- Email: [email protected]
High hydrostatic pressure inactivation of total aerobic bacteria, lactic acid bacteria, yeasts in sour Chinese cabbage
International Journal of Food Microbiology 142 (2010) 180–184
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
High hydrostatic pressure inactivation of total aerobic bacteria, lactic acid bacteria, yeasts in sour Chinese cabbage Lin Li, Lun Feng, Junjie Yi, Cheng Hua, Fang Chen, Xiaojun Liao, Zhengfu Wang, Xiaosong Hu ⁎ College of Food Science and Nutritional Engineering, China Agriculture University, Beijing, 100083, PR China Key Laboratory of Fruits and Vegetables Processing, Ministry of Agriculture, Beijing, 100083, PR China Research Center of Fruits and Vegetables Processing Engineering, Ministry of Education, Beijing, 100083, PR China
a r t i c l e
i n f o
Article history: Received 31 March 2010 Received in revised form 12 June 2010 Accepted 23 June 2010 Keywords: High hydrostatic pressure Inactivation Sour Chinese cabbage Total aerobic bacteria Lactic acid bacteria Yeasts
a b s t r a c t This study investigated the inactivation of total aerobic bacteria (TAB), lactic acid bacteria (LAB), yeasts in sour Chinese cabbage (SCC) treated by high hydrostatic pressure (HHP). The pressure level ranged from 200 to 600 MPa and the treatment time were 10–30 min. All samples were stored at 4, 27 and 37 °C for 90 days. The pressure level of 200 MPa had no significant impact on these microorganisms. The counts of TAB were significantly reduced by 2.7–4.0 log10 CFU/g at 400 MPa and 4.2–4.5 log10 CFU/g at 600 MPa from 6.2 log10 CFU/g; the counts of LAB were also reduced by 2.4–4.3 log10 CFU/g at 400 MPa from 7.0 log10 CFU/g and LAB was completely inactivated at 600 MPa; the counts of yeasts were reduced by 1.5–2.0 log10 CFU/g at 400 and 600 MPa from 4.2 log10 CFU/g. Storage temperatures significantly influenced the microbial proliferation in HHP-treated SCC depending on the pressure levels. The surviving TAB and LAB at 400 MPa equaled initial counts after 15-day storage at 27 and 37 °C, whereas they were inhibited at 4 °C up to 60 days. The surviving TAB at 600 MPa did not grow. Yeasts at 400 and 600 MPa decreased below detectable level after 2 days at all the three storage temperatures. From the microbial safety point of view, the result indicated that HHP at 600 MPa could be used as an alternative preservation method for SCC. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Fermentation is one of the oldest forms of food preservation technologies in the world, which can increase the shelf-life of food, improving their safety, sensory and nutritional properties. Fermented vegetables are very popular throughout the world, such as kimchi from Korea and sauerkraut from Europe. Sour Chinese cabbage (SCC) is a kind of traditional lactic acid fermented vegetable widely consumed in China, especially in the northeast where is too cold to cultivate leafy vegetables in winter. About 1/6 of Chinese consumes SCC in daily diet, according to the data from Chinese Center for Disease Control and Prevention (Cui et al., 2008). These fermented vegetables have been generally considered as safe due to the presence of lactic acid. However, the raw materials are easily contaminated with food-borne pathogens, such as Escherichia coli and Listeria monocytogenes, which tolerate low pH environment (Peñas et al., 2010). Some products such as kimchi are still fermenting after packing, often leading to excessive sourness and pack swelling due to microbial activity during storage (Sohn and Lee, 1999). Other products like sauerkraut are pasteurized. It is known that thermal processing usually causes undesirable organoleptic changes such as ⁎ Corresponding author. 303#, No. 17 Qinghua East Road, Haidian District, Beijing 100083, PR China. Tel./fax: + 86 10 62737464. E-mail address: [email protected] (X. Hu). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.020
texture softening, flavor losing and discoloration. SCC products in Chinese market are various: some are just made in household level and sold in bulk; some are directly packaged without the control of microorganisms like kimchi; most are pasteurized like sauerkraut. SCC has similar problems mentioned above and sometimes even worse. How to produce both safe and tasty SCC is promptly needed. The growing demand for natural, fresh-like and safe food has aroused an increased interest in non-thermal processing technologies (Jordan et al., 2001). High hydrostatic pressure (HHP) is considered as one of the most promising methods because of its successful application in acid foods such as jams, fruit juices and other fruit products. HHP can kill most microorganisms without significantly affecting the sensory and nutritional properties, and so increase the shelf-life of these products. For fermented foods like cheeses, ham and yogurt, HHP has been widely studied. However, there have been only a few reports of HHP in fermented vegetables. Sohn and Lee (1999) firstly reported that in kimchi the count of total aerobic bacteria (TAB) was reduced to 4 log10 CFU/mL at 400 MPa for 10 min, and maintained during the 4-week storage at 20 °C; whereas there was no change in the texture. Lactic acid bacteria (LAB) was significantly reduced from 7 to 3 log10 CFU/mL at 600 MPa for 5 min, which prevented gas production in kimchi and excessive acidity during storage at 10 °C (Lee et al., 2003). Peñas et al. (2010) reported that 300 MPa for 10 min at 4 °C led to a reduction of TAB and LAB in sauerkraut (4–5 log10 CFU/g) immediately after the treatment;
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
although refrigerated storage brought an increase in microorganisms, the counts were still lower than untreated sauerkraut. Although similarities exist, there are also some significant differences between SCC and other lactic acid fermented vegetables in processing method, ingredient and the constituent of microorganisms. For example, SCC is completely anaerobic fermented whereas kimchi is not; the raw material of sauerkraut is white cabbage whereas that of SCC is Chinese cabbage; kimchi is added with high concentration of salt and species whereas SCC is produced with only a little salt and no other seasonings; these differences combined with regional disparities thus lead to the variation of microorganisms. To date, there is little knowledge on the application of HHP in SCC. The objective of this study was to investigate the inactivation of HHP on TAB, LAB, yeasts in SCC and its microbiological stability during 90 days at three storage temperatures.
2. Materials and methods 2.1. SCC preparation The SCC samples were provided by a commercial SCC producer (Beidahuang Co. Ltd., Heilongjiang, China). Two parallel batches of SCC were used for the experiment. The brief process of SCC production was as follows. Chinese cabbages (Brassica rapa L. ssp. pekinensis) harvested from the same field were chosen and their outer leaves were removed. All cabbages were of good quality and free of serious mechanical damage. They were washed in water jet washer and then were blanched with steam for 1 min. After cooling, cabbages were belt-carried into fermentation tank, with a cylinder 2 m in diameter and 6 m in height. Five percents of NaCl were uniformly distributed in cabbages. A mixed starter culture containing Lactobacillus plantarum, Lactobacillus maltaromicus, Leuconostoc meseteroides, Lactobacillus brevis (Northeast Agricultural University, Heilongjiang, China) was inoculated at approximately 6 log10 CFU/g of cabbage. Finally, cabbages were pressed thoroughly to remove air bubbles and the tank was sealed. Fermentation was carried out at 25 °C for 40 days. After the fermentation, SCC with pH 3.8–4.0 was sliced, vacuum packaged (500 g/package) and stored below 10 °C.
181
2.4. Storage study Following the treatment, each sample was divided into three portions and was stored at 4, 27 and 37 °C respectively for 90 days. The samples were taken at 0, 2, 5, 8, 15-day and 15-day intervals thereafter. 2.5. Enumeration of viable cells Each sample (25 g) was aseptically weighted and homogenized with 225 mL of sterile 0.85% NaCl solution for 1.5 min in an electromechanical blender (JYL-B060, Joyong Electric Appliance Co., Shandong, China). After homogenization, the blender was washed using sterile water, sanitized with 50-ppm chlorine dioxide (Beijing Techgreen Co., Ltd., Beijing, China), and then rinsed three times with sterile water. To detect the viable cells in SCC, the total plate count method was used. Sample was serially diluted with sterile 0.85% NaCl solution and 1.0 mL of diluted (or non-diluted) samples was plated into duplicated plates of appropriate agar. The plate count agar (PCA, composition: tryptone 10.0 g/L, yeast extract 2.5 g/L, glucose 1.0 g/L, agar 15.0 g/L, pH 7.0 ± 0.1. Beijing Luqiao Technology Co., Ltd., Beijing, China) was used for detecting the viable cells of TAB. The plates were incubated at 37 °C for 24 ± 2 h. The MRS agar (MRSA, composition: peptone 10.0 g/L, beef extract 5.0 g/L, yeast extract 4.0 g/L, glucose 20.0 g/L, Tween 80 1.0 mL, KH2PO4·7H2O 2.0 g/L, CH3COONa·3H2O 5.0 g/L, triamine citrate 2.0 g/L, MgSO4·7H2O 0.2 g/L, MnSO4·7H2O 0.05 g/L, agar 15.0 g/L, pH 6.2±0.1. Beijing Luqiao Technology Co., Ltd., Beijing, China) was used for detecting the viable cells of LAB. The plates were incubated at 30 °C for 72±2 h. The rose bengal agar (RBA, composition: peptone 5.0 g/L, glucose 10.0 g/L, KH2PO4 1.0 g/L, (NH4)2SO4 0.5 g/L, rose Bengal 0.03 g/L, chloromycetin 0.1 g/L, agar 15.0 g/L, pH 6.0 ± 0.1. Beijing Luqiao Technology Co., Ltd., Beijing, China) was used for detecting the viable cells of yeasts. The plates were incubated at 27 °C for 5 days. After incubation, the colonies were enumerated. The detection limit of TAB and LAB was 1 log10 CFU/g and that of yeasts was 1 CFU/g. Inactivation effect was expressed as log10 N/N0, where N0 was the number of initial count of microorganisms in control, and N was the corresponding viable count after HHP. 2.6. Statistical analysis
2.2. HHP equipment HHP treatment was carried out using a hydrostatic pressurization unit (HHP-650, Baotou Kefa Co., Ltd., Inner Mongolia, China), with a cylinder 11 cm in inner diameter and 30 cm in height and a capacity of 2.8 L. Distilled water was used as the pressure-transmitting fluid. The rate of pressure increase was about 120 MPa/min and the pressure release was immediate. The treatment time reported in this study did not include the pressure increase and release time. The pressure level and treatment time were continuously recorded during the pressurization cycle. The temperature in the pressure vessel rose from 25 to 33 °C during coming-up to 600 MPa by compression heating. Then the temperature decreased to 25 °C for about 5 min during pressure holding.
2.3. HHP treatment Each batch of SCC was mixed thoroughly under aseptic conditions, and then was divided into 560 small packages. Approximately 50 g SCC was transferred into a sterile polyethylene bag and heat-sealed under vacuum. The bags were placed into the pressure vessel and treated at 200, 400 and 600 MPa for 10, 20 and 30 min, respectively. Controls were not pressurized. The experiment was carried out at room temperature.
Analyses of variance (ANOVA) were carried out using the software Microcal Origin 7.5 (Microcal Software, Inc., Northampton, MA, USA). The significance level was 0.05. 3. Results and discussion 3.1. Inactivation of TAB, LAB, yeasts in SCC by HHP Fig. 1a and b showed the effect of pressure levels and treatment time on the inactivation of TAB and LAB in SCC, respectively. The samples treated by HHP at 200 MPa were not significantly different from the control in the counts of the two microorganisms. The counts were significantly reduced at 400 and 600 MPa. A 4-log reduction of TAB was attained at 400 MPa for 30 min while no significant additional reduction (P N 0.05) was observed at 600 MPa, indicating that some bacteria resisting to HHP did exist in SCC. A similar result was reported by Sohn and Lee (1999), showing that the count of TAB in kimchi was reduced from 8 to 4 log10 CFU/mL at 400 MPa for 10 min and 600 MPa did not increase the log reduction. The pressure level of 600 MPa was sufficient to reduce the counts of LAB below detectable level. Whereas, the count of LAB in kimchi at 600 MPa still remained at the level of 3 log10 CFU/mL (Sohn and Lee, 1999; Lee et al., 2003). It has been known that the compositions of food, especially high sugar or salt concentrations, exert very strong baroprotective effect on microorganisms (Cheftel, 1995). The
182
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
inactivation was observed at 400 and 600 MPa. Generally, yeasts are very sensitive to HHP due to their cellular morphology (Cheftel, 1995). For instance, yeasts were completely inactivated at 200 MPa in Gouda and Krupoiwski cheeses (Reps et al., 1998); the yeasts in red wine reduced to null at 300 MPa for 20 min (Mok et al., 2006); they were reduced below detectable level at 400 MPa in cashew apple juice (Lavinas et al., 2008). In this study, the inactivation of yeasts immediately after HHP did not conformed to these previous investigations. Sohn and Lee (1999) reported that the count of yeasts in kimchi was low (about 3 log10 CFU/mL) but remained constant by HHP up to 600 MPa. Smelt et al. (2001) proposed that yeasts are relatively resistant to low pH and barely affected at pH 4.0 when subjected to HHP. The pH of SCC in this study is right about 4, which could explain for the little inactivation effect of HHP on yeasts in SCC. 3.2. Microbiological stability in SCC by HHP
Fig. 1. Inactivation of total aerobic bacteria (a); lactic acid bacteria (b); and yeasts (c); in sour Chinese cabbage by high hydrostatic pressure. The pressure levels are 200 MPa, ■; 400 MPa, ○; and 600 MPa, ▲. Least Significance Difference (5% level) = 0.90. Detection limit for total aerobic bacteria is log10 N/N0 = −6.2; for lactic acid bacteria is log10 N/N0 = −7.0; for yeasts is log10 N/N0 = −4.2. Lines dropping below the x-axis indicate cell counts below the detection limit.
seasonings in kimchi could contribute to the microbial baroprotection, but SCC is produced without these ingredients. Besides, pressure resistance varies considerably between species and among strains within a species (Alpas et al., 1999). These reasons explained for the variation of inactivation effect between SCC and kimchi. As shown in Fig. 1c, the count of yeasts was reduced by 1.5 log10 CFU/g at 200 MPa for 30 min but no significant additional
3.2.1. Population change of LAB by HHP during storage Based on the results in Fig. 1, higher pressure levels (400 and 600 MPa) were selected for a further investigation of microbiological stability in SCC by HHP. Fig. 2 showed the population change of LAB in SCC at 400 MPa for 10, 20 and 30 min during 90-day storage at 37, 27 and 4 °C, respectively. The count (about 7 log10 CFU/g) of LAB in control sample remained statistically constant during storage, indicating that further growth of LAB was inhibited by the accumulated lactic acid. Immediately after HHP, the counts of LAB were significantly reduced to 4.6, 3.9 and 2.7 log10 CFU/g at 400 MPa for 10, 20 and 30 min, respectively. Later, increasing counts were observed and reached to the level of initial count of control sample after 5 days at 37 °C (Fig. 2a). Noticeable increasing counts were also observed during storage at 27 °C (Fig. 2b). The result was similar to earlier studies. Lee et al. (2003) reported that although kimchi at 400 MPa showed a reduction in the counts of TAB and LAB immediately after HHP, after 15-day storage at 10 °C increases occurred in the microbial populations which were finally comparable with that of the control. To date, amount of studies have been reported about the recovery of pathogenic and spoilage bacteria damaged by HHP during storage in various food matrix and buffer solutions (Bozoglu et al., 2004; Bull et al., 2005; Diez et al., 2008; Ellenberg and Hoover, 1999; Koseki and Yamamoto, 2006; O'Reilly et al., 2000). Whereas, the bacterial increase in nutrient-rich media could not be evaluated separately to distinguish between repair of injured cells and bacterial growth after HHP (Koseki and Yamamoto, 2006). Therefore, the increasing counts of LAB in SCC could be due to the growth of surviving cells and the recovery of injured cells. Unlike the obvious increase observed during storage at optimal growth temperatures, the counts of LAB in HHP-treated samples at 4 °C exhibited slight fluctuations and remained statistically constant up to 60 days (Fig. 2c), showing the inhibition effect of low temperature on the proliferation of LAB. But the increasing counts at 400 MPa were finally observed in the last 30 days, which was similar to a previous study on sauerkraut (Peñas et al., 2010). They found that the refrigerated storage of 3 months brought about an increase in the counts of TAB and LAB in sauerkraut after the immediate reduction when subjected to 300 MPa for 10 min at 40 °C. They ascribed the increase of TAB and LAB in sauerkraut during the third month of refrigerated storage after HHP to the recovery of microorganisms that were sublethally injured by HHP. However, several studies have demonstrated that low temperature or low pH could inhibit the microbial recovery (Bozoglu et al. 2004; Bull et al. 2005; Hayman, 2001; Koseki and Yamamoto, 2006; Smelt et al., 2001). Therefore, the microbial increase in the third month could most likely be explained as the growth of surviving cells rather than the recovery of injured cells. The result showed that although low temperature inhibited the proliferation
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
183
3.2.2. Population change of TAB by HHP during storage In SCC where the predominant microflora is LAB, the population change of TAB by HHP during storage was therefore closely correspondent with that of LAB. Fig. 3a showed the change of TAB at 400 MPa during storage at 27 °C. Similar to that of LAB, the counts of TAB increased to the initial count of control sample after 15 days. The change of TAB stored at 37 °C was similar (data not shown). The counts of TAB stored at 4 °C experienced slight fluctuations and finally the increase occurred in the last month of storage, which was also analogical to that of LAB at 4 °C. The counts of TAB at 600 MPa remained relatively constant during 90 days, keeping lower than 2.2 log10 CFU/g at the three storage temperatures (data not shown). It showed that although some bacteria extremely resistant to HHP still existed in SCC, their growth was effectively inhibited by the low pH. The result was in agreement with the earlier study of HHP in kimchi (Lee et al., 2003), in which the count of TAB at 600 MPa maintained at about 3 log10 CFU/mL for at least 24 days. The result showed that SCC treated by HHP at 600 MPa exhibited a better microbiological stability.
3.2.3. Population change of yeasts by HHP during storage Generally yeasts do not cause food-borne disease, but they play an important role in the spoilage of food, especially in acid foods (Lavinas et al., 2008). Yeasts can produce gas in SCC and often causes pack inflation. Although the medium used to detect yeasts is rose bengal agar on which both yeast and molds could be cultivated, no
Fig. 2. Population dynamics of lactic acid bacteria in sour Chinese cabbage treated by 400 MPa during storage at 37 °C (a); 27 °C (b); and 4 °C (c). The treatment times are 0 min, ■; 10 min, ○; 20 min, ▲; and 30 min, . Least Significance Difference (5% level) = 0.90. Detection limit is 1 log10 CFU/g.
◇
of LAB to some extent, the inhibition effect was limited and could not last very long. Like other bacteria, LAB evolves defense mechanisms against cold-shock and it can adapt to a temperature downshift (Van de Guchte et al. 2002). Thus, LAB in SCC at 4 °C began to proliferate after 2-month adaptation of cold. For LAB at 600 MPa, it was not detected during 90 days at all the three storage temperatures (data not shown), indicating that the LAB in this case was totally inactivated.
Fig. 3. Population dynamics of total aerobic bacteria in sour Chinese cabbage treated by 400 MPa during storage at 27 °C (a) and 4 °C (b). The treatment times are 0 min, ■; 10 min, ○; 20 min, ▲; and 30 min, . Least Significance Difference (5% level) = 0.90. Detection limit is 1 log10 CFU/g.
◇
184
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
mold in the samples was detected through microscopy and observation of colony morphology. As shown in Fig. 4, there were two phases for population change of yeasts in the control and HHP-treated samples during 90-day storage, but the patterns were different. For the control sample, the count of yeasts decreased in the initial phase of storage and it began to increase after 30 days. It is because that yeasts were firstly inhibited by the accumulated lactic acid produced by LAB and the non-acid adapted cells gradually died; those survivors consumed acid and increased the pH, becoming the main factor for the spoilage of SCC (Li et al., 2008). The pH analysis in our study supported it: the pH gradually increased from 4.0 to 4.4 during the storage. The counts of yeasts at 400 MPa were sharply reduced below detectable level after 2 days and no resuscitation was observed during the storage at all the three storage temperatures. Combined with the result shown in Fig. 1c, it is indicated that although 400 and 600 MPa did not significantly reduce the counts of yeasts immediately after HHP, these microorganisms were probably injured — a kind of injury defined by Bozoglu et al. (2004) as primary injury which can form visible colonies on non-selective agar. The sharp reduction after 2 days could be interpreted as acid sensitization effect that was a proportion of the cells which survived pressurization were sublethally injured in low pH, the cells were unable to repair the damage, hence lowering their tolerance to the unfavorable pH and organic acids present in the medium (Bayindirli et al., 2006). The result was similar to the study of HHP on mutant of E. coli in fruit juice (Garcia-graells et al., 1998), in which the low level (1.1 log10 CFU/mL) direct reduction of LMM1010 microorganisms in apple juice subjected to 300 MPa for 15 min at 20 °C was reported and was followed by an extensive (almost 5 log10 CFU/mL) further reduction during the first 2-day storage. This indicated that HHP not only injured cells at its own right but also caused changes in the cells that make them more susceptible to subsequent acid injury. Yeasts at 600 MPa showed similar trend during storage as that at 400 MPa (data not shown). It is worth to mention that yeasts in control samples also died out during the storage at 37 °C but it was due to the high temperature unsuitable for yeasts to survive in SCC. Based on the results in this study, it is found that yeasts in SCC is the most sensitive to HHP and the pressure level of 600 MPa is the most effective. It is necessary that the microbiological stability of post-pressurization be taken into consideration in pressure inactivation study since the microbial population could change during storage.
Fig. 4. Population dynamics of yeasts in sour Chinese cabbage by 400 MPa during storage at 27 °C. The treatment times are 0 min, ■; 10 min, ○; 20 min, ▲; and 30 min, . Least Significance Difference (5% level) = 0.90. Detection limit is 1 CFU/g. Lines dropping below the x-axis indicate cell counts below the detection limit.
◇
Acknowledgments This work was funded by project no. 2007AA100405 of 863 HighTech Plan of China. We are grateful to Beidahuang Co. Ltd. for providing the SCC for this study. We also thank Mr. Zhijian Sun for his excellent technical assistance in the HHP treatment and Mr. Jianyong Yi for his kind help in the preliminary work. References Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C.P., Ray, B., 1999. Variation in resistance to hydrostatic pressure among strains of food-borne pathogens, USA. Applied and Environmental Microbiology 65, 4248–4251. Bayindirli, A., Alpas, H., Bozoglu, F., Hızal, M., 2006. Efficiency of high pressure treatment on inactivation of pathogenic microorganisms and enzymes in apple, orange, apricot and sour cherry juices, Turkey. Food Control 17, 52–58. Bozoglu, F., Alpas, H., Kaletunc, G., 2004. Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage, France. FEMS Immunology and Medical Microbiology 40, 243–247. Bull, M.K., Hayman, M.M., Stewart, C.M., Szabo, E.A., Knabel, S.J., 2005. Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk, Australia. International Journal of Food Microbiology 101, 53–61. Cheftel, J.C., 1995. Review: high pressure, microbial inactivation and food preservation, France. Food Science and Technology International 1, 75–90. Cui, Z.H., Zhou, Q., Hu, X.Q., Li, Y.P., Zhai, F.Y., Yang, X.G., Ma, G.S., 2008. Existing condition analysis of Chinese citizen consumption of fruits and vegetables, China. Food and Nutrition in China 34–37. Diez, A.M., Urso, R., Rantsiou, K., Jaime, I., Rovira, J., Cocolin, L., 2008. Spoilage of blood sausages morcilla de Burgos treated with high hydrostatic pressure, Spain. International Journal of Food Microbiology 123, 246–253. Ellenberg, L., Hoover, D.G., 1999. Injury and survival of Aeromondas Hudrophila 7965 and Yersinia Enterocolitica 9610 from high hydrostatic pressure, USA. Journal of Food Safety 19, 263–276. Garcia-Graells, C., Hauben, K., Michels, C.W., 1998. High pressure inactivation and sublethal injury of pressure resistant Escherichia coli mutants in fruit juices, Belgium. Applied and Environmental Microbiology 64, 1566–1568. Hayman, M., 2001. Inactivation of Listeria monocytogenes in milk using ultra high pressure. Honors thesis, Department of Microbiology, University of Sydney. Jordan, S.L., Pascual, C., Bracey, E., Mackey, B.M., 2001. Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices, UK. Journal of Applied Microbilogy 91, 463–469. Koseki, S., Yamamoto, K., 2006. Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure, Japan. International Journal of Food Microbiology 110, 108–111. Lavinas, F.C., Miguel, M.A.L., Lopes, M.L.M., Valente Mesquita, V.L., 2008. Effect of high hydrostatic pressure on cashew apple (Anacardium occidentale L.) juice preservation, Brazil. Journal of Food Science 73, 273–277. Lee, J.W., Cha, D.S., Hwang, K.T., Park, H.J., 2003. Effects of CO2 absorbent and highpressure treatment on the shelf-life of packaged Kimchi products, Korea. International Journal of Food Science and Technology 38, 519–524. Li, W.B., Song, M.L., Tang, Z.W., Wen, Y.Z., Wu, X.L., 2008. Investigation of microflora changes in naturally fermented Paocai, China. Food and Nutrition in China 22–24. Mok, C., Song, K.T., Park, Y.S., Lim, S., Ruan, R., Chen, P., 2006. High hydrostatic pressure pasteurization of red wine, Korea. Journal of Food Science 71, 265–269. O'Reilly, C.E., O'Connor, P.M., Kelly, A.L., Beresford, T.P., Murphy, P.M., 2000. Use of hydrostatic pressure for inactivation of microbial contaminants in cheese, Ireland. Applied and Environmental Microbiology 66, 4890–4896. Peñas, E., Frias, J., Gomez, R., Vidal-Valverde, C., 2010. High hydrostatic pressure can improve the microbial quality of sauerkraut during storage, Spain. Food Control 21, 524–528. Reps, A., Kolakowski, P., Dajnowiec, F., 1998. The effect of high pressure on microorganisms and enzymes of ripening cheeses. In: Isaacs, N.S. (Ed.), High Pressure Food Science. : Bioscience and Chemistry. Royal Society of Chemistry, UK, pp. 265–270. Smelt, J.P., Hellemons, J.C., Patterson, M., 2001. Effects of high pressure on vegetative microorganism. In: Hendrickx, M.E.G., Knorr, D. (Eds.), Ultra High Pressure Treatments of Foods. Kluwer Academic/Plenum, New York, pp. 55–76. Sohn, K.-H., Lee, H.-J., 1999. Effects of high pressure treatment on the quality and storage of kimchi, Japan. International Journal of Food Science and Technology 33, 359–365. Van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrilich, S.D., Maguin, E., 2002. Stress responses in lactic acid bacteria, France. Antonie van Leeuwenhoek 82, 187–216.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
High hydrostatic pressure inactivation of total aerobic bacteria, lactic acid bacteria, yeasts in sour Chinese cabbage Lin Li, Lun Feng, Junjie Yi, Cheng Hua, Fang Chen, Xiaojun Liao, Zhengfu Wang, Xiaosong Hu ⁎ College of Food Science and Nutritional Engineering, China Agriculture University, Beijing, 100083, PR China Key Laboratory of Fruits and Vegetables Processing, Ministry of Agriculture, Beijing, 100083, PR China Research Center of Fruits and Vegetables Processing Engineering, Ministry of Education, Beijing, 100083, PR China
a r t i c l e
i n f o
Article history: Received 31 March 2010 Received in revised form 12 June 2010 Accepted 23 June 2010 Keywords: High hydrostatic pressure Inactivation Sour Chinese cabbage Total aerobic bacteria Lactic acid bacteria Yeasts
a b s t r a c t This study investigated the inactivation of total aerobic bacteria (TAB), lactic acid bacteria (LAB), yeasts in sour Chinese cabbage (SCC) treated by high hydrostatic pressure (HHP). The pressure level ranged from 200 to 600 MPa and the treatment time were 10–30 min. All samples were stored at 4, 27 and 37 °C for 90 days. The pressure level of 200 MPa had no significant impact on these microorganisms. The counts of TAB were significantly reduced by 2.7–4.0 log10 CFU/g at 400 MPa and 4.2–4.5 log10 CFU/g at 600 MPa from 6.2 log10 CFU/g; the counts of LAB were also reduced by 2.4–4.3 log10 CFU/g at 400 MPa from 7.0 log10 CFU/g and LAB was completely inactivated at 600 MPa; the counts of yeasts were reduced by 1.5–2.0 log10 CFU/g at 400 and 600 MPa from 4.2 log10 CFU/g. Storage temperatures significantly influenced the microbial proliferation in HHP-treated SCC depending on the pressure levels. The surviving TAB and LAB at 400 MPa equaled initial counts after 15-day storage at 27 and 37 °C, whereas they were inhibited at 4 °C up to 60 days. The surviving TAB at 600 MPa did not grow. Yeasts at 400 and 600 MPa decreased below detectable level after 2 days at all the three storage temperatures. From the microbial safety point of view, the result indicated that HHP at 600 MPa could be used as an alternative preservation method for SCC. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Fermentation is one of the oldest forms of food preservation technologies in the world, which can increase the shelf-life of food, improving their safety, sensory and nutritional properties. Fermented vegetables are very popular throughout the world, such as kimchi from Korea and sauerkraut from Europe. Sour Chinese cabbage (SCC) is a kind of traditional lactic acid fermented vegetable widely consumed in China, especially in the northeast where is too cold to cultivate leafy vegetables in winter. About 1/6 of Chinese consumes SCC in daily diet, according to the data from Chinese Center for Disease Control and Prevention (Cui et al., 2008). These fermented vegetables have been generally considered as safe due to the presence of lactic acid. However, the raw materials are easily contaminated with food-borne pathogens, such as Escherichia coli and Listeria monocytogenes, which tolerate low pH environment (Peñas et al., 2010). Some products such as kimchi are still fermenting after packing, often leading to excessive sourness and pack swelling due to microbial activity during storage (Sohn and Lee, 1999). Other products like sauerkraut are pasteurized. It is known that thermal processing usually causes undesirable organoleptic changes such as ⁎ Corresponding author. 303#, No. 17 Qinghua East Road, Haidian District, Beijing 100083, PR China. Tel./fax: + 86 10 62737464. E-mail address: [email protected] (X. Hu). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.020
texture softening, flavor losing and discoloration. SCC products in Chinese market are various: some are just made in household level and sold in bulk; some are directly packaged without the control of microorganisms like kimchi; most are pasteurized like sauerkraut. SCC has similar problems mentioned above and sometimes even worse. How to produce both safe and tasty SCC is promptly needed. The growing demand for natural, fresh-like and safe food has aroused an increased interest in non-thermal processing technologies (Jordan et al., 2001). High hydrostatic pressure (HHP) is considered as one of the most promising methods because of its successful application in acid foods such as jams, fruit juices and other fruit products. HHP can kill most microorganisms without significantly affecting the sensory and nutritional properties, and so increase the shelf-life of these products. For fermented foods like cheeses, ham and yogurt, HHP has been widely studied. However, there have been only a few reports of HHP in fermented vegetables. Sohn and Lee (1999) firstly reported that in kimchi the count of total aerobic bacteria (TAB) was reduced to 4 log10 CFU/mL at 400 MPa for 10 min, and maintained during the 4-week storage at 20 °C; whereas there was no change in the texture. Lactic acid bacteria (LAB) was significantly reduced from 7 to 3 log10 CFU/mL at 600 MPa for 5 min, which prevented gas production in kimchi and excessive acidity during storage at 10 °C (Lee et al., 2003). Peñas et al. (2010) reported that 300 MPa for 10 min at 4 °C led to a reduction of TAB and LAB in sauerkraut (4–5 log10 CFU/g) immediately after the treatment;
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
although refrigerated storage brought an increase in microorganisms, the counts were still lower than untreated sauerkraut. Although similarities exist, there are also some significant differences between SCC and other lactic acid fermented vegetables in processing method, ingredient and the constituent of microorganisms. For example, SCC is completely anaerobic fermented whereas kimchi is not; the raw material of sauerkraut is white cabbage whereas that of SCC is Chinese cabbage; kimchi is added with high concentration of salt and species whereas SCC is produced with only a little salt and no other seasonings; these differences combined with regional disparities thus lead to the variation of microorganisms. To date, there is little knowledge on the application of HHP in SCC. The objective of this study was to investigate the inactivation of HHP on TAB, LAB, yeasts in SCC and its microbiological stability during 90 days at three storage temperatures.
2. Materials and methods 2.1. SCC preparation The SCC samples were provided by a commercial SCC producer (Beidahuang Co. Ltd., Heilongjiang, China). Two parallel batches of SCC were used for the experiment. The brief process of SCC production was as follows. Chinese cabbages (Brassica rapa L. ssp. pekinensis) harvested from the same field were chosen and their outer leaves were removed. All cabbages were of good quality and free of serious mechanical damage. They were washed in water jet washer and then were blanched with steam for 1 min. After cooling, cabbages were belt-carried into fermentation tank, with a cylinder 2 m in diameter and 6 m in height. Five percents of NaCl were uniformly distributed in cabbages. A mixed starter culture containing Lactobacillus plantarum, Lactobacillus maltaromicus, Leuconostoc meseteroides, Lactobacillus brevis (Northeast Agricultural University, Heilongjiang, China) was inoculated at approximately 6 log10 CFU/g of cabbage. Finally, cabbages were pressed thoroughly to remove air bubbles and the tank was sealed. Fermentation was carried out at 25 °C for 40 days. After the fermentation, SCC with pH 3.8–4.0 was sliced, vacuum packaged (500 g/package) and stored below 10 °C.
181
2.4. Storage study Following the treatment, each sample was divided into three portions and was stored at 4, 27 and 37 °C respectively for 90 days. The samples were taken at 0, 2, 5, 8, 15-day and 15-day intervals thereafter. 2.5. Enumeration of viable cells Each sample (25 g) was aseptically weighted and homogenized with 225 mL of sterile 0.85% NaCl solution for 1.5 min in an electromechanical blender (JYL-B060, Joyong Electric Appliance Co., Shandong, China). After homogenization, the blender was washed using sterile water, sanitized with 50-ppm chlorine dioxide (Beijing Techgreen Co., Ltd., Beijing, China), and then rinsed three times with sterile water. To detect the viable cells in SCC, the total plate count method was used. Sample was serially diluted with sterile 0.85% NaCl solution and 1.0 mL of diluted (or non-diluted) samples was plated into duplicated plates of appropriate agar. The plate count agar (PCA, composition: tryptone 10.0 g/L, yeast extract 2.5 g/L, glucose 1.0 g/L, agar 15.0 g/L, pH 7.0 ± 0.1. Beijing Luqiao Technology Co., Ltd., Beijing, China) was used for detecting the viable cells of TAB. The plates were incubated at 37 °C for 24 ± 2 h. The MRS agar (MRSA, composition: peptone 10.0 g/L, beef extract 5.0 g/L, yeast extract 4.0 g/L, glucose 20.0 g/L, Tween 80 1.0 mL, KH2PO4·7H2O 2.0 g/L, CH3COONa·3H2O 5.0 g/L, triamine citrate 2.0 g/L, MgSO4·7H2O 0.2 g/L, MnSO4·7H2O 0.05 g/L, agar 15.0 g/L, pH 6.2±0.1. Beijing Luqiao Technology Co., Ltd., Beijing, China) was used for detecting the viable cells of LAB. The plates were incubated at 30 °C for 72±2 h. The rose bengal agar (RBA, composition: peptone 5.0 g/L, glucose 10.0 g/L, KH2PO4 1.0 g/L, (NH4)2SO4 0.5 g/L, rose Bengal 0.03 g/L, chloromycetin 0.1 g/L, agar 15.0 g/L, pH 6.0 ± 0.1. Beijing Luqiao Technology Co., Ltd., Beijing, China) was used for detecting the viable cells of yeasts. The plates were incubated at 27 °C for 5 days. After incubation, the colonies were enumerated. The detection limit of TAB and LAB was 1 log10 CFU/g and that of yeasts was 1 CFU/g. Inactivation effect was expressed as log10 N/N0, where N0 was the number of initial count of microorganisms in control, and N was the corresponding viable count after HHP. 2.6. Statistical analysis
2.2. HHP equipment HHP treatment was carried out using a hydrostatic pressurization unit (HHP-650, Baotou Kefa Co., Ltd., Inner Mongolia, China), with a cylinder 11 cm in inner diameter and 30 cm in height and a capacity of 2.8 L. Distilled water was used as the pressure-transmitting fluid. The rate of pressure increase was about 120 MPa/min and the pressure release was immediate. The treatment time reported in this study did not include the pressure increase and release time. The pressure level and treatment time were continuously recorded during the pressurization cycle. The temperature in the pressure vessel rose from 25 to 33 °C during coming-up to 600 MPa by compression heating. Then the temperature decreased to 25 °C for about 5 min during pressure holding.
2.3. HHP treatment Each batch of SCC was mixed thoroughly under aseptic conditions, and then was divided into 560 small packages. Approximately 50 g SCC was transferred into a sterile polyethylene bag and heat-sealed under vacuum. The bags were placed into the pressure vessel and treated at 200, 400 and 600 MPa for 10, 20 and 30 min, respectively. Controls were not pressurized. The experiment was carried out at room temperature.
Analyses of variance (ANOVA) were carried out using the software Microcal Origin 7.5 (Microcal Software, Inc., Northampton, MA, USA). The significance level was 0.05. 3. Results and discussion 3.1. Inactivation of TAB, LAB, yeasts in SCC by HHP Fig. 1a and b showed the effect of pressure levels and treatment time on the inactivation of TAB and LAB in SCC, respectively. The samples treated by HHP at 200 MPa were not significantly different from the control in the counts of the two microorganisms. The counts were significantly reduced at 400 and 600 MPa. A 4-log reduction of TAB was attained at 400 MPa for 30 min while no significant additional reduction (P N 0.05) was observed at 600 MPa, indicating that some bacteria resisting to HHP did exist in SCC. A similar result was reported by Sohn and Lee (1999), showing that the count of TAB in kimchi was reduced from 8 to 4 log10 CFU/mL at 400 MPa for 10 min and 600 MPa did not increase the log reduction. The pressure level of 600 MPa was sufficient to reduce the counts of LAB below detectable level. Whereas, the count of LAB in kimchi at 600 MPa still remained at the level of 3 log10 CFU/mL (Sohn and Lee, 1999; Lee et al., 2003). It has been known that the compositions of food, especially high sugar or salt concentrations, exert very strong baroprotective effect on microorganisms (Cheftel, 1995). The
182
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
inactivation was observed at 400 and 600 MPa. Generally, yeasts are very sensitive to HHP due to their cellular morphology (Cheftel, 1995). For instance, yeasts were completely inactivated at 200 MPa in Gouda and Krupoiwski cheeses (Reps et al., 1998); the yeasts in red wine reduced to null at 300 MPa for 20 min (Mok et al., 2006); they were reduced below detectable level at 400 MPa in cashew apple juice (Lavinas et al., 2008). In this study, the inactivation of yeasts immediately after HHP did not conformed to these previous investigations. Sohn and Lee (1999) reported that the count of yeasts in kimchi was low (about 3 log10 CFU/mL) but remained constant by HHP up to 600 MPa. Smelt et al. (2001) proposed that yeasts are relatively resistant to low pH and barely affected at pH 4.0 when subjected to HHP. The pH of SCC in this study is right about 4, which could explain for the little inactivation effect of HHP on yeasts in SCC. 3.2. Microbiological stability in SCC by HHP
Fig. 1. Inactivation of total aerobic bacteria (a); lactic acid bacteria (b); and yeasts (c); in sour Chinese cabbage by high hydrostatic pressure. The pressure levels are 200 MPa, ■; 400 MPa, ○; and 600 MPa, ▲. Least Significance Difference (5% level) = 0.90. Detection limit for total aerobic bacteria is log10 N/N0 = −6.2; for lactic acid bacteria is log10 N/N0 = −7.0; for yeasts is log10 N/N0 = −4.2. Lines dropping below the x-axis indicate cell counts below the detection limit.
seasonings in kimchi could contribute to the microbial baroprotection, but SCC is produced without these ingredients. Besides, pressure resistance varies considerably between species and among strains within a species (Alpas et al., 1999). These reasons explained for the variation of inactivation effect between SCC and kimchi. As shown in Fig. 1c, the count of yeasts was reduced by 1.5 log10 CFU/g at 200 MPa for 30 min but no significant additional
3.2.1. Population change of LAB by HHP during storage Based on the results in Fig. 1, higher pressure levels (400 and 600 MPa) were selected for a further investigation of microbiological stability in SCC by HHP. Fig. 2 showed the population change of LAB in SCC at 400 MPa for 10, 20 and 30 min during 90-day storage at 37, 27 and 4 °C, respectively. The count (about 7 log10 CFU/g) of LAB in control sample remained statistically constant during storage, indicating that further growth of LAB was inhibited by the accumulated lactic acid. Immediately after HHP, the counts of LAB were significantly reduced to 4.6, 3.9 and 2.7 log10 CFU/g at 400 MPa for 10, 20 and 30 min, respectively. Later, increasing counts were observed and reached to the level of initial count of control sample after 5 days at 37 °C (Fig. 2a). Noticeable increasing counts were also observed during storage at 27 °C (Fig. 2b). The result was similar to earlier studies. Lee et al. (2003) reported that although kimchi at 400 MPa showed a reduction in the counts of TAB and LAB immediately after HHP, after 15-day storage at 10 °C increases occurred in the microbial populations which were finally comparable with that of the control. To date, amount of studies have been reported about the recovery of pathogenic and spoilage bacteria damaged by HHP during storage in various food matrix and buffer solutions (Bozoglu et al., 2004; Bull et al., 2005; Diez et al., 2008; Ellenberg and Hoover, 1999; Koseki and Yamamoto, 2006; O'Reilly et al., 2000). Whereas, the bacterial increase in nutrient-rich media could not be evaluated separately to distinguish between repair of injured cells and bacterial growth after HHP (Koseki and Yamamoto, 2006). Therefore, the increasing counts of LAB in SCC could be due to the growth of surviving cells and the recovery of injured cells. Unlike the obvious increase observed during storage at optimal growth temperatures, the counts of LAB in HHP-treated samples at 4 °C exhibited slight fluctuations and remained statistically constant up to 60 days (Fig. 2c), showing the inhibition effect of low temperature on the proliferation of LAB. But the increasing counts at 400 MPa were finally observed in the last 30 days, which was similar to a previous study on sauerkraut (Peñas et al., 2010). They found that the refrigerated storage of 3 months brought about an increase in the counts of TAB and LAB in sauerkraut after the immediate reduction when subjected to 300 MPa for 10 min at 40 °C. They ascribed the increase of TAB and LAB in sauerkraut during the third month of refrigerated storage after HHP to the recovery of microorganisms that were sublethally injured by HHP. However, several studies have demonstrated that low temperature or low pH could inhibit the microbial recovery (Bozoglu et al. 2004; Bull et al. 2005; Hayman, 2001; Koseki and Yamamoto, 2006; Smelt et al., 2001). Therefore, the microbial increase in the third month could most likely be explained as the growth of surviving cells rather than the recovery of injured cells. The result showed that although low temperature inhibited the proliferation
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
183
3.2.2. Population change of TAB by HHP during storage In SCC where the predominant microflora is LAB, the population change of TAB by HHP during storage was therefore closely correspondent with that of LAB. Fig. 3a showed the change of TAB at 400 MPa during storage at 27 °C. Similar to that of LAB, the counts of TAB increased to the initial count of control sample after 15 days. The change of TAB stored at 37 °C was similar (data not shown). The counts of TAB stored at 4 °C experienced slight fluctuations and finally the increase occurred in the last month of storage, which was also analogical to that of LAB at 4 °C. The counts of TAB at 600 MPa remained relatively constant during 90 days, keeping lower than 2.2 log10 CFU/g at the three storage temperatures (data not shown). It showed that although some bacteria extremely resistant to HHP still existed in SCC, their growth was effectively inhibited by the low pH. The result was in agreement with the earlier study of HHP in kimchi (Lee et al., 2003), in which the count of TAB at 600 MPa maintained at about 3 log10 CFU/mL for at least 24 days. The result showed that SCC treated by HHP at 600 MPa exhibited a better microbiological stability.
3.2.3. Population change of yeasts by HHP during storage Generally yeasts do not cause food-borne disease, but they play an important role in the spoilage of food, especially in acid foods (Lavinas et al., 2008). Yeasts can produce gas in SCC and often causes pack inflation. Although the medium used to detect yeasts is rose bengal agar on which both yeast and molds could be cultivated, no
Fig. 2. Population dynamics of lactic acid bacteria in sour Chinese cabbage treated by 400 MPa during storage at 37 °C (a); 27 °C (b); and 4 °C (c). The treatment times are 0 min, ■; 10 min, ○; 20 min, ▲; and 30 min, . Least Significance Difference (5% level) = 0.90. Detection limit is 1 log10 CFU/g.
◇
of LAB to some extent, the inhibition effect was limited and could not last very long. Like other bacteria, LAB evolves defense mechanisms against cold-shock and it can adapt to a temperature downshift (Van de Guchte et al. 2002). Thus, LAB in SCC at 4 °C began to proliferate after 2-month adaptation of cold. For LAB at 600 MPa, it was not detected during 90 days at all the three storage temperatures (data not shown), indicating that the LAB in this case was totally inactivated.
Fig. 3. Population dynamics of total aerobic bacteria in sour Chinese cabbage treated by 400 MPa during storage at 27 °C (a) and 4 °C (b). The treatment times are 0 min, ■; 10 min, ○; 20 min, ▲; and 30 min, . Least Significance Difference (5% level) = 0.90. Detection limit is 1 log10 CFU/g.
◇
184
L. Li et al. / International Journal of Food Microbiology 142 (2010) 180–184
mold in the samples was detected through microscopy and observation of colony morphology. As shown in Fig. 4, there were two phases for population change of yeasts in the control and HHP-treated samples during 90-day storage, but the patterns were different. For the control sample, the count of yeasts decreased in the initial phase of storage and it began to increase after 30 days. It is because that yeasts were firstly inhibited by the accumulated lactic acid produced by LAB and the non-acid adapted cells gradually died; those survivors consumed acid and increased the pH, becoming the main factor for the spoilage of SCC (Li et al., 2008). The pH analysis in our study supported it: the pH gradually increased from 4.0 to 4.4 during the storage. The counts of yeasts at 400 MPa were sharply reduced below detectable level after 2 days and no resuscitation was observed during the storage at all the three storage temperatures. Combined with the result shown in Fig. 1c, it is indicated that although 400 and 600 MPa did not significantly reduce the counts of yeasts immediately after HHP, these microorganisms were probably injured — a kind of injury defined by Bozoglu et al. (2004) as primary injury which can form visible colonies on non-selective agar. The sharp reduction after 2 days could be interpreted as acid sensitization effect that was a proportion of the cells which survived pressurization were sublethally injured in low pH, the cells were unable to repair the damage, hence lowering their tolerance to the unfavorable pH and organic acids present in the medium (Bayindirli et al., 2006). The result was similar to the study of HHP on mutant of E. coli in fruit juice (Garcia-graells et al., 1998), in which the low level (1.1 log10 CFU/mL) direct reduction of LMM1010 microorganisms in apple juice subjected to 300 MPa for 15 min at 20 °C was reported and was followed by an extensive (almost 5 log10 CFU/mL) further reduction during the first 2-day storage. This indicated that HHP not only injured cells at its own right but also caused changes in the cells that make them more susceptible to subsequent acid injury. Yeasts at 600 MPa showed similar trend during storage as that at 400 MPa (data not shown). It is worth to mention that yeasts in control samples also died out during the storage at 37 °C but it was due to the high temperature unsuitable for yeasts to survive in SCC. Based on the results in this study, it is found that yeasts in SCC is the most sensitive to HHP and the pressure level of 600 MPa is the most effective. It is necessary that the microbiological stability of post-pressurization be taken into consideration in pressure inactivation study since the microbial population could change during storage.
Fig. 4. Population dynamics of yeasts in sour Chinese cabbage by 400 MPa during storage at 27 °C. The treatment times are 0 min, ■; 10 min, ○; 20 min, ▲; and 30 min, . Least Significance Difference (5% level) = 0.90. Detection limit is 1 CFU/g. Lines dropping below the x-axis indicate cell counts below the detection limit.
◇
Acknowledgments This work was funded by project no. 2007AA100405 of 863 HighTech Plan of China. We are grateful to Beidahuang Co. Ltd. for providing the SCC for this study. We also thank Mr. Zhijian Sun for his excellent technical assistance in the HHP treatment and Mr. Jianyong Yi for his kind help in the preliminary work. References Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C.P., Ray, B., 1999. Variation in resistance to hydrostatic pressure among strains of food-borne pathogens, USA. Applied and Environmental Microbiology 65, 4248–4251. Bayindirli, A., Alpas, H., Bozoglu, F., Hızal, M., 2006. Efficiency of high pressure treatment on inactivation of pathogenic microorganisms and enzymes in apple, orange, apricot and sour cherry juices, Turkey. Food Control 17, 52–58. Bozoglu, F., Alpas, H., Kaletunc, G., 2004. Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage, France. FEMS Immunology and Medical Microbiology 40, 243–247. Bull, M.K., Hayman, M.M., Stewart, C.M., Szabo, E.A., Knabel, S.J., 2005. Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk, Australia. International Journal of Food Microbiology 101, 53–61. Cheftel, J.C., 1995. Review: high pressure, microbial inactivation and food preservation, France. Food Science and Technology International 1, 75–90. Cui, Z.H., Zhou, Q., Hu, X.Q., Li, Y.P., Zhai, F.Y., Yang, X.G., Ma, G.S., 2008. Existing condition analysis of Chinese citizen consumption of fruits and vegetables, China. Food and Nutrition in China 34–37. Diez, A.M., Urso, R., Rantsiou, K., Jaime, I., Rovira, J., Cocolin, L., 2008. Spoilage of blood sausages morcilla de Burgos treated with high hydrostatic pressure, Spain. International Journal of Food Microbiology 123, 246–253. Ellenberg, L., Hoover, D.G., 1999. Injury and survival of Aeromondas Hudrophila 7965 and Yersinia Enterocolitica 9610 from high hydrostatic pressure, USA. Journal of Food Safety 19, 263–276. Garcia-Graells, C., Hauben, K., Michels, C.W., 1998. High pressure inactivation and sublethal injury of pressure resistant Escherichia coli mutants in fruit juices, Belgium. Applied and Environmental Microbiology 64, 1566–1568. Hayman, M., 2001. Inactivation of Listeria monocytogenes in milk using ultra high pressure. Honors thesis, Department of Microbiology, University of Sydney. Jordan, S.L., Pascual, C., Bracey, E., Mackey, B.M., 2001. Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices, UK. Journal of Applied Microbilogy 91, 463–469. Koseki, S., Yamamoto, K., 2006. Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure, Japan. International Journal of Food Microbiology 110, 108–111. Lavinas, F.C., Miguel, M.A.L., Lopes, M.L.M., Valente Mesquita, V.L., 2008. Effect of high hydrostatic pressure on cashew apple (Anacardium occidentale L.) juice preservation, Brazil. Journal of Food Science 73, 273–277. Lee, J.W., Cha, D.S., Hwang, K.T., Park, H.J., 2003. Effects of CO2 absorbent and highpressure treatment on the shelf-life of packaged Kimchi products, Korea. International Journal of Food Science and Technology 38, 519–524. Li, W.B., Song, M.L., Tang, Z.W., Wen, Y.Z., Wu, X.L., 2008. Investigation of microflora changes in naturally fermented Paocai, China. Food and Nutrition in China 22–24. Mok, C., Song, K.T., Park, Y.S., Lim, S., Ruan, R., Chen, P., 2006. High hydrostatic pressure pasteurization of red wine, Korea. Journal of Food Science 71, 265–269. O'Reilly, C.E., O'Connor, P.M., Kelly, A.L., Beresford, T.P., Murphy, P.M., 2000. Use of hydrostatic pressure for inactivation of microbial contaminants in cheese, Ireland. Applied and Environmental Microbiology 66, 4890–4896. Peñas, E., Frias, J., Gomez, R., Vidal-Valverde, C., 2010. High hydrostatic pressure can improve the microbial quality of sauerkraut during storage, Spain. Food Control 21, 524–528. Reps, A., Kolakowski, P., Dajnowiec, F., 1998. The effect of high pressure on microorganisms and enzymes of ripening cheeses. In: Isaacs, N.S. (Ed.), High Pressure Food Science. : Bioscience and Chemistry. Royal Society of Chemistry, UK, pp. 265–270. Smelt, J.P., Hellemons, J.C., Patterson, M., 2001. Effects of high pressure on vegetative microorganism. In: Hendrickx, M.E.G., Knorr, D. (Eds.), Ultra High Pressure Treatments of Foods. Kluwer Academic/Plenum, New York, pp. 55–76. Sohn, K.-H., Lee, H.-J., 1999. Effects of high pressure treatment on the quality and storage of kimchi, Japan. International Journal of Food Science and Technology 33, 359–365. Van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrilich, S.D., Maguin, E., 2002. Stress responses in lactic acid bacteria, France. Antonie van Leeuwenhoek 82, 187–216.