Heat Treatment for the Control of Bacillus cereus Spores in Foods

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1 February , 1 2 Heat Treatment for the Control of Bacillus cereus Spores in Foods Keiko T6C6@6 1,,Hirofumi MDID> 1 and Yukiko H6G6-KJ9D 2 ( 1 Nisshin Seifun Group Inc., Research Center for Basic Science: 5 3 1, Tsurugaoka, Oi-machi, Iruma-gun, Saitama , Japan; 2 National Institute of Health Science, Division of Microbiology: , Kamiyoga, Setagaya-ku, Tokyo , Japan; Corresponding author) The e#ect of heat treatments on food products related to soybean curd contaminated with Bacillus cereus spores was investigated for the purpose of preventing food-poisoning outbreaks by B. cereus. Inthe case of B. cereus strains isolated from foods, heating foods inoculated with the spores for 20 min at 70, 5min at 75, 2min at 100, or10sec with a microwave oven plus reheating after standing for 2 hr at 25 reduced the number of surviving cells within the foods to less than one-hundredth. In the case of a heat-resistant strain isolated from a food poisoning outbreak, heating for 20 min at 70 plus reheating after standing for hrat35, heating for 10 min at 75 plus reheating after standing for hrat35, orheating for 2 min at 100 plus reheating after standing for 4 hr at 25 was e#ective. There was not much di#erence between the flavor components in foods with and without heat treatment at 70, asanalyzed by gas chromatography mass spectrometry. These results indicate that these heat treatments are available to control B. cereus spores, without a#ecting the sensory quality of the foods. Key words: Bacillus cereus; spore; germination; heat treatment; food; delicatessen; soybean curd Bacillus cereus 1) 2), 3) 4) 5) 6) 7) 8) ), 10) 11)

2 cfu/g 2. BD BBL CRYSTAL GP (BC-1) (BC-2) BA06ce Taylor Gilbert s H- 3 BA- CILLUS DIARRHOEAL ENTEROTOXIN VISUAL IMMUNOASSAY (TECRA) BC-1 BC-2 BA06ce BC-1 BC-2 BA06ce (PBS) 1 ml 12,000 rpm 5 2 PBS cfu/g 10 g 25 0, 1, 2 9 PBS PBS NGKG 0.1 ml Trypticase soy broth (TSB) (Difco) cfu/ml 0.1 ml TSB 10 ml ,000 rpm 5 PBS Vol. 46, No g cfu/g 9 PBS PBS 0.1 ml NGKG cfu/g g cfu/g (1.4 kw) 10 NGKG g cfu/g (1.4 kw) 10 BC-1 BC BA06ce 25 2 BA06ce PBS NGKG 8. (GC/MS) 2 (GC/MS) 10 g Takmer Headspace Auto-

3 February Fig. 1. Influence of heat treatment on the stability of Bacillus cereus spores in food (n 1) sampler 7000 (Takmer) GC Model 6890 (Agilent) Automass SUN 200S (JEOL) DB mm i.d. 30 m 1.0 mm, J&WScientific 50 (3 min) 4 /min 260 (15 min) ml/min; 70 ev; TIC: m/z Fig Fig ) ) Fig. 2. E#ect of heat treatment on spores of Bacillus cereus in 10-fold dilutions of food (n 3) BC-1: Bacillus cereus spores isolated from Shira-ae; BC-2: Bacillus cereus spores isolated from Unohana; BA06ce: Bacillus cereus spores isolated from foodpoisoning outbreak ) 14) Fig. 3 1,

4 4 Vol. 46, No. 1 Fig. 3. E#ect of heat treatment on vegetative cells of Bacillus cereus in food (n 3) BC-1: Bacillus cereus spores isolated from Shira-ae; BC-2: Bacillus cereus spores isolated from Unohana; BA06ce: Bacillus cereus spores isolated from food-poisoning outbreak Fig. 4. E#ect of various heat treatments on Bacillus cereus spores isolated from Shira-ae and Unohana (n 3) BC-1: Bacillus cereus spores isolated from Shira-ae; BC-2: Bacillus cereus spores isolated from Unohana; BA06ce: Bacillus cereus spores isolated from food-poisoning outbreak. I: Initial number of inoculated spores; Step 1: the first heat treatment; Step 2: standing for 2 hr at 25 or 4 ; Step 3: the second heat treatment

5 February Fig , , (Fig. 4(a)) (Fig. 4(b)) (Fig. 5) 25 2 Fig. 5. E#ect of various heat treatments on Bacillus cereus spores isolated from patients with food poisoning (n 3) I: Initial number of inoculated spores; Step 1: the first heat treatment; Step 2: standing for 2 hr at 25 ; Step 3: the second heat treatment Fig. 6. E#ect of various heat treatments on Bacillus cereus spores isolated from patients with food poisoning (n 3) NT: not tested; I: Initial number of inoculated spores; Step 1: the first heat treatment; Step 2: standing; Step 3: the second heat treatment

6 6 Vol. 46, No. 1 Fig. 7. GC/MS Chromatograms of Shira-ae and Unohana The numbering of peaks indicates retention times. (Fig. 6) GC/MS

7 February (Fig. 7(a)) (Fig. 7(b)) ) Kusunoki, J., Ikejima, N., Arai, T., Jin, M., Irikura, Y., Tomonari, M., Itoh, T., Sakai, S., The contamination of Bacillus cereus in various food and biochemical characteristics and heat resistance of spores isolated strains. Tokyo Eiken Nenpou (Ann. Rep. Tokyo Metr. Res. Lab. P.H.), 28, (1977) 2) 1988, p (ISBN ) 3) Sakazaki, R. ed., Shokusuikeikansensyou To Saikinsei Shokuchudoku, Tokyo, Chuou-houki Shuppan, 1991, p (ISBN ) 4) Ueda, S., Shokuhinchu no shokuchudokukin kensahou 14 sereusukin (Detection of food-poisoning bacteria in foods 14, Bacillus cereus). Boukin Boubai, (2002) 5) 12 Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 42, J-160 J-166 (2001) 6) Agata, N., Ohta, M., Yokoyama, K., Production of Bacillus cereus emetic toxin (cereulide) in various foods. Int. J. Food Microbiol., 73, (2002). 7) Shimizu, U. et al. eds., Shokuhin Kigai Biseibutsu Handobukku Tokyo, Science Forum, 1998, p (ISBN ) 8) Shinagawa, K., Kunita, N., Sasaki, Y., Okamoto, A., Biochemical characteristics and heat tolerance of strains of Bacillus cereus isolated from uncooked and cooked rice after food poisoning outbreaks. Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 20, (1979). 9) Finlay, W. J. J., Logan, N. A., Sutherland, A. D., Bacillus cereus produces most emetic toxin at lower temperatures. Lett. Appl. Microbiol., 31, (2000). 10) Choma, C., Guinebretiere, M. H., Carlin, F., Schmitt, P., Vilge, P., Granum, P. E., Nguyen-The, C., Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables. J. Appl. Microbiol., 88, (2000). 11) Uemura, T., Food poisoning caused by Clostridium perfringens and Bacillus cereus. Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 40, J-9 J-14 (1999). 12) 1990, p ) Kondo, M. et al. eds., Supoa Jikken Manyuaru Biseibutu No Gahou Housi No Kisokenkyu Kara Ouyoukenkyu Made, Tokyo, Gihodo Shuppan, 1995, p (ISBN ) 14) Shimizu, U., Shokuhin Biseibutsu Kisohen Shokuhin Biseibutsu No Kagaku, Tokyo, Saiwai Shobo, 2001, p (ISBN )

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