438 Journal of Food Protection, Vol. 48, No. 5, Pages 438-441 (May 1985) Copyright11 International Association of Milk, Food, and Environmental Sanitarians Evaluation of the Microbiological Safety of Tempeh Made from Unacidified Soybeans NOBUMASA TANAKA, SUSAN K. KOVATS, JEAN A. GUGGISBERG, LOUISE M. MESKE and MICHAEL P. DOYLE* The Food Research Inslilute, University of Wisconsin-Madison, 1925 Willow Drive, Madison, Wisconsin 53706 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/5/438/1651486/0362-028x-48_5_438.pdf by guest on 28 September 2021 (Received for publication October 3, 1984) ABSTRACT an expected shelf life of several weeks if handled prop­ Studies were done to evaluate the safety of tempeh made erly. However, the product is often displayed on a pro­ from unacidifed soybeans and inoculated with different bacterial duce counter with little refrigeration or sometimes on a pathogens. Pathogens were added to either the soybeans before display table without refrigeration. Little is known about fermentation by Rhizopus oligosporus or the tempeh after fer­ the microbiological safety of tempeh during production mentation and steaming. In the latter method, the inoculated or at the retail and consumer level. The purpose of this products were incubated at several different temperatures (5, study was to determine the ability of foodborne bacterial 10, 15 and 25°C). Clostridium botulinum (types A and/or B) pathogens to grow or produce toxin in tempeh during toxin was produced in 2 d during the fermentation and within production and when the heat-treated, packaged product 5 d at 25°C or 4 wk at 15°C in tempeh inoculated and incubated in vacuum packages after fermentation and steaming. is held at different storage temperatures. Staphylococcus aureus grew very well (>6-log10 CFU/g in­ crease) in 2 d during the fermentation, and grew from ca. 103 MATERIALS AND METHODS CFU/g to 108 CFU/g in 7 d at 25°C and 21 d at 15°C in tempeh Bacterial cultures and methods of enumeration inoculated after fermentation and steaming. Staphylococcal en- Four foodborne bacterial pathogens were studied. These in­ terotoxins were detected in some of these samples. Salmonella cluded: Clostridium botulinum, Staphylococcus aureus, Sal­ typhimurium also grew well during the fermentation (>6-logio monella typhimurium and Yersinia enterocolitica. CFU/g increase in Id), but grew relatively slowly at 25 and 15°C in tempeh inoculated after fermentation and steaming. A ten-strain spore mixture of C. botulinum, consisting of five strains each of types A and B (8) in water, was used. Im­ Yersinia enterocolitica grew very well (>6-log10 CFU/g in­ crease) in 1 d during the fermentation, and also grew well in mediately before inoculating tempeh, the spores were heat- tempeh inoculated after fermentation and steaming, with a >6 shocked at 80°C for 15 min. Immediately after inoculation, C. botulinum (three samples per batch) was enumerated using a logl0 CFU/g increase in 2 d at 25 or 15°C and 5 d at 10"C. Results of these studies indicate the need for maintaining: (a) 5-tube most probable number (MPN) method with Trypticase- a high level of sanitary practices during production and (b) peptone-glucose-yeast extract broth as the growth medium (4). good refrigeration (=S5°C) of the product following fermentation 5. aureus strains 196 E (enterotoxin A and D producer) and until it is used. 361 (enterotoxin C2 producer) were kindly provided by M. S. Bergdoll, University of Wisconsin-Madison. Cultures were stored in brain heart infusion (BHI; Difco) agar slants at 4°C. For each experiment, cultures were grown overnight in BHI Tempeh is usually made of soybeans fermented with broth at 37°C, harvested by centrifugation, washed and resus- Rhizopus oligosporus (9), although it could be made of pended in 0.01 M phosphate-buffered saline, pH 7.5 (PBS). other grains (6). The beans are cooked, mixed with The cell suspensions were diluted appropriately with PBS and spores of R. oligosporus, and incubated at about 30°C mixed to give an equal number mixture of the two strains. 5. until dense, white mycelia of the mold grow and bind aureus was enumerated by plate count on Baird-Parker agar the beans together to make a cake- or patty-like product (Difco) incubated at 37°C for 48 h. Typical 5. aureus colonies (6,9). Tempeh is one of the most important soybean (black, shiny and convex surrounded by clear zone) were foods in Indonesia (6) and is becoming increasingly counted and randomly chosen isolates were confirmed for coagulase activity. popular in the United States. Tempeh is so easy to make that it is commonly made at home. Commercially, in the 5. typhimurium strains 40252 and 40467 were obtained from the Wisconsin State Laboratory of Hygiene, Madison, WI. Each United States, the fermented tempeh is fried or steamed, strain was stored on Trypticase soy agar (TSA; BBL Microbiol­ and packaged in plastic film. ogy Systems) at 4°C. For each experiment, cultures were grown Because tempeh is made of cooked whole soybeans, overnight in Trypticase soy broth (TSB;BBL Microbiology Sys­ which is a very nutritious medium, it is quite susceptible tems) at 37°C. Cells of each strain were harvested by centrifu­ to microbial growth. In the United States, tempeh has gation, washed and resuspended in PBS, then diluted and mixed JOURNAL OF FOOD PROTECTION, VOL. 48, MAY 1985 MICROBIOLOGICAL SAFETY OF TEMPEH 439 to give an equal number mixture of the two strains. Hektoen and after ca. 36 h parts of the tempeh became black indicating enteric agar (Difco) was used for enumeration of 5. the development of some Rhizopus spores. Method B consisted typhimurium (24 h at 37°C). Randomly selected colonies were of spraying test bacteria on fermented, steamed tempeh patties, confirmed as S. typhimurium by agglutination with the appropri­ vacuum packaging the tempeh in an oxygen impermeable film ate antisera (Difco). (Curwood, Inc., New London, WI; oxygen transfer rate less Y. enterocolitica strains 34/IB and FRI-YE16 were used. Y. than 0.5 cm3/l atm/100 cm2), and incubating at several differ­ enterocolitica 34/1B (serotype 0:8), kindly provided by C. C. ent temperatures. Details of the procedure are as follows. Tem­ G. Aulisio, Food and Drug Administration, Washington, DC, peh patties (covered by R. oligosporus mycelia following ca. was originally isolated from unchlorinated water used in the 24 to 28 h of incubation at 30°C) were removed from petri dis­ manufacture and packaging of tofu which caused food-poison­ hes, wrapped in sterile aluminum foil, and steamed in a ing incidents (1). Y. enterocolitica FRI-YE16 (serotype 0:3) is chamber with flowing steam for 30 min. The steamed patties a virulent strain originally isolated from a porcine tongue (2). were cooled to room temperature and inoculated with the test Cultures were stored on TSA slants at 4°C, and for each experi­ bacteria (each type of pathogen was tested individually) by an ment, overnight cultures were grown in TSB at 25°C. Cells of aerosol delivered through a plant sprayer. The sprayer was ad­ each strain were harvested by centrifugation, washed and resus- justed to discharge an average of 0.2 ml of aerosol per applica­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/5/438/1651486/0362-028x-48_5_438.pdf by guest on 28 September 2021 pended in PBS, then an equal number mixture of the two tion. For safety considerations, tempeh was inoculated by the strains was made. Cells were enumerated on yersinia selective aerosol procedure in a disposable glove box. The inoculated agar base containing yersinia selective supplement (Oxoid) incu­ patties were individually vacuum packaged with a laboratory bated at 25°C for 48 h. Randomly selected colonies were con­ vacuum packaging unit (Model 24 V, Packaging Aid Corp., firmed as virulent Y. enterocolitica by agglutination with anti­ San Francisco, CA), and incubated at 5, 10, 15 or 25°C. serum (WA-SAA; 3). Biochemical characteristics of these iso­ Analysis of samples lates were determined by the API-20E diagnostic system An entire patty of tempeh was blended for 2 min with two (Analytab Products, Plainview, NY). volumes of double distilled water using a Stomacher Lab Blen­ der (Model 400, Seward UAG, London). For samples inocu­ Preparation of tempeh lated with C. botulinum, the blended tempeh was centrifuged Tempeh was prepared according to a procedure described by (5,000 xg, 5 min) and the supernatant fluid was used for the H. L. Wang, Northern Regional Research Center, U.S. Depart­ mouse toxicity assay (4). Samples inoculated with other or­ ment of Agriculture, Peoria, IL (personal communication), ganisms were blended, diluted appropriately in PBS, and which is as follows. Dry, cracked soybeans were soaked in cold plated. Triplicate samples were analyzed for each treatment at tap water for ca. 30 min, then washed and floating hulls were each sampling interval, and an average of these results was re­ removed. The washed soybeans were boiled on an electric stove ported. in about three volumes of water for 30 min. During this time, Some samples containing greater than 106 cells of S. aureus/g hulls that floated to the surface were removed with a ladle. were tested for the presence of staphylococcal enterotoxins (A, The cooked soybeans were then drained, spread as a thin layer C and D) by an enzyme-linked immunosorbent assay (5). (1 to 2 cm thick) over a double layer of sterile cheesecloth, 2 These samples were stored frozen (-20°C) until enterotoxin as­ covered with another double layer of sterile cheesecloth, and says were performed. excess moisture was removed by blotting with paper toweling.