70 Vol. 49, No. 2 19 10 23 1 1 1 2 3 2, Survival of Salmonella in Spices and Growth in Cooked Food Yurie UG67: 1,Yuji M>C6> 1,Minoru H6<6 1, Yoshiko SJ<>I6-KDC>H=> 2,Atsushi IH=><JGD 3 and Yukiko H6G6-KJ9D 2, 1 Tamagawa University, 6 1 1 Tamagawa-gakuen, Machida, Tokyo 194 8610, Japan; 2 National Institute of Health Sciences, Division of Microbiology: 1 18 1 Kamiyoga, Setagaya-ku, Tokyo 158 8501 Japan; 3 Donq, 3 19 14 Tanakamachi, Higashinada-ku, Kobe-shi, Hyogo 658 0081, Japan; Corresponding author Contamination of spices with pathogens has been reported worldwide, and Salmonella might result in foodborne infections, In this study, we investigated the survival of Salmonella in black pepper and red pepper, and the growth of the surviving Salmonella in cooked food. Salmonella Enteritidis, Salmonella Weltevreden and Salmonella Senftenberg were inoculated into spices, and their survival during storage was examined. In black pepper, S. Enteritidis was no longer viable after storage for 28 days, but S. Weltevreden and S. Senftenberg remained viable. In red pepper, S. Weltevreden and S. Senftenberg survived for 28 days although S. Enteritidis was not viable after 7 days. Salmonella Weltevreden and Salmonella Senftenberg were inoculated into cooked food, and their survival during storage was determined. In potato salad, egg salad, namul and kimchi as cooked foods, both pathogens grew at 30, butnot at 10. Our results indicate that cooked food should be stored at low temperature after addition of spices, such as black pepper and red pepper, following the cooking. (Received October 23, 2007) Key words: food spice; Salmonella; survival; growth; cooked 1 1), 2) 3) 5) 1g 10 3 10 5 1 194 8610 6 1 1 2 158 8501 1 18 1 3 658 0081 3 19 4 6) 7) 9) 1g 1,000 1 1g 10 6 10)
April 2008 71 1. Salmonella Enteritidis SE315 Salmonella Weltevreden SEC 442 Salmonella Senftenberg SEC443 2. (1,050 g) 10 (140 g) (105 g) (700 g) (105 g) (1,360 g) 20 1,140 g (220 g) (1,390 g) 10 (1,146 g) (72 g) (72 g) (1,238 g) 10 (1,180 g) (50 g) (8 g) 25 g 0.1 g 0.25 g 1g 3. (A w ) ph A w ph AW GSI ph IQ240 TOHO 4. 1 Trypticase soy broth (TSB, Oxoid, UK) 10 ml 37 18 2,000 15 PBS 10 ml PBS 10 ml PBS 10 7 10 0.1 ml Trypticase soy agar (TSA, Oxoid) 37, 18 5. S. Enteritidis, S. Weltevreden S. Senftenberg 10 g S. Enteritidis, S. Weltevreden S. Senftenberg 10 6 CFU/g 90 ml PBS GSI 15 PBS 10 5 CFU/g 10 Xylose Lysine Desoxycholate agar (XLD, Oxoid) 0.1 ml 10 37 18 XLD 2 Bu#ered Peptone Water (BPW, Oxoid) 100 ml 37, 18 XLD 37 18 XLD Oxoid 25 g 8 6. S. Weltevreden 0.1 g S. Weltevreden 10 3 CFU/0.1 g 28 0.1 g 10CFU 25 g 10 30 4 PBS 225 ml 1 PBS 10 XLD 0.1 ml 10 37 18 2 BPW 250 ml 37 18 XLD 37 18 XLD Oxoid 25 g
72 7. S. Senftenberg 0.25 g 1g S. Senftenberg 10 2.4 CFU/0.25 g 10 3 CFU/g 7 0.25 g 1g 10CFU 0.25 g 1g 25 g 10 30 6 25 g 8 Table 1. Properties of food samples Potato salad Egg salad Namul Kimchi A w 0.985 0.962 0.982 0.930 ph 5.35 6.67 5.73 6.20 Energy (kj/100 g) 569 1,004 385 67 Protein (g/100 g) 0.8 12.2 9.2 0.8 Lipid (g/100 g) 9.2 20.8 1.2 0 Carbohydrates (g/100 g) 13.2 1.2 2.8 3.6 Sodium (mg/100 g) 82.8 219.6 34.2 4.0 Calculated from the values in ref. 15 Vol. 49, No. 2 8. Polymerase chain reaction (PCR) 5, 6 7 inva PCR 0.1 ml 4 10 (10,000 ) 50 mm NaOH 0.1 ml 100 10 1MTris HCl (ph7.0) 16 ml 4 10 (10,000 ) Template DNA inva inva 139 inva 141 11) 1 PCR 10 PCR bu#er 5 ml, dntp mixture 4 ml, Primer inva 139 (40 pmol/ml) 0.5 ml, Primer inva 141 (40 pmol / ml) 0.5 ml Ex Taq polymerase (5 U/mL) 0.25 ml 34.75 ml Template DNA 5 ml 50 ml (DNA Engine PTC-200, MJ Research, USA) 95 1 95 30 64 30 72 30 35 72 4 PCR PCR inva (284 bp) 1. A w ph A w (0.930) (0.962) 0.98 (Table 1) ph Fig. 1. Survival of Salmonella Enteritidis, Salmonella Weltebreden and Salmonella Senftenberg inoculated onto the surface of black pepper. IL: inoculation level, ND: not detected by plating, but detected in enrichment culture, NDE: not detected in enrichment culture. 6.0 2. S. Enteritidis, S. Weltevreden S. Senftenberg 10 6.1 CFU/g S. Enteritidis 10 2.7 CFU/g 1 10 CFU/g 28 (Fig. 1) S. Weltevreden 10 4.1 CFU/g 56 84 S. Senftenberg 10 3.5 CFU/g 7 S. Enteritidis 10 4.0 CFU/g 7 (Fig. 2) S. Weltevreden S. Senftenberg 10 3.6 10 4.3 CFU/g 7 10 CFU/g 28
April 2008 73 Fig. 2. Survival of Salmonella Enteritidis, Salmonella Weltevreden and Salmonella Senftenberg inoculated onto the surface of red pepper. IL: inoculation level, ND: not detected by plating, but detected in enrichment culture, NDE: not detected in enrichment culture. Fig. 4. Growth of Salmonella Senftenberg in namul and kimchi stored at 10 and 30 IL: inoculation level, ND: not detected by plating, but detected in enrichment culture, NDE: notdetected in enrichment culture, : Significant di#erence from data at 0 h (Student s t-test: p 0.005) Fig. 3. Growth of Salmonella Weltevreden in potato salad and egg salad stored at 10 and 30 IL: inoculation level, ND: not detected by plating, but detected in enrichment culture, NDE: not detected in enrichment culture, : Significant di#erence from data at 0 h (Student s t-test: p 0.005). 3. S. Weltevreden S. Weltevreden 10 5.8 CFU/0.1 g 28 10 2 CFU/0.1 g 30 24 10 7.9 CFU/g, 10 2.4 CFU/g (Fig. 3a) 10 48 10 CFU/g (Fig. 3b) 4. S. Senftenberg S. Senftenberg 10 6.2 CFU/0.25 g 10 6.8 CFU/g 7 10 CFU/0.25 g 1 g 30 24 10 7.4 CFU/g (Fig. 4a) 10 48 (Fig. 4b) 0 10 CFU/g 12) 14)
74 S. Weltevreden S. Senftenberg S. Enteritidis S. Weltevreden S. Senftenberg 84 28 S. Weltevreden S. Senftenberg S. Enteritidis S. Weltevreden S. Senftenberg S. Weltevreden 0.1 g 10CFU S. Senftenberg 0.25 1 g 10 CFU 30 6 24 10 8 CFU/g 6 10 CFU/ g 24 10 7 CFU/g ph 4 (ph 5.35) A w (A w 0.930) A w ph 6.67 ph A w 0.962 A w (Table 1) A w 0.982 A w ph 5.73 ph 15) 16) (Table 1) A w ph PCR PCR PCR Vol. 49, No. 2 ph S. Weltevreden S. Senftenberg S. Enteritidis 10 30 24 1) Aikawa, K., Murakami, H., Inomata, K., Maruyama, T., Fujisawa, T., Takahashi, T., Yamai, S. Influence of the conditions of storage and cooking on growth, invasion and survival of Salmonella Enteritidis in eggs. Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 43, 178 184 (2002). 2) Duguid, J. P., North, R. A. Eggs and Salmonella foodpoisoning: an evaluation. J. Med. Microbiol., 34, 65 72. (1991). 3) 16 56, 62 133 (2004). 4) 17 57, 78 153 (2005). 5) 18 58, 88 163 (2006) 6) Satchell, F. B., Bruce, V. R., Allen, G., Andrews, W. H., Gerber, H. R. Microbiological survey of selected imported spices and associated fecal pellet specimens. J. AOAC Int., 72, 632 637 (1989). 7) D Aoust, J. Y. Salmonella and the international food trade. Int. J. Food Microbiol., 24, 11 31 (1994). 8) Vij, V., Ailes, E., Wolyniak, C., Angulo, F. J., Klontz, K. C. Recalls of spices due to bacterial contamination monitored by the U.S. Food and Drug Administration: the predominance of Salmonellae. J. Food Prot., 69, 233-237 (2006). 9) Heintz, M. L., Ruble, R. D., Wagner, D. E., Tatini, S. R. Incidence of Salmonella in fish and seafood. J. Food Prot., 63, 579 592 (2000). 10) Hara-Kudo, Y., Ohtsuka, K., Onoue, Y., Otomo, Y., Furukawa, I., Yamaji, A., Segawa, Y., Takatori, K. Salmo-
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