Bacterial Contamination of Soybean Curd (Tofu) Sold in Thailand

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1 Food Sci. Technol. Res., 18 (6), , 2012 Bacterial Contamination of Soybean Curd (Tofu) Sold in Thailand Chiraporn Ananchaipattana 1,2,4, Yukie Hosotani 1, Susumu Kawasaki 1, Sirikhae Pongswat 4, Bari Md. Latiful 3, Seiichiro Isobe 1,2 and Yasuhiro Inatsu 1* 1 National Food Research Institute, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, , Japan 2 Graduate School of Life and Environmental Science, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, , Japan 3 Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh 4 Department of Biology, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, 39 Muh1, Thanyaburi Pathum Thani, 12110, Thailand Received April 16, 2012; Accepted July 25, 2012 Soybean curd (Tofu) is a popular food for many people, especially those with health concerns. The objective of this study, therefore, was to evaluate the contamination rate and types of foodborne pathogens and food spoilage bacteria in packaged and unpackaged tofu sold in Thai markets. A total of 133 tofu samples (59 packaged tofu samples and 74 unpackaged tofu samples) were collected from open markets and supermarkets in Thailand. The predominantly identified Gram-negative bacteria were coliforms (67% in tested samples), Pseudomonas spp. (56%) and Escherichia coli (28%) and the predominantly identified Gram-positive bacteria were Enterococcus spp. (77%), lactic acid bacteria (68%), Bacillus cereus (41%) and Staphylococcus spp. (26%). No Listeria spp. were found in any of the tofu samples. The contamination rates of E. coli, Salmonella spp. and Enterococcus spp. in unpackaged tofu were significantly higher than those in packaged tofu (P < 0.05). Three kinds of Gram-negative pathogenic bacteria, Cronobacter sakazakii, Salmonella spp. and Yersinia enterocolitica (serotype 0:5), were found only in unpackaged tofu samples. Of 54 B. cereus isolates, four isolates from three unpackaged tofu samples and one packaged tofu sample were found to produce diarrheal enterotoxin. The most common Enterococcus spp. isolates were Enterococcus faecium and Enterococcus faecalis. These results suggested that the improvement of hygienic practices might be required for reducing the risk of food poisoning and food spoilage of tofu in Thailand. Keywords: tofu, bacterial contamination, Thailand Introduction Tofu (soybean curd) is considered a healthy part of a well-balanced diet, as it is low in calories and rich in protein, iron, calcium, magnesium, and B-vitamins (Pontecorvo and Bourne, 1978; Prestamo and Fontecha, 2007). The low cost of soybean products makes them a suitable alternative for reducing malnutrition problems in poor people living on grain-centered diets (Shurtleff and Aoyagi, 1983). Tofu has been produced for centuries using the same basic methods. Soybeans are soaked until completely hydrated, rinsed and ground. The liquid and solid portions are then separated. The soymilk is heated to the boiling point, filtered and then *To whom correspondence should be addressed. inatu@affrc.go.jp precipitated with coagulants such as calcium or magnesium sulfate, calcium or magnesium chloride. The resultant curd is formed and pressed into cakes of tofu (Kovats et al., 1984; Shurtleff and Aoyagi, 1983). Tofu is available in several types: soft tofu is approximately 84-89% water, firm tofu is approximately 79 87% water and has a firm texture, and dried tofu is the firmest variety, with a moisture content of 60 82%. In Thailand, two categories of tofu (packaged tofu and unpackaged tofu) are commonly sold in open markets and supermarkets. Usually, packaged tofu is sold in supermarkets, where it is normally found refrigerated (4 10 ). Unpackaged tofu is sold only in open markets, typically by independent vendors who display the product unwrapped or covered by banana leaf, laid on a tray or soaked in a large bucket filled with water at ambient temperature, and where

2 844 hands can directly touch it. Tofu is easily spoiled in warm environments by microbial growth due to its characteristics: high protein, high water content, and neutral ph (Asharaf et al., 1999; Tuitemwong and Fung, 1991; van Kooij and de Boer, 1985). Thus, its shelf life is short and storage / distribution is limited. Spoilage of tofu is characterized by a sour taste and is associated with bacterial growth (Dotson et al., 1977); however, limited information has been available on the major bacteria responsible for its spoilage. Joo et al. (1998) reported that Acinetobacter calcoaceticus, Bacillus cereus, Klebsiella pneumoniae and Xenorhabdus luminescens were major causes of tofu spoilage. Dotson et al. (1977) developed criteria for measuring spoilage and attributed lactic acid bacteria to the phenomenon. In addition, several outbreaks of yersiniosis have been implicated in tofu; a case in Seattle (Aulisio et al., 1983) and one in Washington State between December 1981 and February 1982 (Tacket et al., 1985) were reported. Several incidents of tofu recalled due to Listeria monocytogenes contamination were also reported (Associated Press, 1992). The lack of good sanitary practices in tofu manufacturing processes may result in the purchase and consumption of unacceptable tofu products. The evaluation of contamination rates and types of contaminating bacteria in tofu could provide important information for tracing the source of bacterial contamination, as well as in the development of suitable food hygiene measures for tofu production. The objective of this study was to evaluate the contamination rates and types of foodborne pathogens / spoilage bacteria, including coliform bacteria, E. coli, Salmonella spp., Yersinia spp., C. sakazakii, Pseudomonas spp., Listeria spp., B. cereus, Staphylococcus spp., Enterococcus spp., and lactic acid bacteria (LAB), in packaged and unpackaged tofu sold in Thai markets. Materials and Methods Collection of Thai tofu samples A total of 133 tofu samples, including 74 unpackaged tofu samples from open markets and 59 packaged tofu samples from supermarkets, were purchased randomly from 12 different open markets and 7 supermarkets in Bangkok (Pathum Thani and Chiang Rai areas) from September 2010 to February All samples were transported to the laboratory of Rajamangala University of Technology Thanyaburi in an insulated box with ice to maintain the temperature at 4 6. Tofu samples were homogenized with distilled water (1:2, wt/v) and ph values were measured using an electric ph meter (F-55 ph meter, Horiba, Japan). Isolation of foodborne pathogens and spoilage bacteria Each 25 g tofu sample (cake and immersion solution) was C. Ananchaipattana et al. homogenized in 225 ml of enrichment buffer corresponding to each kind of bacteria: EEM enrichment broth (Nissui Co., Ltd., Tokyo, Japan) for Salmonella spp.; phosphate-buffered saline (PBS, ph 7.0) for Yersinia spp.; Mossel-Bouillon enrichment medium (Merck, Darmstadt, Germany) for C. sakazakii, coliform bacteria and E. coli; Listeria enrichment broth (Oxoid Ltd., Basingstoke, England) for Listeria spp.; buffered peptone water (Merck) for Bacillus spp., Staphylococcus spp., Pseudomonas spp. and Enterococcus spp.; and de Man, Rogosa and Sharpe (MRS) broth (Difco, NH, USA) for LAB. Enrichment cultivation of Listeria and Yersinia was performed at 30 for 48 h and 10 for 7 days, respectively. The other enrichment cultivations were performed at for 24 h. A loopful of each enrichment culture was streaked onto the corresponding selective media [PBS (ph 7.0) cultures for Yersinia spp. were streaked onto Yersinia selective agar (Oxoid Ltd) with Yersinia selective supplement SR0109 (Oxoid Ltd), Mossel-Bouillon enrichment cultures were streaked onto Chromocult Enterobacter Sakazakii agar (Merck) and MacConkey Sorbitol agar (Nissui Co., Ltd., Tokyo, Japan), Listeria enrichment was streaked onto Listeria selective agar (Oxoid Ltd) supplemented with SR0140E (Oxoid Ltd), buffered peptone water was streaked onto NGKG agar (Nissui Co., Ltd) with 20% egg yolk, mannitol salt agar (Nissui Co.) with 20% egg yolk and NAC agar (Nissui Co.) and MRS broth was streaked on MRS agar], and the plates were incubated at for h. Portions (0.5 and 1.0 ml) of the EEM-enriched samples were transferred to 10 ml of Rappaport-Vassiliadis (RV) broth (Eiken Co., Ltd., Tokyo, Japan) (incubated at 42 for 20 h) and 10 ml Hajna tetrathionate (TT) broth (Eiken Co.) (incubated at 35 for 20 h), respectively. To isolate Salmonella strains, the cultures were streaked onto DHL agar (Nissui Co.), Bismuth sulfide agar (Difco) and MLCB agar (Nissui Co.) and incubated at 35 for 20 h. Ten milliliters of buffered peptone water culture was added to 10 ml of two times concentration (2x) of AC medium (Nissui Co.) and incubated at 35 for 20 h. After incubation, one loopful of culture was inoculated into a 10 ml of standard concentration (1x) of AC medium and incubated at 42 for 20 h. To isolate Enterococcus strains, a loopful of 1x AC culture was streaked onto EF agar (Nissui Co.). Aliquots (10 ml) of the Mossel-Bouillon enrichment cultures were transferred to 10 ml of two times concentration (2x) of EC medium (Nissui Co.) and incubated at 35 for 20 h. One loopful of culture was inoculated into a 10 ml of standard concentration (1x) of EC medium with Durham fermentation tubes and incubated at 42 for 20 h. To isolate E. coli strains, a loopful of 1x EC culture broth from a gas producing tube was streaked onto EMB agar (Nissui Co.). After incubation, strains were isolated by visual observation

3 Bacteria in Thai Tofu of colony shape and color, and stored at 4 until further studies. Identification of bacterial isolates Characterization of the isolates was based on colony morphology, shape of microscopic features after Gram straining, biochemical characteristics in the TSI/LIM test (Nissui Co.) and API diagnostic system (BioMérieux, Marcy l Etoile, France). Suspected strains of Salmonella spp. and Listeria spp. were picked and subjected to further immunological tests using a Salmonella LA Seiken latex-agglutination test kit (Denka Seiken Co., Ltd., Tokyo, Japan) and a NH Immunochromato Listeria kit (Cosmo Bio Co., Ltd., CA, USA), respectively. Identification of Yersinia spp., C. sakazakii, Listeria spp. and S. aureus was performed using 16S ribosomal DNA sequencing. Production of diarrheal enterotoxin and the existence of the cereulide synthetic enzyme (CRS) gene of B. cereus were determined with a commercially available CRET-RPLA Kit (Denka Seiken Co., Ltd) and a PCR detection kit (Takara Bio Inc., Tokyo, Japan), respectively. The antiserum type of isolated Y. enterocolitica strains was fixed by a Yersinia enterocolitica O-Antisera SEIKEN set (Denka Seiken Co., Ltd). Suspected colonies of E. coli serotype O26, O157 or O111 were picked from XM-EHEC agar (Nissui Co., Ltd) and CT- MacConkey Sorbitol agar (Nissui Co., Ltd) and confirmed by biochemical characteristics in TSI/LIM tubes (Nissui Co., Ltd). The O26, O157 or O111 antisera (Denka Seiken Co., Ltd) were used for serotype confirmation. The possible production of Shiga-like toxin (stx) 1 and Shiga-like toxin (stx) 2 was assayed by NH Immunochromato VT1/2 (Cosmo Bio Co., Ltd., CA, USA) and O-157 VT1/2 PCR Typing Set Plus (Takara Biotechnology Co., Ltd). Statistical analysis Differences between the two categories of tofu were evaluated using the Student s t-test. Mi- crosoft EXCEL 2007 was used for the data analysis. 845 Results and Discussion Tofu is recognized as a very good source of protein and numerous other nutrients necessary for good health. However, there are few published reports about the contamination rate or prevalence of foodborne pathogens / spoilage bacteria in tofu products. In this study, 11 kinds of bacteria (coliform bacteria, E. coli, Salmonella spp., Yersinia spp., C. sakazakii, Pseudomonas spp., Listeria spp., B. cereus, Staphylococcus spp., Enterococcus spp., and LAB) were isolated and identified from 133 Thai tofu samples, including 74 unpackaged samples from 38 vendors and 59 packaged samples manufactured by 17 commercial brands, purchased from September 2010 to February The ph range of collected tofu samples was recorded as 4 6 in both tofu categories. A low ph level in tofu is reported to be associated with the growth of Gram-positive organisms, primarily Streptococcus sp. and Pediococcus sp. (Tuitemwong and Fung, 1991). The contamination rate of bacteria in the collected 133 tofu samples is summarized in Table 1. The contamination rate of Gram-negative bacteria was 67% for coliform bacteria, 56% for Pseudomonas spp., 28% for E. coli, 4% for Salmonella spp., 2% for Yersinia spp. and 1% for C. sakazakii. Nine E. coli O26, O157 and O111 suspected isolates did not harbor stx1/2 genes or produce Shiga-like toxins. The identified bacteria isolated from each category of tofu sample are shown in Table 2. In the 32 types of Gram-negative isolates, 12 kinds of them were found in both tofu sample categories (Citrobacter braakii, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cancerogenus, Enterobacter cloacae, E. coli, Klebsiella oxytoca, Pantoea spp., Rahnella aquatilis, Serratia marcescens, Serratia odorifera and Pseudomonas Pathogens Table 1. Contamination rate of bacteria in Thai tofu. Tofu samples packaged tofu (%) unpackage tofu (%) total tofu (%) Coliform bacteria* 26/ / / Escherichia coli* 1/ / / Salmonella spp.* 0/59 0 5/74 7 5/133 4 Yersinia spp. 1/59 2 1/74 1 2/133 2 Cronobacter sakazakii 0/59 0 1/74 1 1/133 1 Pseudomonas spp. 35/ / / Listeria spp. 0/59 0 0/74 0 0/133 0 Bacillus spp. 24/ / / Staphylococcus spp. 14/ / / Enterococcus spp.* 37/ / / Lactic acid bacteria 40/ / / The * indicates a significant difference (P<0.05) in the contamination rate of each bacterium between the two categories of tofu.

4 846 C. Ananchaipattana et al. Table 2. Identified Gram-negative and Gram-positive bacteria in Thai tofu. Categories of tofu Gram-negative bacteria Gram-positive bacteria packaged tofu Acinetobacter baumannii Aeromonas hydrophila Citrobacter braakii Citrobacter freundii Enterobacter aerogenes Enterobacter amnigenus Enterobacter cancerogenus Enterobacter cloacae Escherichia coli Klebsiella oxytoca Pantoea spp. Rahnella aquatilis Serratia marcescens Serratia odorifera Yersinia intermedia Pseudomonas spp. Aneurinibacillus aneurinilyticus Bacillus cereus Brevibacillus laterosporus Bacillus mycoides Staphylococcus hominis Staphylococcus lentus Staphylococcus sciuri Staphylococcus xylosus Aerococcus viridans Enterococcus faecalis Enterococcus faecium Enterococcus durans Lactobacillus acidophilus Lactobacillus brevis Lactobacillus collonoides Lactobacillus fermentum Lactobacillus plantarum Leuconostoc lactis Leuconostoc mesenteroides ssp. mesenteroides/dextranicum Pediococcus pentosaceus Weissella confusa Categories of tofu Gram-negative bacteria Gram-positive bacteria unpackaged tofu Buttiauxella agrestis Citrobacter braakii Citrobacter freundii Citrobacter koseri Citrobacter youngae Cronobacter sakazakii Enterobacter aerogenes Enterobacter asburiae Enterobacter cancerogenus Enterobacter cloacae Enterobacter hormaechei Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae ssp. pneumoniae Kluyvera spp. Pantoea spp. Pasturella pneumotropica Proteus mirabilis Proteus penneri Proteus vulgaris Rahnella aquatilis Salmonella spp. Serratia fonticola Serratia liquefaciens Serratia marcescens Serratia odorifera Yersinia enterocolitica (serotype 0:5) Pseudomonas spp. Bacillus cereus Bacillus mycoides Staphylococcus capitis Staphylococcus lentus Staphylococcus sciuri Staphyloccous xylosus Micrococcus spp. Microbacterium spp. Enterococcus avium Enterococcus faecalis Enterococcus faecium Lactobacillus acidophilus Lactobacillus brevis Lactobacillus collinoides Lactobacillus delbrueckii ssp. delbrueckii Lactobacillus fermentum Lactobacillus plantarum Leuconostoc lactis Leuconostoc lactis ssp. lactis Leuconostoc mesenteroides ssp. mesenteroides/dextranicum Pediococcus pentosaceus Weissella confusa

5 Bacteria in Thai Tofu spp). Four kinds of Gram-negative bacteria were found only in packaged tofu (Acinetobacter baumannii, Aeromonas hydrophila, Enterobacter amnigenus and Yersinia intermedia). Sixteen kinds of Gram-negative bacteria were found in only unpackaged tofu (Buttiauxella agrestis, Citrobacter koseri, Citrobacter youngae, Cronobacter sakazakii, Enterobacter asburiae, Enterobacter hormaechei, Klebsiella pneumoniae ssp. pneumoniae, Kluyvera spp., Pasteurella pneumotropica, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Salmonella spp., Serratia fonticola, Serratia liquefaciens and Y. enterocolitica). A high coliform bacteria contamination rate (67%) in Thai tofu was found in this study. Rehberger (1984) reported that coliform bacteria were found often in fresh tofu. On the other hand, a substantially higher E. coli contamination rate was observed in unpackaged tofu (49%) than in packaged tofu (2%) in this study. E. coli contamination of commercial foods is typically of human or animal origin, and grows under inappropriate temperature control. Differences in hygienic conditions of these markets relates to the rate of E. coli contamination. A previous outbreak of yersiniosis in Seattle was thought to be associated with the water used in the manufacturing processes (Aulisio et al., 1983). The other case of yersiniosis in Washington State was associated with contaminated tofu (Tacket et al., 1985). Moreover, Y. enterocolitica was detected in 11% of 154 collected tofu samples sold in the Netherlands (van Kooij and Boer, 1983). In this study, out of two Yersinia spp. isolates (2% contamination rate in tested samples), one was Y. intermedia from packaged tofu and the other one was Y. enterocolitica (serotype 0:5) from unpackaged tofu. According to Kovats et al. (1984), Y. enterocolitica can increase to 4.0 to 6.0 log cycles within 14 days at 5, but sufficient heat treatment of tofu after packaging can inactivate this bacterium. This is the first report that shows the contamination of Salmonella spp. into commercially distributed tofu. Salmonella spp. were found only in unpackaged tofu (7%) purchased from open markets. All of these contaminated unpackaged tofu samples were sold in close proximity to meat, suggesting that the display of unwrapped tofu products in these areas and/or the direct touching of tofu after touching meat may increase the risk of contamination with pathogenic bacteria. C. sakazakii contamination was also found only in 847 unpackaged tofu. The source of C. sakazakii contamination is probably similar to that of Salmonella spp. The contamination rate of Gram-positive bacteria in collected tofu samples was 77% for Enterococcus spp., 68% for LAB, 41% for B. cereus and 26% for Staphylococcus spp., while no Listeria spp. were found in any of the collected samples. Fouad and Hegeman (1993) observed that major LAB species were found in all aged tofu samples and major Enterococcus spp. were found in all aged tofu samples except for 1 day-old lot of tofu. Sixteen of 26 kinds of Grampositive bacteria isolated from the 133 tofu samples were found in both categories of tofu samples (B. cereus, Bacillus mycoides, Staphylococcus lentus, Staphylococcus sciuri, Staphylococcus xylosus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus collonoides, Lactobacillus fermentum, Lactobacillus plantarum, Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides, Pediococcus pentosaceus and Weissella confuse). Five kinds of Gram-positive bacteria were found only in packaged tofu (Aneurinibacillus aneurinilyticus, Brevibacillus laterosporus, Staphylococcus hominis, Aerococcus viridans and Enterococcus durans). Five kinds of Gram-positive bacteria were found in only unpackaged tofu (Staphylococcus capitis, Micrococcus spp., Microbacterium spp., Enterococcus avium and Lactobacillus delbrueckii ssp. delbrueckii). Of 54 B. cereus isolates, 4 isolates from 3 unpackaged and 1 isolate from packaged tofu samples produced diarrheal enterotoxin. No isolated B. cereus strains harbored the CRS gene (Table 3). No statistically significant differences in the contamination rate of B. cereus in packaged tofu (36%) and that in unpackaged tofu (36%) were observed. The source of contamination might be the presence of B. cereus spores on soybean seeds, which survived the heat treatment step of soy milk production. In this study, S. aureus strains were not found in any of the tested tofu samples. However, 26% of tested tofu samples were contaminated with Staphylococcus spp. This might be due to inadequate sanitary practices during production and inappropriate storage conditions. The contamination rates of coliform bacteria, E. coli, Salmonella spp. and Enterococcus spp. in unpackaged tofu samples were significantly higher than those in packed tofu Table 3. Production of B. cereus diarrheal enterotoxins in Thai tofu. Categories of tofu Number of tested samples Number of isolated B. cereus Number of positive enterotoxin production Packaged tofu Unpackaged tofu Total

6 848 samples (P < 0.05) (Table1). This study confirmed the concerns regarding the safety of unpackaged tofu sold in openmarkets. Results of this microbiological survey of two categories of tofu highlight the inadequacy of sanitary practices during tofu production and the exposure of tofu to inappropriate open-market storage conditions, leading to contamination. The presence of Y. enterocolitica, Salmonella spp., C. sakazakii or the higher contamination rate of E. coli and B. cereus in foods constitutes or indicates a potential health hazard. Thus, good manufacturing practices (GMP) during the production process, hygienic distribution practices and proper storage conditions are necessary for producing high quality tofu. Moreover, increasing public awareness of the proper manufacturing, storage and hygienic handling of tofu is recommended. Acknowledgements This work was supported by a grant from the Ministry of Agriculture, the Forestry and Fisheries of Japan (research project for ensuring food safety from farm to table DI-7301). References Asharaf, H.R., White, M. and Klubek, B. (1999). Microbiology survey of tofu sold in a rural Illinois county. J. Food Protect., 62, Associated Press. 9 February Possible contaminant prompts firm to recall tofu. San Francisco Examiner. B7. Aulisio, C.C.G., Stanfield, J.T., Weagant, S.D. and Hill, W.E. (1983). Yersiniosis associated with tofu consumption: serological, biochemical and pathogenicity studies of Yersinia enterocolitica C. Ananchaipattana et al. isolates. J. Food Protect., 46, Dontson, C.R., Frank, HA. and Cavaletto, C.G. (1977). Indirect methods as criteria of spoilage in tofu (soybean curd). J. Food Sci., 42, Fouad, H.K. and Hegeman, G.D. (1993). Microbial spoilage of tofu (soybean curd). J. Food Protect., 56, Joo, G.J., Hur, S.S., Choi, Y.H. and Rhee, I.K. (1998). Characterization and identification of bacteria from putrefying soybean curd. Korean J. Postharvest Sci. Technol., 5, Kovats, S.K., Doyle, M.P. and Tanaka, N. (1984). Evaluation of the microbiological safety of tofu. J. Food Protect., 47, Pontecorvo, A.J. and Bourne, M.C. (1978). Simple methods for extending the shelf life of soy curd (tofu) in tropical areas. J. Food Sci., 43, Prestamo, G. and Fontecha, J. (2007). High pressure treatment on the tofu fatty acids and acylglycerols content. Innovat. Food Sci. Emerg. Tech., 8, Rehberger, T.G., Wilson, L. A. and Glatz, B.A. (1984). Microbiological quality of commercial tofu. J. Food Protect., 47, Shurtleff, W. and Aoyagi, A. (1983). The book of Tofu. Ten Speed Press, Berkeley, CA. Tacket, C.O., Harris, N., Allard, J., Nolan, C., Nissinen, A., Quan, T. and Cohen, M.L. (1985). An outbreak of Yersinia enterocolitica infections caused by contaminated tofu (Soybean curd). Am. J. Epidemiol., 121, Tuitemwong, K. and Fung, D.Y.C. (1991). Microbiological study of tofu. J. Food Protect., 54, van Kooij, J. A. and de Boer, E. (1985). A survey of the microbiological quality of commercial tofu in the Netherlands. Int. J. Food Microbiol., 2,