at 4 C on LuriaeBertani (LB) ager and activated by transferring a loop of inocula into 150 mL nutrient broth and cultured for 14 h at 37 C. Cultures were harvested by centrifugation (9000 rpm, 5 min), and pellets were washed twice in 150 mL of sterile 0.1% peptone water. Finally, the pellets were re-suspended in 3 mL sterile 0.1% peptone water. The suspension was poured into a sterile Petri dish and put in a desiccator for 3 days to ensure aw of below 0.10. The dried cells were ground with a mortar and pestle. The dried inoculum (0.01 g) was mixed with 0.99 g of equilibrated skim milk powder to provide a 1.00 g sample in a polypropylene vial. It was assumed that the addition of inoculum did not influence aw of the milk powder because the dose was too small to be significant. The samples were placed in retort pouches, then vacuum packaged. The vacuum packaged samples were stored in desiccators at their corresponding relative humidity in an incubator at 37 C. The samples were taken at: 0, 3, 6, 12, 30 and 60 days for survival experiments. 2.5. Enumeration of cultivable Salmonella The plate count method was adopted to enumerate Salmonella populations. Each inoculated sample (1.00 g) was assayed for population of Salmonella at selected time intervals. All samples were respectively dissolved in 9 mL sterile saline and homogenized for 1 min with a vortex (Lab Dancer, IKA, Germany). After homogenization, 1 mL dissolved sample was serially diluted using sterile test tubes containing 9 mL sterile saline to obtain the appropriate degree of dilution. Then 1 mL appropriate dilution was transferred to a Petri dish, and soon afterwards approximately 15 mL nutrient ager, with the temperature about 46 C, was added. When the nutrient agar (Sinopharm Chemical Reagent, China) solidified, the petri dishes were inverted and placed in an incubator. The plates were enumerated after incubation at 37 C for 24 h. 2.6. Statistical analysis and curve fitting Cultivability of S. enteric for each combination of water activity and water mobility was performed. Each experiment was carried out at least in triplicate. Data was analyzed in Microsoft Excel 2010 (Microsoft Corporation, US). Initial data (CFU/g) was converted into log of the survival of S. enteric at a given time. Comparison was made by SPSS software. Curves were fitted using the Curve Expert software (Version 1.3). This software contained 60 models. The most appropriate model was chosen to describe the curves.
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3. Results and discussion 3.1. The survival of S. enteric influenced by water activity The viability results for S. enteric in skim milk powder at three levels of aw at 37 C are shown in Fig. 1. After adjusting to the targeted aw and inoculating with S. enteric, the initial populations were 9.90, 10.01 and 10.19 logs, in samples with aw of 0.33, 0.53 and 0.81, respectively. A reduction of about 3.96 logs occurred within the first month of storage, and about 0.27 logs in the second month at aw 0.33. At aw 0.53, the reduction population during the first month was 3.89 logs, and it was similar to that at aw 0.33. Reduction during the second month was 0.8 logs, and this was significantly higher than at aw 0.33 (p < 0.05). Reduction populations at aw of 0.81 were 4.69 and 1.16 logs, respectively. They were both significantly higher than that at aw 0.33 and 0.53 (p < 0.05). This indicates that the death rate during the first month was higher than the second, at the three aw of skim milk powder. This phenomenon is in agreement with observations by Licari and Potter (1970). These researchers reported that Salmonella in skim milk powder died in a two-stage process: rapid death during the first several weeks followed by slow death during the remaining shelf life. After storage for two months, the reduction populations were 4.23, 4.69 and 5.85 logs at aw levels of 0.33, 0.53 and 0.81, respectively. This is similar with the results of Jung and Beuchat (1999), who reported that after storage of two months at 37 C, log populations in all the samples were less than 1.00 log with an initial population of about 5.00 logs. It also indicates that survival is enhanced as the aw of skim milk powder decreases. Vesterlund, Salminen, and Salminen (2012) studied the effect of aw on the storage stability of probiotics included in crushed flaxseed to identify the right aw level for storage at room temperature. They observed that at the highest aw tested (0.43), the probiotic product was unstable. Within 4 months of storage, the reduction of viable cells was 3.70, 0.81 and 0.29 logs at aw of 0.43, 0.22 and 0.11, respectively, demonstrating that lower aw is more suitable for the survival of microorganisms in dried foods. 3.2. Preparation of samples with different water mobility by ultrahigh pressure Ultra-high pressure is classified as non-thermal processing technique, using the fluid medium (water or oil) for transmitting pressure. Proteins or complex biopolymers subjected to ultra-high
Fig. 2. Continuous relaxation time spectra of samples at three aw and six water mobility (water mobility 1: control, water mobility 2: protein denatured at 100 MPa, water mobility 3: protein denatured at 200 MPa, water mobility 4: protein denatured at 300 MPa, water mobility 5: protein denatured at 400 MPa, water mobility 5: protein denatured at 500 MPa). Fig. 1. Viability of S. enteric at 37 C and 3 water activities (aw) during 2 months of storage in skim milk powder.
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pressure may have their tertiary and secondary structures modified by disrupting hydrophobic and electrostatic interactions (Molina, Papadopoulou, & Ledward, 2001). It has been widely used in the modification of proteins. In this study, water mobility of skim milk powder was manipulated using ultra-high pressure to change the tertiary structure of proteins, thus changing watereprotein interactions, that is water mobility. Skim milk powder with different water mobility at similar aw levels and other conditions (such as solutes) were obtained to evaluate the influence of aw and water mobility on the survival of Salmonella in low-aw foods. LF 1H NMR has been used to investigate water mobility in materials and foods (Choi & Kerr, 2003; Fan et al., 2013). LF 1H NMR can measure proton relaxation, thus can be used to study changes in water mobility (Carneiro et al., 2013). Continuously distributed exponential curve fitting was performed on the obtained T2 relaxation time from samples. Dependence of the nature of skim milk powder, the faster relaxation component which ranges from 0 to 10 ms in the following is called T21. In the present study, T21 represents the water which is closely associated with macromolecules and microbial cells (Li et al., 2012). Fig. 2 shows the distributed T2 relaxation time spectra of skim milk powder submitted to different treatments with ultra-high pressure. Table 1 shows the values of T21 detected by LF 1H NMR according to the different treatments of skim milk powder. Comparison of the continuous distributed curves, at three aw levels of 0.33, 0.53 and 0.81, reveals visible differences between the treated skim milk powder and the control. The treated samples mostly tended to exhibit a broader T21 distribution than the control, which is likely due to the differences in water distribution caused by ultra-high pressure. Fig. 2 shows significant differences in the distribution of water mobility between different treated samples. As shown in Fig. 2(a), a significant increase (p < 0.05) in relaxation time (T21) was observed in the samples treated with pressure of 100, 200, 300 and 400 MPa. The T21 distribution of the sample treated with 500 MPa pressure was broader than the control. However, it was narrower than the rest of the samples. This phenomena indicates that pressure ranging from 100 to 500 MPa affects the T21 distribution at aw 0.33. As shown in Fig. 2(b), the curve of the sample at aw 0.53 is similar to that at aw
Table 1 Range of the LF 1H NMR obtained in skim milk powder, according to the different treatments. aw
Water mobility a
0.33 ± 0.05
1 2b 3c 4d 5e 6f 1 2 3 4 5 6 1 2 3 4 5 6
0.53 ± 0.04
0.81 ± 0.06
a b c d e f
Control. Protein denatured Protein denatured Protein denatured Protein denatured Protein denatured
at at at at at
100 200 300 400 500
MPa. MPa. MPa. MPa. MPa.
T21 (ms) 0.187 0.756 0.498 0.572 0.498 0.248 0.327 0.658 0.572 0.658 0.756 0.572 0.756 1.000 1.000 1.000 1.000 0.376
Fig. 3. Survival of S. enteric in samples at three aw and six water mobility (water mobility 1: control, water mobility 2: protein denatured at 100 MPa, water mobility 3: protein denatured at 200 MPa, water mobility 4: protein denatured at 300 MPa, water mobility 5: protein denatured at 400 MPa, water mobility 6: protein denatured at 500 MPa).
F. Lian et al. / Food Control 47 (2015) 1e6
0.33. The samples treated with pressure ranging from 100 to 400 MPa had similar T21 distributions, indicating that they had similar water mobility. Fig. 2(c) shows the T2 distribution of skim milk powder at aw 0.81. It was also discovered that the samples treated with pressure ranging from 100 to 400 MPa had similar T21 distributions, and as shown in Table 1, the T21 values of these samples were the same. The other significant difference is that the T21 value of the sample treated with 500 MPa is lower than the control, which suggests that its water mobility is lower. 3.3. The cultivability of S. enteric influenced by water mobility Fig. 3 shows the cultivability of S. enteric in skim milk powder treated at different pressure at three aw. It was observed that about 5.00 logs of S. enteric were inactive after two months storage in all the samples from Fig. 3(a). This indicates that water mobility has little influence on the cultivability of S. enteric compared with the control at aw 0.33 (p > 0.05). This was also demonstrated by Farakos et al., (2013). They revealed that water activity significantly influenced the survival of Salmonella in low-aw foods (aw < 0.60), while water mobility had no effect independent of aw. Vittadini et al. (2005) investigated the correlation of microbial response in model food systems with physico-chemical and “mobility” descriptors of the media. They studied the role of aw, “macromolecular” mobility and water molecular dynamics in multicomponent model food (ranging from solid to liquid) systems in their correlation with microbial activity. It was found that a correlation between cell death and increased 1H and 2H NMR mobility in low-aw foods (solid and semisolid media), indicating NMR molecular mobility is a possible tool to describe water availability in solid and semisolid systems (dry and intermediate moisture foods), while in a high moisture system, the use of water activity remains a valid indicator for microbial activity. However, it is difficult to differentiate the action of water mobility from that of aw because different water mobility with different aw were employed to correlate with microbial response. To further discriminate the influence of molecular mobility on microbial survival behavior in low-aw foods, the present study prepared skim milk powder with different water mobility at similar aw levels to evaluate how aw and water mobility influence the survival of Salmonella in low-aw foods. As shown in Fig. 3(b), all samples had similar reduction during the two months storage period, which was similar at aw 0.33. It also indicates that
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water mobility has little influence on the cultivability of S. enteric at this level of aw (p > 0.05). The cultivability of S. enteric at aw 0.81 after storage at 37 C was assayed in the treated and control skim milk powder for up to two months (Fig. 3(c)). During the first month, the curve for the sample with water mobility 6 was similar to others'. However, in the second month, the S. enteric in the sample with water mobility 6 had a reduction of 0.39 logs which was significantly lower (p < 0.05) than the reduction of approximately 1.28 logs for the other samples. The spectra of LF 1H NMR (Fig. 2(c)) has indicates that water mobility of the sample treated with 500 MPa is weaker (p < 0.05) than the rest. These results suggest that water mobility has little influence on the survival of S. enteric at low aw (aw < 0.53). However, it could influence the cultivability of S. enteric in skim milk powder at aw 0.81 (Fig. 3(c)). After two months storage, the decrease of microorganisms in the sample with water mobility 6 was about 5.00 logs. The rest of the samples had about 6.00 logs decrease. This indicates that water mobility may exert its effect at higher aw in low moisture content foods and the cultivability population of S. enteric is higher in the sample with lower water mobility. This level of aw is similar with the aw (0.75e0.97) used by Pham, Vittadini, Levin, and Chinachoti (1999), who reported that the germination time of the L-sorbose resistant strain of Aspergillus nidulans was highly dependent on solid composition and aw (0.75e0.95) but correlation was best with water mobility as indicated by 2H NMR T2 relaxation time. Vittadini, Dickinson, and Chinachoti (2002) reported that a strong mannitolewater interaction, leading to a decreased mobility, played a role in protecting cells from death. The published literature to date indicates little attempt to reveal molecular mechanisms involved in cell survival to discriminate water mobility from aw. It was speculated that a higher water molecular mobility might have allowed initiation but not completion of metabolic reactions leading to cells death (Vittadini et al., 2005). Further studies are needed to understand the metabolic response of the cell to water mobility and aw. 3.4. Fitting of inactivation models of S. enteric in samples with different water mobility and aw Farakos et al. (2013) developed models for non-fat food systems. They observed that the survival data at 36 C could be described by all models with the exception of the log-linear model. In this study,
Table 2 Statistical parameter, standard error (S) and relation index (r) of the model of Logistic model and Exponential association. Water activity
0.33 ± 0.05
0.53 ± 0.04
0.81 ± 0.06
a, b, c
Configuration
0.187 0.756 0.498 0.572 0.498 0.248 0.327 0.658 0.572 0.658 0.756 0.572 0.756 1.000 1.000 1.000 1.000 0.376
Logistic model
Exponential association
a
b
c
r
S
a
b
r
S
4.060 4.354 4.376 4.392 4.226 4.198 4.151 4.644 4.395 4.451 4.370 4.623 5.267 5.135 5.088 5.376 5.521 4.458
11.972 14.567 44.382 41.470 107.747 17.982 13.728 14.282 21.277 6.285 14.973 7.286 6.749 5.626 8.560 10.328 7.675 23.223
0.282 0.284 0.352 0.345 0.448 0.300 0.293 0.318 0.343 0.244 0.300 0.165 0.216 0.188 0.223 0.240 0.117 0.272
0.986 0.995 0.997 0.997 0.999 0.994 0.990 0.984 0.987 0.968 0.987 0.985 0.966 0.950 0.961 0.979 0.968 0.992
0.377 0.253 0.200 0.188 0.106 0.259 0.329 0.470 0.413 0.592 0.408 0.410 0.722 0.843 0.776 0.610 0.723 0.323
4.333 4.703 5.012 5.025 4.739 4.573 4.479 5.046 4.784 4.597 4.716 4.988 5.634 5.553 5.676 5.802 6.204 5.173
0.076 0.068 0.05 0.050 0.054 0.0646 0.072 0.075 0.0696 0.100 0.071 0.057 0.076 0.071 0.062 0.067 0.039 0.047
0.992 0.993 0.983 0.982 0.964 0.988 0.990 0.988 0.979 0.991 0.997 0.997 0.991 0.980 0.987 0.991 0.992 0.989
0.254 0.256 0.399 0.430 0.592 0.323 0.289 0.356 0.463 0.274 0.229 0.157 0.325 0.468 0.388 0.341 0.316 0.332
the parameters in equation, r relation index,
S
standard error.
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the Curve Expert software was used to find a suitable model with time as X-axis and log (N0/Nt) as Y-axis. The Logistic model (Equation (1)) and Exponential association (Equation (2)) were more suitable for the survival of S. enteric in comparison with other models. (1) Logistic model
y¼
a 1 þ becx
(1)
(2) Exponential association
y ¼ a 1 ebx
(2)
Table 2 shows the parameter details for these two models for all samples. It was observed that Exponential association is more suitable for the inactivation of S. enteric for all samples compared with Logistic model. Exponential association was therefore selected as the model to describe the inactivation of S. enteric. It was also observed that the parameters for the sample with aw 0.81 and T21 0.376 are different from other samples with aw 0.81. This may indicate that water mobility may influence the survival of S. enteric at aw 0.81. 4. Conclusions S. enteric could survive for a long period of time in skim milk powder. The survival could be influenced by water activity and water mobility. The higher the aw of skim milk powder, the lower the S. enteric survival. When aw was at 0.33 and 0.53, water mobility had little effect. However, water mobility also influenced the survival of S. enteric when aw was increased to 0.81. The survival population of S. enteric was higher in the sample with low water mobility. Acknowledgments The authors gratefully acknowledge the financial support provided by Project 31101376 of the National Natural Science Foundation of China, the Program for New Century Excellent Talents in University of Ministry of Education of China (Grant No. NCET-130834) and the Fundamental Research Funds for the Central Universities (JUSRP51406A). References Awuah, G. B., Ramaswamy, H. S., Economides, A., & Mallikarjunan, K. (2005). Inactivation of Escherichia coli K-12 and Listeria innocua in milk using radio
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