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Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure
Food Control 28 (2012) 273e278
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Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure Baowei Yang a, Ying Shi a, Xiaodong Xia a, Meili Xi a, Xin Wang a, Baoyi Ji a, Jianghong Meng a, b, * a b
College of Food Science and Engineering, Northwest A&F University, 28# Xinong Road, Yangling 712100, PR China Joint Institute for Food Safety and Applied Nutrition, and Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 November 2011 Received in revised form 11 April 2012 Accepted 25 April 2012
Plate counting, viability test, pulse field gel electrophoresis (PFGE) and transmission electron microscopy (TEM) were used to investigate the effect of high hydrostatic pressure (HHP) on Salmonella, Escherichia coli, Shigella and Staphyloccocus aureus in raw milk in order to determine the optimal inactivation conditions, and further understand the mechanisms of HHP on pathogens inactivation in food. The results exhibited that 300 Mpa treatment with 30 min duration at 25 C was the optimal condition for Salmonella, E. coli, Shigella and S. aureus inactivation. Damage of the cell wall, cell membrane and cytoplasmic components by high pressure treatment can be observed in TEM micrographs. The injured cells could not be recovered, the growth rate of survivors was much lower than that of the untreated cells. PFGE showed neither corresponding DNA bands with same molecular weight nor DNA bands with same brightness could be found in the lanes between HHP treated pathogens and untreated ones. The results indicated that HHP processing can be applied to inactivate pathogens in food, the inactivation is mainly due to cell membrane damage, cell wall rupture and chromosome DNA degradation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: High hydrostatic pressure Pathogens Inactivation
1. Introduction High hydrostatic pressure (HHP) processing has been considered as a promising alternative to conventional thermal pasteurization for food preservation due to its potential to inactivate spoilage and pathogenic bacteria, however, causing minimal loss of vitamins and flavor compounds, and maintaining the quality attributes of food products (Kaletunc, Lee, Alpas, & Bozoglu, 2004; Kimura et al., 1994; Lee & Kaletunc, 2010). Over a century ago, hydrostatic pressure treatment was demonstrated an effective method to extend shelf life of milk and other foods products, and the first commercial HHP treated product appeared on the market in 1991 in Japan (Patterson & Kilpatrick, 1998; Porretta, Birzi, Ghizzoni, & Vicini, 1995; Smelt, 1998; Trujillo, 2002). HHP processing is now being used for processing many types of food such as fruit juices, jams, sauces, rice, cakes and desserts. Most studies showed HHP causes a number of changes in morphology, cell wall, thermotropic phase in cell membrane lipids, dissociation of ribosomes, biochemical reactions, and loss of genetic functions of the microorganisms (Cheftel, 1995; Ritz, Freulet, Orange, & Federighi, 2000; Wouters, Glaasker, & Smelt, 1998), these all are proposed as possible reasons and mechanisms that caused inactivation of microorganisms subjected to HHP (Alpas, Lee, Bozoglu, & * Corresponding author. College of Food Science and Engineering, Northwest A&F University, 28# Xinong Road, Yangling 712100, PR China. Tel./fax: þ86 29 87091391. E-mail address: [email protected] (J. Meng). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.04.030
Kaletunc, 2003; Kaletunc et al., 2004; Mackey, Forestiere, Isaacs, Stenning, & Brooker, 1994). However, microbial resistance to HHP varies among bacteria, their physiological state, and food composition at the time of pressurization. The exact mechanism of inactivation caused by HHP is still not clearly understood (Kaletunc et al., 2004; Patterson, Quinn, Simpson, & Gilmour, 1995; Robey et al., 2001). Salmonella, Escherichia coli, Shigella and Staphyloccocus aureus are significant foodborne pathogens and commonly found in raw milk. Interest in HHP application to milk pasteurization has recently increased (Trujillo, 2002). When milk is treated under HHP, not only the pathogens can be inactivated, but the quality characteristics, such as taste, flavour, vitamins, and nutrients can be improved (Johnston, Austin, & Murphy, 1992; LopezFandino, Carrascosa, & Olano, 1996; Trujillo, 2002). The advantages of HHP merit further exploring its application for milk and other food processing and preservation. The objective of this study was to determine optimal conditions for inactivating Salmonella, E. coli, Shigella and S. aureus in raw milk by HHP, and to investigate HHP-induced pathogens morphological changes, growth conditions, and chromosome variations. 2. Materials and methods 2.1. Bacterial culture preparation Salmonella typhimurium LT2, E. coli ATCC25922, Shigella dysenteriae and S. aureus ATCC29213 were obtained from State Food and Drug Administration, Beijing, China. The cultures were stored at
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80 C in 40% (vol/vol) sterile glycerol. The cultures were streaked on LuriaeBertani (Beijing Land Bridge Technology Co., Ltd, Beijing, China) plates and incubated for 24 h at 37 C. A single typical colony was selected and inoculated into 90 ml of LuriaeBertani broth (Beijing Land Bridge Technology Co., Ltd, Beijing, China). After incubation at 37 C for 18 h with shaking at 150 rpm, the cells at stationary phase were harvested by centrifugation (10 min at 2400 g) and washed once in phosphate-buffered saline (PBS; 0.01 M phosphate, 0.137 M NaCl, pH 7.3). The washed cells were resuspended in sterile milk (purchased from cattle farm of Northwest A&F University, China) to a bacterial concentration of approximately 1 108 CFU$ml1. Inoculated milk samples were stored at 4 C for 24 h prior to HHP treatment to allow the cells to acclimatize to the new environment. 2.2. HHP treatment After 24 h storage at 4 C, the milk samples were placed in sterile polyethylene bags (Yangling Sanhe S&T Co., Ltd, Yangling, China) for HHP treatment. Air was removed from the bags prior to heat sealing. Each bag was placed in a second bag and sealed again to prevent contamination of the HHP equipment if the primary bag broke. The HHP equipment (Baotou Kefa High Pressure Technology Limited Company, Baotou, China) is capable of operating up to 750 Mpa was employed to apply pressure to the inoculated milk samples. The pressure transmission fluid was castor oil, and the temperature increase due to adiabatic heating was approximately 2 C per 100 Mpa. Pressure come-up rate was approximately 4200 Mpa per min and pressure release time was approximately 20 s. Pressurization time reported in this study excluded the time for pressure increase and release times. Pressure level and time of pressurization were set manually. Samples containing each kind of bacteria were pressured in 3-duplicate at designed pressure and time, and unpressured strains were used as controls. Both pressure treated and untreated cell suspensions were centrifuged (J2-21, Beckman, Palo Alto, Calif.) at 10,000 g for 10 min at 4 C to form pellets prior to transmission electron microscopy analysis.
pathogens, respectively. Cell pellets were prepared from untreated and pressure treated cell suspensions by centrifugation at 4000 g for 10 min at 4 C and washed once with 50 ml of sterile distilled water. Cell pellets were transferred to sterile centrifuge tube (1.5 ml) and resuspended in 1 ml of 0.1 M phosphate buffer at pH 7.4. After the suspension was centrifuged, the pellet was embedded in 2% agarose. The agarose was cut into 1 mm3 pieces and postfixed for 1 h in 1% osmium tetroxide (OsO4) in phosphate buffer, then the samples were rinsed in distilled water and stained for 1 h in 1% aqueous uranyl acetate. After dehydration through an ascending series of ethanol solutions (50, 70, 95, and 100%), cells in agar were transferred to propylene oxide, then were infiltrated and embedded in spurr’s resin, and sections (70 nm) were obtained with an ultramicrotome and stained with Reynolds’ lead citrate prior to examination under a transmission electron microscope (JEMd1230, Japan Electronics Co., Ltd.) at 60 kV. 2.6. Pulse field gel electrophoresis (PFGE) PFGE was used to assess the level of DNA injuries caused by HHP treatment (Ribot et al., 2006). Briefly, agarose-embedded DNA was digested with 50 U of XbaI (TaKaRa, Dalian, China) for 1.5e2 h in a water bath at 37 C. The restriction fragments were separated by electrophoresis in 0.5 TBE buffer at 14 C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA) with pulse times of 2.16 se63.8 s. The gels were stained with ethidium bromide, and DNA bands were visualized with UV transillumination (Bio-Rad). The initial cell numbers of HHP treated and non-treated were identical before HHP treatment, which can be acquired by adding same volume of bacterial suspension to the same volume of sterilized milk. 2.7. Statistical analysis Inactivation rates of the four pathogens under different pressure treatments and duration times were analyzed using Chi-Square tests with SPSS software (version 18 (SPSS, Chicago, IL, USA)).
2.3. Enumeration of cells 3. Results To determinate the initial number of cells before pressure treated and the number of treated milk samples, 1 ml of each sample was used to prepare 10-fold dilutions in buffered peptone water (Beijing Land Bridge Technology Co., Ltd, Beijing, China), subsequently, 1 ml of these dilutions were plated in duplicate onto LuriaeBertani agar plates and incubated at 37 C for 24 h. Plates containing 30e300 colonies were selected for counting. Results were expressed as CFU per ml, the lethality rate was calculated as the percentage of difference between the colony counts of the untreated and treated samples in untreated ones. 2.4. Viability test The viability test was performed according to the previous methods (Brinez, Roig-Sagues, Herrero, & Lopez, 2007; Patterson et al., 1995) with some modifications. Briefly, the diluted milk samples were prepared and plated in duplicate in LB, TSA-YE (Beijing Land Bridge Technology Co, Ltd.,), TSA-YE þ NaCl (3%) and TSA-YE þ NaCl (10%) plates, and incubated at 37 C for 24 h, the morphology of the colonies was monitored to assess the lethality and the level of injury caused by HHP treatment. 2.5. Electron microscopy E. coli ATCC25922 and S. aureus ATCC29213 were the representatives of Gram negative (G) and Gram positive (Gþ)
3.1. Effect of duration time on inactivation of the pathogens Overall, HHP inactivation of the pathogens was more effective against G than Gþ bacteria. Under the same pressure treatment condition, the longer the treatment was the better the inactivation could be achieved. The inactivation rates differed among the three G pathogens under the same pressure and duration time treatment; for example, under 300 Mpa for 10 min treatment, Shigella exhibited the highest inactivation rate (94.6%) while Salmonella had the lowest inactivation rate (93.1%) among the pathogens. When the samples were treated under 300 Mpa for 20, 30, 40, or 50 min), the inactivation rate for Salmonella was the highest whereas S. aureus was the lowest (Fig. 1). Significant differences (p < 0.05) were observed among the inactivation rates of the four pathogens during HHP treatment. Inactivation rates of E. coli, Salmonella and Shigella were relative high in prophase (from 10 min to 20 min) under 300 Mpa pressure treatment, although the inactivation rate of E. coli was lower than that of Salmonella and Shigella. The inactivation rate of the three pathogens decreased slowly when the duration time was increased from 30 min to 50 min, inactivation rate for Salmonella has no apparent change (Fig. 1). Different from the three G pathogens, the inactivation rate of S. aureus increased slowly during the whole HHP treatment, almost all cells could be inactivated after 50 min treatment (Fig. 1). Thus, 30 min was considered as a satisfactory duration time for HHP treatment.
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In a c t i v a t i o n R a t e (%)
in milk (Fig. 3). The results indicated that 300 Mpa treatment with 30 min duration at 25 C was the optimal condition for pathogens inactivation. 3.3. Viability test E.Coli Salmonella Staphyloccocus aureus Shigella
Time (Unit: Min) Notes: The abscissa axis indicates pressure treatment time, the ordinate axis indicates inactivation rate under 300 Mpa treatment. Min: Minute Fig. 1. Effect of duration time on inactivation rates of 4 pathogens at 25 C under 300 Mpa.
3.2. Effect of pressure on inactivation of the pathogens
In a c tiv a tio n R a te (% )
Significant differences (p < 0.05) were observed between inactivation rates among the four pathogens under different pressurization (Fig. 2). When treated at 25 C for 30 min under different pressures, the general trend of inactivation rate of the four pathogens exhibited higher inactivation rates with higher pressure, and the inactivation rates of G was higher than that of Gþ ones (Fig. 2). The inactivation rates of Salmonella and E. coli increased slowly and showed no significant change when the pressure increased from 100 Mpa to 200 Mpa, in contrast, the inactivation rate of Shigella and S. aureus increased rapidly. The inactivation rates increased significantly and almost all live cells were inactivated when the pressure increased from 200 Mpa to 300 Mpa. The inactivation rate of all pathogens exhibited little variation when the pressure increased from 300 Mpa to 500 Mpa. Consequently, 300 Mpa was considered as the satisfactory pressure (Fig. 2). Significant differences were observed among different pressures, duration time and pathogens when different pressures (300, 350 and 400 Mpa), duration times (5, 10, 20 and 30 min) and pathogens were chosen to analyze interactions among pressure, duration time and pathogen species to determine optimal conditions in inactivating pathogens
Salmonella E.Coli Staphyloccocus aureus Shigella
Pressure (Unit: M pa) Notes: The abscissa axis indicates different pressure, the ordinate axis indicates inactivation rate under HHP treatment. Mpa: Million Pascal Fig. 2. Effect of pressure on inactivation rates of 4 pathogens at 25 C with 30 min duration.
The effect of HHP treatment on the viability of the pathogens was determined using colony enumeration by direct plating. When the untreated milk sample, milk samples treated under 100 Mpa30 min and 400 Mpa-30 min were spread on LB plates with the same volume and pathogens concentration, respectively, and incubated at 37 C for 24 h, the colony numbers of untreated cells were found more than that of the treated samples, and the colonies were bigger than that of the pressure treated ones. Viable colonies of 400 Mpa-30 min treatment cells were smaller than that of 100 Mpa-30 min treated ones. Since LB medium is nutrient rich and non-selective, and therefore severely injured cells may not have been recovered, and the growth speed of recovered cells was much slower than the normal ones. When the pressure treated E. coli, Salmonella and Shigella were inoculated onto TSA-YE and TSA-YE þ NaCl (3%) plates, pressure treated S. aureus was inoculated onto TSA-YE and TSA-YE þ NaCl (10%) plates, respectively, and were incubated at 37 C for 24 h, the tiny colonies of treated pathogens could be found on TSA-YE medium, while very few colonies could be observed on TSAYE þ NaCl medium, either the concentration of NaCl was 3% or 10%. Since these media were somewhat selective osmotic pressure, the NaCl may penetrate the broken cell membrane and therefore injured cells could not be recovered and grow on these media. 3.4. Effect of pressure on chromosome DNA of the pathogens Effect of pressure on chromosome DNA of E. coli and Shigella was shown in Fig. 4. After the chromosome DNA of the untreated and 500 Mpa-30 min treated E. coli was digested with XbaI and separated by electrophoresis, respectively, different number and brightness of DNA bands can be found in the lane of 500 Mpa30 min treated and untreated E. coli. Furthermore, there was no corresponding band with same molecular weight in the two lanes even if the initial cells were the same. At the same time, the DNA bands of the 500 Mpa-30 min treated E. coli were not as bright as the untreated ones, although the concentration of the cells was identical. Same result could be found when untreated and pressure treated Shigella cells were digested and electrophoresesed. The reason may be that HHP treatment damaged the integrity of the cells and the chromosome DNA of the pathogens. After HHP treatment, bacteria cell wall, cell membrane, and some DNA hydrogen bonds and covalent bonds are broken, the intact DNA chain was destroyed. Therefore, some small DNA fragments were lost after a 20 h electrophoresis. 3.5. Electron microscopy Untreated E. coli and S. aureus cells exhibited intact cell wall, cell membrane, uniform cell cytoplasm, and electron-transparent regions of nucleoid in electron micrographs (Fig. 5a, b). However, application of HHP leads to morphological changes in the internal and external structures. The most visible changes with application of HHP were a double-track bilayer structure of cell membrane instead of a single-thick-layer appearance, and the enlargement of electron-transparent ranges in the cell cytoplasm. Treatment of HHP at 500 Mpa for 30 min resulted in expanded nucleoid regions and compacted interior regions (Fig. 5a’, b’). Most of lower pressure treated cells maintained a distinct membrane and cell wall, whereas, in higher pressure (such as 500 Mpa) treated cells,
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A 100.0% 99.0% 98.0%
a
97.0% 96.0% 95.0% 94.0%
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d
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30
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Pressure (Mpa) Effect of Pressure on Inactivation Rate to the Pathogens with 30 min Duration
Inactivation Ratio (%)
b
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93.0%
C
100.0% 99.0%
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Inactivation Ratio (%)
Inactivation Ratio (%)
B
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a 100.0%
10 Time (Min)
Effect of Time on Inactivation Rate to the Pathogens with 300Mpa
c b
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Notes: A: The abscissa axis indicates different pressure, the ordinate axis indicates inactivation rate under HHP treatment to the four pathogens.
99.0% 98.0%
B: The abscissa axis indicates different treat time, the ordinate axis indicates inactivation rate under 300Mpa treatment to the four pathogens.
97.0% 96.0%
E.Coli
Shigella
Salmonella
94.0%
Pathogen
Staphyloccocus aureus
95.0%
C: The abscissa axis indicates four pathogens, the ordinate axis indicates inactivation rate under the given conditions. Min: Minute Mpa: Million Pascal
Inactivation Rate of Each Pathogens under tha Given Pressure and Time
Fig. 3. Interactions among pressure, duration time and pathogen species on inactivation rate.
breakdown of the peptidoglycan layer was evident, parts of the outer layer appeared to slough off (Fig. 5a’, b’). Aggregation of cytoplasmic material and enlarged electron-transparent region of nucleoid with a fibrous appearance were also observed (Fig. 5a’, b’). 4. Discussion
Fig. 4. DNA profiles of E. coli and Shigella after PFGE.
E. coli, Salmonella, Shigella and S. aureus are foodborne pathogens of concern for the dairy industry, and raw milk has been identified as an important vehicle for the transmission of these pathogens and been implicated in outbreaks (Brinez et al., 2007; Chapman, 1993; Rajeev Kumar, 2010; Rodriguez, Arques, Nunez, Gaya, & Medina, 2005; Tambekar DH., 2006). Heat treatment is one of the most efficient and economical processes for achieving microbial inactivation in milk, however, it cannot be used to treat heat-labile food and compounds. Furthermore, high temperature treatments may result in undesirable effects in milk, such as offflavor, non-enzymatic browning and denaturation of certain vitamins and proteins (Brinez et al., 2007; Diels, Callewaert, Wuytack, Masschalck, & Michiels, 2005; Kheadr, Vachon, Paquin, & Fliss, 2002). The application of HHP on milk and dairy products has been shown to be an effective technology to inactivate microorganisms including most infectious foodborne pathogens. In addition to microbial destruction, it has been reported that HHP improves rennet of acid coagulation of milk without detrimental effects on important quality characteristics (Brinez et al., 2007; Johnston et al., 1992; Trujillo, 2002). The present study showed a higher inactivation rate to Salmonella, Shigella and E. coli by high pressure treatment at 300 Mpa than that to S. aureus. Among three G pathogens, Shigella was more
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Fig. 5. Cell structures of untreated and 500 Mpa-30 min treated E. coli and S. aureus under TEM.
resistant to high pressure than Salmonella and E. coli. The work reported here is in agreement with previous researches that showed when treated by HHP, variations of microorganisms in resistance to high pressure are common between and within species, and Gþ bacteria appears to be more pressure resistant than G one (Benito, Ventoura, Casadei, Robinson, & Mackey, 1999; Patterson et al., 1995; Rivalain, Roquain, & Demazeau, 2010), and S. aureus is significantly more resistant than other microorganisms such as S. enteric serovar Typhimurium and E. coli when inoculated and treated under different conditions (between 100 and 300 Mpa of pressure and between 5 and 40 C of inlet temperature) (Diels, Wuytack, & Michiels, 2003; Wuytack et al., 2003). This study and previous studies suggest that S. aureus may be assigned as a potential indicator bacterium when HHP is adopted for pathogens inactivation in milk production. At the same time, our results also indicated that when milk was treated with HHP, at lower pressure, the inactivation rate of S. aureus and Shigella increased faster than that of E. coli and Salmonella, this hence suggested that relative lower pressure treatment to milk is a good choice for pathogens inactivation. When comparing the grow condition of the four untreated and pressure treated strains on LB agar, TSA-YE agar and TSA-YE-NaCl agar, respectively, normal and uniform colonies could be found on LB, TSA-YE and TSA-YE with NaCl (3%) media, which were nonor less-selective, when the untreated bacteria were inoculated. However, on TSA-YE-NaCl media with low NaCl concentration (3%), even on the LB and TSA-YE media, the HHP treated strains grew very slowly with tiny colonies, and those that subjected to higher pressure and longer duration time could not grow on LB and TSA-YE media, which are not selective. At the same time, the colony
number of treated strains was less than that of the untreated one even if the same inoculation. On TSA-YE with higher NaCl concentration (10%), the HHP treated cells could hardly grow, which indicated that the strains were sublethally injured or injured after HHP treatment and our results were in agreement with the previous studies (Ponce, Pla, Capellas, Guamis, & Mor-Mur, 1998; Ponce, Pla, Sendra, Guamis, & Mor-Mur, 1999). Cell membrane is often considered as the first site of damage in pressure injured bacteria, the sublethal damage or damage of the membrane and changes in membrane fluidity by HHP treatment could facilitate the access of NaCl to the cytoplasm of bacteria (Hauben, Wuytack, Soontjens, & Michiels, 1996; Ritz, Tholozan, Federighi, & Pilet, 2002; Rodriguez et al., 2005). At the same time, the cytoplasma and other intracellular substances may outflow, suggesting that the injured survivors became sensitive to NaCl after pressurization, even for the NaCl tolerant S. aureus. The TEM images also demonstrated the membrane and cell wall were damaged after HHP treatment (Fig. 5a’, b’). HHP treatment could cause alterations of membrane bound protein and enzyme functionality as well (Kato & Hayashi, 1999), after treatment at 350 Mpa or 400 Mpa, outer and internal membrane protein contents of Salmonella typhimurium were drastically modified, some of proteins almost entirely disappeared (Ritz et al., 2000). The degradation of some proteins impaired the ability of cells to reproduce and develop colonies on non-selective media, such as LB and TSA-YE. Previous studies on mechanism of inactivation of microorganisms by HHP focused on perturbation of the cell membrane, the damaged cell wall and cell membrane function, inhibition or inactivation of essential enzyme systems, the destruction of
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ribosome and the changes of cell morphologies (Cheftel, 1995; Earnshaw, Appleyard, & Hurst, 1995; Kaletunc et al., 2004; Lee & Kaletunc, 2010; Ritz et al., 2000; Smelt, 1998). Our results were in agreement with some of the previous studies, and further confirmed that the cell membrane and cell wall of the E. coli and S. aureus were totally damaged after different pressure treatment (Fig. 5a’, b’). Although few investigations observed that the thermal stability of DNA peak transition happened using differential scanning calorimetry during pressure treatment (Alpas et al., 2003; Dubins, Lee, Macgregor, & Chalikian, 2001; Kaletunc et al., 2004). This study is the first one using PFGE to analyze DNA alteration before and after bacteria was pressure treated. In our survey, PFGE results showed genomic difference between HHP untreated and treated E. coli and Shigella cells, as well as the difference of DNA concentration, which indicated that the DNA of bacteria was damaged during pressure treatment, and this should be one of the most important potential mechanisms of inactivation by HHP. On summary, this work has shown that HHP processing can be used to inactivate some of the common pathogens in food, such as Salmonella, Shigella and E. coli, and even the pressure resistant S. aureus. The optimal condition for pathogens inactivation in milk by using of HHP was under 300 Mpa at 25 C with duration time of 30 min. Mechanisms for inactivation of pathogens are possibly due to cell membrane damage, cell wall rupture and chromosome DNA degradation. Acknowledgement The research work was supported in part by the Houji Scholar Program, and the Principal Fund of Northwest A&F University, Yangling, Shaanxi, China. We also appreciate Drs Shuangkui Du and Min Sheng in Northwest A&F University for data analysis by using SPSS software. References Alpas, H., Lee, J., Bozoglu, F., & Kaletunc, G. (2003). Evaluation of high hydrostatic pressure sensitivity of Staphylococcus aureus and Escherichia coli O157: H7 by differential scanning calorimetry. International Journal of Food Microbiology, 87, 229e237. Benito, A., Ventoura, G., Casadei, M., Robinson, T., & Mackey, B. (1999). Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses. Applied and Environmental Microbiology, 65, 1564e1569. Brinez, W. J., Roig-Sagues, A. X., Herrero, M. M. H., & Lopez, B. G. (2007). Inactivation of Staphylococcus spp. strains in whole milk and orange juice using ultra high pressure homogenisation at inlet temperatures of 6 and 20 degrees C. Food Control, 18, 1282e1288. Chapman, P. A. (1993). Untreated milk as a source of verotoxigenic Escherichia-Coli O157 (Vol 133, Pg 171, 1993). Veterinary Record, 133, 252. Cheftel, J. C. (1995). Review: high-pressure, microbial inactivation and food preservation. Food Science and Technology International, 1, 75e90. Diels, A. M. J., Callewaert, L., Wuytack, E. Y., Masschalck, B., & Michiels, C. W. (2005). Inactivation of Escherichia coli by high-pressure homogenisation is influenced by fluid viscosity but not by water activity and product composition. International Journal of Food Microbiology, 101, 281e291. Diels, A. M. J., Wuytack, E. Y., & Michiels, C. W. (2003). Modelling inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation at different temperatures. International Journal of Food Microbiology, 87, 55e62. Dubins, D. N., Lee, A., Macgregor, R. B., & Chalikian, T. V. (2001). On the stability of double stranded nucleic acids. Journal of the American Chemical Society, 123, 9254e9259. Earnshaw, R. G., Appleyard, J., & Hurst, R. M. (1995). Understanding physical inactivation processes: combined preservation opportunities using heat, ultrasound and pressure. International Journal of Food Microbiology, 28, 197e219. Hauben, K. J. A., Wuytack, E. Y., Soontjens, C. C. F., & Michiels, C. W. (1996). High-pressure transient sensitization of Escherichia coli to lysozyme and nisin
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International Journal of Dairy Sciences, 1, 32e35. Trujillo, A. J. (2002). Applications of high-hydrostatic pressure on milk and dairy products. High Pressure Research, 22, 619e626. Wouters, P. C., Glaasker, E., & Smelt, J. P. P. M. (1998). Effects of high pressure on inactivation kinetics and events related to proton efflux in Lactobacillus plantarum. Applied and Environmental Microbiology, 64, 509e514. Wuytack, E. Y., Phuong, L. D. T., Aertsen, A., Reyns, K. M. F., Marquenie, D., & De Ketelaere, B. (2003). Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments. Journal of Food Protection, 66, 31e37.
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Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure Baowei Yang a, Ying Shi a, Xiaodong Xia a, Meili Xi a, Xin Wang a, Baoyi Ji a, Jianghong Meng a, b, * a b
College of Food Science and Engineering, Northwest A&F University, 28# Xinong Road, Yangling 712100, PR China Joint Institute for Food Safety and Applied Nutrition, and Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 November 2011 Received in revised form 11 April 2012 Accepted 25 April 2012
Plate counting, viability test, pulse field gel electrophoresis (PFGE) and transmission electron microscopy (TEM) were used to investigate the effect of high hydrostatic pressure (HHP) on Salmonella, Escherichia coli, Shigella and Staphyloccocus aureus in raw milk in order to determine the optimal inactivation conditions, and further understand the mechanisms of HHP on pathogens inactivation in food. The results exhibited that 300 Mpa treatment with 30 min duration at 25 C was the optimal condition for Salmonella, E. coli, Shigella and S. aureus inactivation. Damage of the cell wall, cell membrane and cytoplasmic components by high pressure treatment can be observed in TEM micrographs. The injured cells could not be recovered, the growth rate of survivors was much lower than that of the untreated cells. PFGE showed neither corresponding DNA bands with same molecular weight nor DNA bands with same brightness could be found in the lanes between HHP treated pathogens and untreated ones. The results indicated that HHP processing can be applied to inactivate pathogens in food, the inactivation is mainly due to cell membrane damage, cell wall rupture and chromosome DNA degradation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: High hydrostatic pressure Pathogens Inactivation
1. Introduction High hydrostatic pressure (HHP) processing has been considered as a promising alternative to conventional thermal pasteurization for food preservation due to its potential to inactivate spoilage and pathogenic bacteria, however, causing minimal loss of vitamins and flavor compounds, and maintaining the quality attributes of food products (Kaletunc, Lee, Alpas, & Bozoglu, 2004; Kimura et al., 1994; Lee & Kaletunc, 2010). Over a century ago, hydrostatic pressure treatment was demonstrated an effective method to extend shelf life of milk and other foods products, and the first commercial HHP treated product appeared on the market in 1991 in Japan (Patterson & Kilpatrick, 1998; Porretta, Birzi, Ghizzoni, & Vicini, 1995; Smelt, 1998; Trujillo, 2002). HHP processing is now being used for processing many types of food such as fruit juices, jams, sauces, rice, cakes and desserts. Most studies showed HHP causes a number of changes in morphology, cell wall, thermotropic phase in cell membrane lipids, dissociation of ribosomes, biochemical reactions, and loss of genetic functions of the microorganisms (Cheftel, 1995; Ritz, Freulet, Orange, & Federighi, 2000; Wouters, Glaasker, & Smelt, 1998), these all are proposed as possible reasons and mechanisms that caused inactivation of microorganisms subjected to HHP (Alpas, Lee, Bozoglu, & * Corresponding author. College of Food Science and Engineering, Northwest A&F University, 28# Xinong Road, Yangling 712100, PR China. Tel./fax: þ86 29 87091391. E-mail address: [email protected] (J. Meng). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.04.030
Kaletunc, 2003; Kaletunc et al., 2004; Mackey, Forestiere, Isaacs, Stenning, & Brooker, 1994). However, microbial resistance to HHP varies among bacteria, their physiological state, and food composition at the time of pressurization. The exact mechanism of inactivation caused by HHP is still not clearly understood (Kaletunc et al., 2004; Patterson, Quinn, Simpson, & Gilmour, 1995; Robey et al., 2001). Salmonella, Escherichia coli, Shigella and Staphyloccocus aureus are significant foodborne pathogens and commonly found in raw milk. Interest in HHP application to milk pasteurization has recently increased (Trujillo, 2002). When milk is treated under HHP, not only the pathogens can be inactivated, but the quality characteristics, such as taste, flavour, vitamins, and nutrients can be improved (Johnston, Austin, & Murphy, 1992; LopezFandino, Carrascosa, & Olano, 1996; Trujillo, 2002). The advantages of HHP merit further exploring its application for milk and other food processing and preservation. The objective of this study was to determine optimal conditions for inactivating Salmonella, E. coli, Shigella and S. aureus in raw milk by HHP, and to investigate HHP-induced pathogens morphological changes, growth conditions, and chromosome variations. 2. Materials and methods 2.1. Bacterial culture preparation Salmonella typhimurium LT2, E. coli ATCC25922, Shigella dysenteriae and S. aureus ATCC29213 were obtained from State Food and Drug Administration, Beijing, China. The cultures were stored at
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80 C in 40% (vol/vol) sterile glycerol. The cultures were streaked on LuriaeBertani (Beijing Land Bridge Technology Co., Ltd, Beijing, China) plates and incubated for 24 h at 37 C. A single typical colony was selected and inoculated into 90 ml of LuriaeBertani broth (Beijing Land Bridge Technology Co., Ltd, Beijing, China). After incubation at 37 C for 18 h with shaking at 150 rpm, the cells at stationary phase were harvested by centrifugation (10 min at 2400 g) and washed once in phosphate-buffered saline (PBS; 0.01 M phosphate, 0.137 M NaCl, pH 7.3). The washed cells were resuspended in sterile milk (purchased from cattle farm of Northwest A&F University, China) to a bacterial concentration of approximately 1 108 CFU$ml1. Inoculated milk samples were stored at 4 C for 24 h prior to HHP treatment to allow the cells to acclimatize to the new environment. 2.2. HHP treatment After 24 h storage at 4 C, the milk samples were placed in sterile polyethylene bags (Yangling Sanhe S&T Co., Ltd, Yangling, China) for HHP treatment. Air was removed from the bags prior to heat sealing. Each bag was placed in a second bag and sealed again to prevent contamination of the HHP equipment if the primary bag broke. The HHP equipment (Baotou Kefa High Pressure Technology Limited Company, Baotou, China) is capable of operating up to 750 Mpa was employed to apply pressure to the inoculated milk samples. The pressure transmission fluid was castor oil, and the temperature increase due to adiabatic heating was approximately 2 C per 100 Mpa. Pressure come-up rate was approximately 4200 Mpa per min and pressure release time was approximately 20 s. Pressurization time reported in this study excluded the time for pressure increase and release times. Pressure level and time of pressurization were set manually. Samples containing each kind of bacteria were pressured in 3-duplicate at designed pressure and time, and unpressured strains were used as controls. Both pressure treated and untreated cell suspensions were centrifuged (J2-21, Beckman, Palo Alto, Calif.) at 10,000 g for 10 min at 4 C to form pellets prior to transmission electron microscopy analysis.
pathogens, respectively. Cell pellets were prepared from untreated and pressure treated cell suspensions by centrifugation at 4000 g for 10 min at 4 C and washed once with 50 ml of sterile distilled water. Cell pellets were transferred to sterile centrifuge tube (1.5 ml) and resuspended in 1 ml of 0.1 M phosphate buffer at pH 7.4. After the suspension was centrifuged, the pellet was embedded in 2% agarose. The agarose was cut into 1 mm3 pieces and postfixed for 1 h in 1% osmium tetroxide (OsO4) in phosphate buffer, then the samples were rinsed in distilled water and stained for 1 h in 1% aqueous uranyl acetate. After dehydration through an ascending series of ethanol solutions (50, 70, 95, and 100%), cells in agar were transferred to propylene oxide, then were infiltrated and embedded in spurr’s resin, and sections (70 nm) were obtained with an ultramicrotome and stained with Reynolds’ lead citrate prior to examination under a transmission electron microscope (JEMd1230, Japan Electronics Co., Ltd.) at 60 kV. 2.6. Pulse field gel electrophoresis (PFGE) PFGE was used to assess the level of DNA injuries caused by HHP treatment (Ribot et al., 2006). Briefly, agarose-embedded DNA was digested with 50 U of XbaI (TaKaRa, Dalian, China) for 1.5e2 h in a water bath at 37 C. The restriction fragments were separated by electrophoresis in 0.5 TBE buffer at 14 C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA) with pulse times of 2.16 se63.8 s. The gels were stained with ethidium bromide, and DNA bands were visualized with UV transillumination (Bio-Rad). The initial cell numbers of HHP treated and non-treated were identical before HHP treatment, which can be acquired by adding same volume of bacterial suspension to the same volume of sterilized milk. 2.7. Statistical analysis Inactivation rates of the four pathogens under different pressure treatments and duration times were analyzed using Chi-Square tests with SPSS software (version 18 (SPSS, Chicago, IL, USA)).
2.3. Enumeration of cells 3. Results To determinate the initial number of cells before pressure treated and the number of treated milk samples, 1 ml of each sample was used to prepare 10-fold dilutions in buffered peptone water (Beijing Land Bridge Technology Co., Ltd, Beijing, China), subsequently, 1 ml of these dilutions were plated in duplicate onto LuriaeBertani agar plates and incubated at 37 C for 24 h. Plates containing 30e300 colonies were selected for counting. Results were expressed as CFU per ml, the lethality rate was calculated as the percentage of difference between the colony counts of the untreated and treated samples in untreated ones. 2.4. Viability test The viability test was performed according to the previous methods (Brinez, Roig-Sagues, Herrero, & Lopez, 2007; Patterson et al., 1995) with some modifications. Briefly, the diluted milk samples were prepared and plated in duplicate in LB, TSA-YE (Beijing Land Bridge Technology Co, Ltd.,), TSA-YE þ NaCl (3%) and TSA-YE þ NaCl (10%) plates, and incubated at 37 C for 24 h, the morphology of the colonies was monitored to assess the lethality and the level of injury caused by HHP treatment. 2.5. Electron microscopy E. coli ATCC25922 and S. aureus ATCC29213 were the representatives of Gram negative (G) and Gram positive (Gþ)
3.1. Effect of duration time on inactivation of the pathogens Overall, HHP inactivation of the pathogens was more effective against G than Gþ bacteria. Under the same pressure treatment condition, the longer the treatment was the better the inactivation could be achieved. The inactivation rates differed among the three G pathogens under the same pressure and duration time treatment; for example, under 300 Mpa for 10 min treatment, Shigella exhibited the highest inactivation rate (94.6%) while Salmonella had the lowest inactivation rate (93.1%) among the pathogens. When the samples were treated under 300 Mpa for 20, 30, 40, or 50 min), the inactivation rate for Salmonella was the highest whereas S. aureus was the lowest (Fig. 1). Significant differences (p < 0.05) were observed among the inactivation rates of the four pathogens during HHP treatment. Inactivation rates of E. coli, Salmonella and Shigella were relative high in prophase (from 10 min to 20 min) under 300 Mpa pressure treatment, although the inactivation rate of E. coli was lower than that of Salmonella and Shigella. The inactivation rate of the three pathogens decreased slowly when the duration time was increased from 30 min to 50 min, inactivation rate for Salmonella has no apparent change (Fig. 1). Different from the three G pathogens, the inactivation rate of S. aureus increased slowly during the whole HHP treatment, almost all cells could be inactivated after 50 min treatment (Fig. 1). Thus, 30 min was considered as a satisfactory duration time for HHP treatment.
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In a c t i v a t i o n R a t e (%)
in milk (Fig. 3). The results indicated that 300 Mpa treatment with 30 min duration at 25 C was the optimal condition for pathogens inactivation. 3.3. Viability test E.Coli Salmonella Staphyloccocus aureus Shigella
Time (Unit: Min) Notes: The abscissa axis indicates pressure treatment time, the ordinate axis indicates inactivation rate under 300 Mpa treatment. Min: Minute Fig. 1. Effect of duration time on inactivation rates of 4 pathogens at 25 C under 300 Mpa.
3.2. Effect of pressure on inactivation of the pathogens
In a c tiv a tio n R a te (% )
Significant differences (p < 0.05) were observed between inactivation rates among the four pathogens under different pressurization (Fig. 2). When treated at 25 C for 30 min under different pressures, the general trend of inactivation rate of the four pathogens exhibited higher inactivation rates with higher pressure, and the inactivation rates of G was higher than that of Gþ ones (Fig. 2). The inactivation rates of Salmonella and E. coli increased slowly and showed no significant change when the pressure increased from 100 Mpa to 200 Mpa, in contrast, the inactivation rate of Shigella and S. aureus increased rapidly. The inactivation rates increased significantly and almost all live cells were inactivated when the pressure increased from 200 Mpa to 300 Mpa. The inactivation rate of all pathogens exhibited little variation when the pressure increased from 300 Mpa to 500 Mpa. Consequently, 300 Mpa was considered as the satisfactory pressure (Fig. 2). Significant differences were observed among different pressures, duration time and pathogens when different pressures (300, 350 and 400 Mpa), duration times (5, 10, 20 and 30 min) and pathogens were chosen to analyze interactions among pressure, duration time and pathogen species to determine optimal conditions in inactivating pathogens
Salmonella E.Coli Staphyloccocus aureus Shigella
Pressure (Unit: M pa) Notes: The abscissa axis indicates different pressure, the ordinate axis indicates inactivation rate under HHP treatment. Mpa: Million Pascal Fig. 2. Effect of pressure on inactivation rates of 4 pathogens at 25 C with 30 min duration.
The effect of HHP treatment on the viability of the pathogens was determined using colony enumeration by direct plating. When the untreated milk sample, milk samples treated under 100 Mpa30 min and 400 Mpa-30 min were spread on LB plates with the same volume and pathogens concentration, respectively, and incubated at 37 C for 24 h, the colony numbers of untreated cells were found more than that of the treated samples, and the colonies were bigger than that of the pressure treated ones. Viable colonies of 400 Mpa-30 min treatment cells were smaller than that of 100 Mpa-30 min treated ones. Since LB medium is nutrient rich and non-selective, and therefore severely injured cells may not have been recovered, and the growth speed of recovered cells was much slower than the normal ones. When the pressure treated E. coli, Salmonella and Shigella were inoculated onto TSA-YE and TSA-YE þ NaCl (3%) plates, pressure treated S. aureus was inoculated onto TSA-YE and TSA-YE þ NaCl (10%) plates, respectively, and were incubated at 37 C for 24 h, the tiny colonies of treated pathogens could be found on TSA-YE medium, while very few colonies could be observed on TSAYE þ NaCl medium, either the concentration of NaCl was 3% or 10%. Since these media were somewhat selective osmotic pressure, the NaCl may penetrate the broken cell membrane and therefore injured cells could not be recovered and grow on these media. 3.4. Effect of pressure on chromosome DNA of the pathogens Effect of pressure on chromosome DNA of E. coli and Shigella was shown in Fig. 4. After the chromosome DNA of the untreated and 500 Mpa-30 min treated E. coli was digested with XbaI and separated by electrophoresis, respectively, different number and brightness of DNA bands can be found in the lane of 500 Mpa30 min treated and untreated E. coli. Furthermore, there was no corresponding band with same molecular weight in the two lanes even if the initial cells were the same. At the same time, the DNA bands of the 500 Mpa-30 min treated E. coli were not as bright as the untreated ones, although the concentration of the cells was identical. Same result could be found when untreated and pressure treated Shigella cells were digested and electrophoresesed. The reason may be that HHP treatment damaged the integrity of the cells and the chromosome DNA of the pathogens. After HHP treatment, bacteria cell wall, cell membrane, and some DNA hydrogen bonds and covalent bonds are broken, the intact DNA chain was destroyed. Therefore, some small DNA fragments were lost after a 20 h electrophoresis. 3.5. Electron microscopy Untreated E. coli and S. aureus cells exhibited intact cell wall, cell membrane, uniform cell cytoplasm, and electron-transparent regions of nucleoid in electron micrographs (Fig. 5a, b). However, application of HHP leads to morphological changes in the internal and external structures. The most visible changes with application of HHP were a double-track bilayer structure of cell membrane instead of a single-thick-layer appearance, and the enlargement of electron-transparent ranges in the cell cytoplasm. Treatment of HHP at 500 Mpa for 30 min resulted in expanded nucleoid regions and compacted interior regions (Fig. 5a’, b’). Most of lower pressure treated cells maintained a distinct membrane and cell wall, whereas, in higher pressure (such as 500 Mpa) treated cells,
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A 100.0% 99.0% 98.0%
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Inactivation Ratio (%)
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Notes: A: The abscissa axis indicates different pressure, the ordinate axis indicates inactivation rate under HHP treatment to the four pathogens.
99.0% 98.0%
B: The abscissa axis indicates different treat time, the ordinate axis indicates inactivation rate under 300Mpa treatment to the four pathogens.
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C: The abscissa axis indicates four pathogens, the ordinate axis indicates inactivation rate under the given conditions. Min: Minute Mpa: Million Pascal
Inactivation Rate of Each Pathogens under tha Given Pressure and Time
Fig. 3. Interactions among pressure, duration time and pathogen species on inactivation rate.
breakdown of the peptidoglycan layer was evident, parts of the outer layer appeared to slough off (Fig. 5a’, b’). Aggregation of cytoplasmic material and enlarged electron-transparent region of nucleoid with a fibrous appearance were also observed (Fig. 5a’, b’). 4. Discussion
Fig. 4. DNA profiles of E. coli and Shigella after PFGE.
E. coli, Salmonella, Shigella and S. aureus are foodborne pathogens of concern for the dairy industry, and raw milk has been identified as an important vehicle for the transmission of these pathogens and been implicated in outbreaks (Brinez et al., 2007; Chapman, 1993; Rajeev Kumar, 2010; Rodriguez, Arques, Nunez, Gaya, & Medina, 2005; Tambekar DH., 2006). Heat treatment is one of the most efficient and economical processes for achieving microbial inactivation in milk, however, it cannot be used to treat heat-labile food and compounds. Furthermore, high temperature treatments may result in undesirable effects in milk, such as offflavor, non-enzymatic browning and denaturation of certain vitamins and proteins (Brinez et al., 2007; Diels, Callewaert, Wuytack, Masschalck, & Michiels, 2005; Kheadr, Vachon, Paquin, & Fliss, 2002). The application of HHP on milk and dairy products has been shown to be an effective technology to inactivate microorganisms including most infectious foodborne pathogens. In addition to microbial destruction, it has been reported that HHP improves rennet of acid coagulation of milk without detrimental effects on important quality characteristics (Brinez et al., 2007; Johnston et al., 1992; Trujillo, 2002). The present study showed a higher inactivation rate to Salmonella, Shigella and E. coli by high pressure treatment at 300 Mpa than that to S. aureus. Among three G pathogens, Shigella was more
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Fig. 5. Cell structures of untreated and 500 Mpa-30 min treated E. coli and S. aureus under TEM.
resistant to high pressure than Salmonella and E. coli. The work reported here is in agreement with previous researches that showed when treated by HHP, variations of microorganisms in resistance to high pressure are common between and within species, and Gþ bacteria appears to be more pressure resistant than G one (Benito, Ventoura, Casadei, Robinson, & Mackey, 1999; Patterson et al., 1995; Rivalain, Roquain, & Demazeau, 2010), and S. aureus is significantly more resistant than other microorganisms such as S. enteric serovar Typhimurium and E. coli when inoculated and treated under different conditions (between 100 and 300 Mpa of pressure and between 5 and 40 C of inlet temperature) (Diels, Wuytack, & Michiels, 2003; Wuytack et al., 2003). This study and previous studies suggest that S. aureus may be assigned as a potential indicator bacterium when HHP is adopted for pathogens inactivation in milk production. At the same time, our results also indicated that when milk was treated with HHP, at lower pressure, the inactivation rate of S. aureus and Shigella increased faster than that of E. coli and Salmonella, this hence suggested that relative lower pressure treatment to milk is a good choice for pathogens inactivation. When comparing the grow condition of the four untreated and pressure treated strains on LB agar, TSA-YE agar and TSA-YE-NaCl agar, respectively, normal and uniform colonies could be found on LB, TSA-YE and TSA-YE with NaCl (3%) media, which were nonor less-selective, when the untreated bacteria were inoculated. However, on TSA-YE-NaCl media with low NaCl concentration (3%), even on the LB and TSA-YE media, the HHP treated strains grew very slowly with tiny colonies, and those that subjected to higher pressure and longer duration time could not grow on LB and TSA-YE media, which are not selective. At the same time, the colony
number of treated strains was less than that of the untreated one even if the same inoculation. On TSA-YE with higher NaCl concentration (10%), the HHP treated cells could hardly grow, which indicated that the strains were sublethally injured or injured after HHP treatment and our results were in agreement with the previous studies (Ponce, Pla, Capellas, Guamis, & Mor-Mur, 1998; Ponce, Pla, Sendra, Guamis, & Mor-Mur, 1999). Cell membrane is often considered as the first site of damage in pressure injured bacteria, the sublethal damage or damage of the membrane and changes in membrane fluidity by HHP treatment could facilitate the access of NaCl to the cytoplasm of bacteria (Hauben, Wuytack, Soontjens, & Michiels, 1996; Ritz, Tholozan, Federighi, & Pilet, 2002; Rodriguez et al., 2005). At the same time, the cytoplasma and other intracellular substances may outflow, suggesting that the injured survivors became sensitive to NaCl after pressurization, even for the NaCl tolerant S. aureus. The TEM images also demonstrated the membrane and cell wall were damaged after HHP treatment (Fig. 5a’, b’). HHP treatment could cause alterations of membrane bound protein and enzyme functionality as well (Kato & Hayashi, 1999), after treatment at 350 Mpa or 400 Mpa, outer and internal membrane protein contents of Salmonella typhimurium were drastically modified, some of proteins almost entirely disappeared (Ritz et al., 2000). The degradation of some proteins impaired the ability of cells to reproduce and develop colonies on non-selective media, such as LB and TSA-YE. Previous studies on mechanism of inactivation of microorganisms by HHP focused on perturbation of the cell membrane, the damaged cell wall and cell membrane function, inhibition or inactivation of essential enzyme systems, the destruction of
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ribosome and the changes of cell morphologies (Cheftel, 1995; Earnshaw, Appleyard, & Hurst, 1995; Kaletunc et al., 2004; Lee & Kaletunc, 2010; Ritz et al., 2000; Smelt, 1998). Our results were in agreement with some of the previous studies, and further confirmed that the cell membrane and cell wall of the E. coli and S. aureus were totally damaged after different pressure treatment (Fig. 5a’, b’). Although few investigations observed that the thermal stability of DNA peak transition happened using differential scanning calorimetry during pressure treatment (Alpas et al., 2003; Dubins, Lee, Macgregor, & Chalikian, 2001; Kaletunc et al., 2004). This study is the first one using PFGE to analyze DNA alteration before and after bacteria was pressure treated. In our survey, PFGE results showed genomic difference between HHP untreated and treated E. coli and Shigella cells, as well as the difference of DNA concentration, which indicated that the DNA of bacteria was damaged during pressure treatment, and this should be one of the most important potential mechanisms of inactivation by HHP. On summary, this work has shown that HHP processing can be used to inactivate some of the common pathogens in food, such as Salmonella, Shigella and E. coli, and even the pressure resistant S. aureus. The optimal condition for pathogens inactivation in milk by using of HHP was under 300 Mpa at 25 C with duration time of 30 min. Mechanisms for inactivation of pathogens are possibly due to cell membrane damage, cell wall rupture and chromosome DNA degradation. Acknowledgement The research work was supported in part by the Houji Scholar Program, and the Principal Fund of Northwest A&F University, Yangling, Shaanxi, China. We also appreciate Drs Shuangkui Du and Min Sheng in Northwest A&F University for data analysis by using SPSS software. References Alpas, H., Lee, J., Bozoglu, F., & Kaletunc, G. (2003). Evaluation of high hydrostatic pressure sensitivity of Staphylococcus aureus and Escherichia coli O157: H7 by differential scanning calorimetry. International Journal of Food Microbiology, 87, 229e237. Benito, A., Ventoura, G., Casadei, M., Robinson, T., & Mackey, B. (1999). Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses. Applied and Environmental Microbiology, 65, 1564e1569. Brinez, W. J., Roig-Sagues, A. X., Herrero, M. M. H., & Lopez, B. G. (2007). Inactivation of Staphylococcus spp. strains in whole milk and orange juice using ultra high pressure homogenisation at inlet temperatures of 6 and 20 degrees C. Food Control, 18, 1282e1288. Chapman, P. A. (1993). Untreated milk as a source of verotoxigenic Escherichia-Coli O157 (Vol 133, Pg 171, 1993). Veterinary Record, 133, 252. Cheftel, J. C. (1995). Review: high-pressure, microbial inactivation and food preservation. Food Science and Technology International, 1, 75e90. Diels, A. M. J., Callewaert, L., Wuytack, E. Y., Masschalck, B., & Michiels, C. W. (2005). Inactivation of Escherichia coli by high-pressure homogenisation is influenced by fluid viscosity but not by water activity and product composition. International Journal of Food Microbiology, 101, 281e291. Diels, A. M. J., Wuytack, E. Y., & Michiels, C. W. (2003). Modelling inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation at different temperatures. International Journal of Food Microbiology, 87, 55e62. Dubins, D. N., Lee, A., Macgregor, R. B., & Chalikian, T. V. (2001). On the stability of double stranded nucleic acids. Journal of the American Chemical Society, 123, 9254e9259. Earnshaw, R. G., Appleyard, J., & Hurst, R. M. (1995). Understanding physical inactivation processes: combined preservation opportunities using heat, ultrasound and pressure. International Journal of Food Microbiology, 28, 197e219. Hauben, K. J. A., Wuytack, E. Y., Soontjens, C. C. F., & Michiels, C. W. (1996). High-pressure transient sensitization of Escherichia coli to lysozyme and nisin
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