February 2005 1 15 11 7 1, 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 356 8511, Japan; 2 National Institute of Health Science, Division of Microbiology: 1 18 1, Kamiyoga, Setagaya-ku, Tokyo 158 8501, 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 1.5 2 hrat35, heating for 10 min at 75 plus reheating after standing for 1.5 2 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 10 9), 10) 11) 10 1 2 356 8511 5 3 1 158 8501 1 18 1 1. 50
2 20 15 10 2 2 1 50 10 10 10 5 10 3 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 3. 35 2 1 1 (PBS) 1 ml 12,000 rpm 5 2 PBS 70 20 10 4 cfu/g 10 g 25 0, 1, 2 9 PBS 10 70 20 PBS NGKG 0.1 ml 35 24 10 10 4. Trypticase soy broth (TSB) (Difco) 35 18 10 8 cfu/ml 0.1 ml TSB 10 ml 35 6 12,000 rpm 5 PBS Vol. 46, No. 1 5. 10 g 4. 10 6 cfu/g 9 PBS 10 100 10 75 30 70 20 PBS 0.1 ml NGKG 3. 10 6 cfu/g 10 100 10 75 30 70 20 6. 10 g 4. 10 5 cfu/g 70 20 75 5 100 30 100 2 (1.4 kw) 10 NGKG 7. 2 10 g 3. 3 10 5 cfu/g 70 20 75 5 100 30 100 2 (1.4 kw) 10 BC-1 BC-2 25 4 2 BA06ce 25 2 BA06ce 70 20 75 10 100 2 35 1.5 2 25 4 1 1 2 2 4 9 PBS 10 70 20 NGKG 8. (GC/MS) 2 (GC/MS) 10 g 70 20 100 20 25 2 65 40 Takmer Headspace Auto-
February 2005 3 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-5 0.25 mm i.d. 30 m 1.0 mm, J&WScientific 50 (3 min) 4 /min 260 (15 min) 160 1.2 ml/min; 70 ev; TIC: m/z 30 300 1. 70 20 Fig. 1 2 2. 10 Fig. 2 100 10 12) 75 30 4) 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. 10 70 20 10 10 70 20 3. 60 80 13) 14) 60 80 70 75 100 Fig. 3 1,000 1 75 5 100 2
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 2 4. 2 3. 5 2
February 2005 5 Fig. 4 1 25 70 20 75 5 100 1 100 2 25 2 1,000 1 10 1,000 1 100 30 (Fig. 4(a)) 4 25 70 75 (Fig. 4(b)) 25 2 5. 2 (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 Vol. 46, No. 1 Fig. 7. GC/MS Chromatograms of Shira-ae and Unohana The numbering of peaks indicates retention times. (Fig. 6) 75 5 75 10 25 2 35 1.5 2 25 4 70 20 35 1.5 2 75 10 35 1.5 2 100 2 25 4 100 1 100 1 6. 70 75 GC/MS
February 2005 7 8.1 8.4 12.5 100 70 20 115 93 92 100 20 255 209 131 70 100 (Fig. 7(a)) 12.5 100 70 126 100 165 100 100 70 8.1 (Fig. 7(b)) 70 2 70 20 75 5 100 2 10 25 2 100 1 70 20 35 1.5 2 75 10 35 1.5 2 100 2 25 4 100 1 70 20 1) 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, 11 14 (1977) 2) 1988, p. 86 100 (ISBN 4-8058-0495-5) 3) Sakazaki, R. ed., Shokusuikeikansensyou To Saikinsei Shokuchudoku, Tokyo, Chuou-houki Shuppan, 1991, p. 304 335. (ISBN 4-8058-0834-9) 4) Ueda, S., Shokuhinchu no shokuchudokukin kensahou 14 sereusukin (Detection of food-poisoning bacteria in foods 14, Bacillus cereus). Boukin Boubai, 30 511 524 (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, 23 27 (2002). 7) Shimizu, U. et al. eds., Shokuhin Kigai Biseibutsu Handobukku Tokyo, Science Forum, 1998, p. 220 222. (ISBN 4-916164-18-0) 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, 431 436 (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, 385 389 (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, 617 625 (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. 105. 13) Kondo, M. et al. eds., Supoa Jikken Manyuaru Biseibutu No Gahou Housi No Kisokenkyu Kara Ouyoukenkyu Made, Tokyo, Gihodo Shuppan, 1995, p. 73 81. (ISBN 4-7655-0229-5) 14) Shimizu, U., Shokuhin Biseibutsu Kisohen Shokuhin Biseibutsu No Kagaku, Tokyo, Saiwai Shobo, 2001, p. 145 146. (ISBN4-7821-0179-1)