Survival and Growth of Foodborne Pathogens during Cooking and Storage of Oriental-Style Rice Cakes

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1 3037 Journal of Food Protection, Vol. 69, No. 12, 2006, Pages Copyright, International Association for Food Protection Research Note Survival and Growth of Foodborne Pathogens during Cooking and Storage of Oriental-Style Rice Cakes SUN-YOUNG LEE, 1 HYUN-JUNG CHUNG, 2 JOONG-HAN SHIN, 1 RICHARD H. DOUGHERTY, 1 AND DONG-HYUN KANG 1 * 1 Department of Food Science and Human Nutrition and 2 Department of Biological Systems Engineering, Washington State University, Pullman, Washington 99164, USA MS : Received 10 February 2006/Accepted 10 July 2006 ABSTRACT Fresh cooked rice cakes for retail sale are typically held at room temperature because refrigeration dramatically reduces their quality. Room temperature, high water activity, and a ph of 4.6 provided an environment conducive to pathogen growth. To date, no studies have been published regarding survival and growth of foodborne pathogens in fresh cooked rice cakes. This study was undertaken to investigate the effect of steam cooking on foodborne pathogens and their subsequent growth in five varieties of rice cakes made from flours of regular rice, sweet rice, white rice, tapioca, and mung bean. Bacillus cereus spores were detected in white rice, tapioca, and mung bean samples. The rice cake flours were inoculated with non spore-forming foodborne pathogens (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Staphylococcus aureus) or spore-forming bacteria (Bacillus cereus) and steam cooked (100C) for 30 min. Steam cooking significantly reduced (6 log CFU/g) non spore-forming foodborne pathogens in all samples and inactivated spores of B. cereus by 1 to 2 log CFU/g. Although spores of B. cereus survived steam cooking and germinated during 3 days of storage at room temperature, populations in most rice cakes remained below 10 6 CFU/g, which is the threshold for producing toxin. Rice cakes made from mung bean flour supported growth and germination of B. cereus spores above that critical level. In mung bean rice cakes, enterotoxin production was detected by the second day, when B cereus cell populations reached about 6.9 log CFU/g. The toxin concentration increased with storage time. However, our results suggest that rapid growth of total mesophilic microorganisms by more than 7 to 8 log CFU/ml during the first day of storage produced off flavors and spoilage before B. cereus was able to grow enough to produce toxins. Therefore, steam-cooked rice cakes made from a variety of flours including mung bean flour are safe for sale for up to 1 day after storage at room temperature and are free of B. cereus toxins. Bacillus cereus is a gram-positive aerobic or facultative anaerobic rod-shaped spore-forming bacterium. It is widespread in the environment, and spores are frequently isolated from rice, cereals, milk, and spices (3). This microorganism produces an emetic or diarrheal syndrome induced by an emetic toxin and an enterotoxin, respectively (7). B. cereus is a cause of food poisoning frequently associated with consumption of rice-based dishes (5 7, 20). In the United States, from 1988 to 1997, 35 outbreaks were caused by B. cereus and 1,124 patients were affected (2, 17). In Asia, fresh rice cakes are very popular food items, and a variety of flours, such as regular rice, sweet rice, white rice, tapioca, and beans, are major ingredients. Currently, several hundred varieties of Korean, Vietnamese, and Philippine style rice cakes are available in markets. Rice cakes can include various ingredients such as red beans, green beans, mung beans, and sesame seeds. * Author for correspondence. Tel: ; Fax: ; dhkang@wsu.edu. Present address: Department of Food and Nutrition, Choong-Ang University, An-Sung, South Korea. Rice cakes are cooked by steaming, frying, or boiling in water. Steam cooking is the most convenient and popular method for manufacturing a variety of rice cakes (13, 23). Cooking of the starch results in the swelling and gelatinization of starch granules, which are slowly reformed into a helical structure during cooling and storage, a process known as staling or retrogradation. Retrogradation causes hardness in starch-based foods. The rate of starch retrogradation is at its maximum at about 5C and declines with increasing temperature (19). In the baking industry, about 3% of bread is returned due to retrogradation (11). Thus, it is not recommended to store rice cakes under refrigeration conditions (14). Rice cakes for sale are held at room temperature for only 1 day and are discarded the following day. The state of California permits the sale of Korean rice cakes held at room temperature for up to 24 h and requires that manufacturers provide the date and time when the cakes were made (1). This study was undertaken with the assumption that steam cooking for 30 min completely destroys vegetative cells of pathogenic microorganisms and spores present in the raw ingredients and that it is microbiologically safe to

2 3038 LEE ET AL. J. Food Prot., Vol. 69, No. 12 offer for sale cooked rice cakes held for 1 day at room temperature. However, to date, there has been no scientific information to support this assumption. From a food safety viewpoint, there are several important microbiological factors that should be addressed. First, steam cooking is the only thermal processing involved in rice cake manufacturing and thus is considered a critical point for controlling bacterial growth. However, steam cooking may not be effective for destroying foodborne pathogens during the manufacture of rice cakes. Second, the ingredients are not free from pathogenic bacteria or spore-forming bacteria such as B. cereus. Surviving pathogenic bacteria might multiply during storage of cakes at room temperature, resulting in illness in consumers. Third, spore-forming bacteria such as B. cereus might survive steam cooking, and spores may germinate, grow, and produce toxin during storage. In this study, we focused on steam cooking procedures, with the assumption that good manufacturing practices were used. The purpose of this study was to evaluate the effect of steam cooking on the survival and growth of foodborne pathogens and B. cereus spores in manufactured rice cakes. The objectives were (i) to determine the level of contamination in a variety of rice cake ingredients, (ii) to investigate the effect of steam cooking on foodborne pathogens and B. cereus spores in rice cakes, and (iii) to determine the effect of growth temperature on the survival and growth of B. cereus spores and their toxin production. MATERIALS AND METHODS bacteria and spores of B. cereus: samples. Four kinds of rice flour were evaluated for the concentration of total mesophilic microorganisms and the presence of B. cereus spores: regular rice flour (RR) and sweet rice flour (SR) were used in manufacturing Korean style rice cakes, and white rice flour (WR) and tapioca flour (TP) were used in manufacturing Vietnamese style rice cakes. Mung bean flour (MB) also was evaluated. RR, SR, and MB were obtained from Han-Yang Rice Cake Manufacturers (Seattle, Wash.) and WR and TP were purchased from a local store (Pullman, Wash.). Samples were procured on about 10 separate occasions and used throughout the study. bacteria and spores of B. cereus: enumeration of total mesophilic microorganisms. Twenty-five grams of each ingredient was placed in a stomacher bag, mixed with 50 ml of sterile buffered peptone water (ph 7.0), and homogenized for 2 min. Samples were serially diluted 10-fold with 9 ml of sterile buffered peptone water. One milliliter of appropriate dilutions was seeded onto Aerobic Count Plate Petrifilm (3M Co., St. Paul, Minn.) and incubated at 32C for 48 h. bacteria and spores of B. cereus: isolation of B. cereus. B. cereus was isolated by direct plating or by plating after enrichment. For direct plating, serial dilutions from stomached samples were spread plated in duplicate onto B. cereus selective agar (BCSA), which contains 25 ml/liter egg yolk emulsion and 10 5 U/liter B. cereus selective supplements (Oxoid, Ogdensburg, N.Y.). The plates were incubated at 37C for 24 h. Colonies with the typical appearance of B. cereus were counted as B. cereus. For enrichment, stomached samples were sealed and incubated at 37C for 24 h. incubation, 10 l of sample was plated onto BCSA using sterile loops. Another 1 ml of stomached sample was transferred to 9 ml of tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, Md.) and incubated at 37C for 24 h. Tubes with positive growth were plated onto BCSA using sterile loops. bacteria and spores of B. cereus: confirmation of B. cereus. Colonies isolated from BCSA were subjected to a typical staining method and phase contrast microscopy (1,000) to evaluate shapes and positions of B. cereus spores for confirmation. This method was developed by combining a spore stain with an intracellular lipid stain (12). Films on slides were made from the center of 1-day-old colonies or from the edge of 2-day-old colonies. Films were air dried and fixed with minimal flaming. Slides were placed over boiling water, flooded with 5% malachite green (wt / vol) for 2 min, washed, blotted, stained with 0.3% Sudan black B in 70% ethyl alcohol (wt /vol) for 15 min, washed in xylol for 5 s, blotted, and counterstained with 0.5% safranin (wt /vol) for 20 s (12). Effect of steam cooking on foodborne pathogens in rice cakes: cultures and cell suspension. Three American Type Culture Collection (ATCC) strains each of Escherichia coli O157:H7 (ATCC 35150, 42890, and 43889), Salmonella Typhimurium (ATCC 13311, 14028, and 19585), L. monocytogenes (ATCC 7644, 19113, and 19114), and Staphylococcus aureus (ATCC 6538, 12692, and 13565) were obtained from the Food Science and Human Nutrition culture collection at Washington State University (Pullman) and used to inoculate rice cake ingredients prior to steam cooking. Individual strains were cultured in TSB at 37C for 24 h, and cells were harvested by centrifugation at 4,000 g for 20 min at 4C and washed three times with buffered peptone water. Final pellets were resuspended in buffered peptone water, corresponding to approximately 10 8 to 10 9 CFU/ml. Strains of all of these foodborne pathogens were pooled to construct mixed culture cocktails. Effect of steam cooking on foodborne pathogens in rice cakes: inoculation, manufacturing, and steam cooking. Five milliliters of mixed culture cocktails was inoculated into g of rice flour or mung bean flour at 10 6 to 10 7 CFU/g and mixed thoroughly with gloved hands for 5 min. Inoculated rice cake ingredients were placed in a sieve lined with cheesecloth over a pot containing boiling water and steam cooked for 30 min (minimum time for cooking rice cakes with our apparatus). cooking, rice cakes were cooled to room temperature and divided into g slices with a flame-sterilized knife. Effect of steam cooking on foodborne pathogens in rice cakes: bacterial enumeration. Raw or cooked rice cakes (50 g) were placed in stomacher bags containing 100 ml of buffered peptone water and homogenized for 2 min. Samples were serially diluted 10-fold with 9 ml of sterile buffered peptone water, and diluted samples were spread plated onto each selective medium. MacConkey sorbitol agar (Difco, Becton Dickinson), xylose lysine desoxycholate agar (Difco, Becton Dickinson), Oxford agar base with antimicrobial supplement (Difco, Becton Dickinson), and Baird-Parker agar (Difco, Becton Dickinson) were used for enumeration of E. coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and S. aureus, respectively. All plates were incubated at 37C for 24 to 48 h under aerobic conditions. Effect of storage temperature on the growth of B. cereus in rice cakes: preparation of B. cereus spores. Two strains of

3 J. Food Prot., Vol. 69, No. 12 MICROBIAL STUDIES OF ORIENTAL-STYLE RICE CAKES 3039 B. cereus (ATCC and 13061) and two strains (WR1 and WR2) isolated from ingredients and confirmed as B. cereus in experiment 1 were used for the preparation of B. cereus spores. Overnight cultures of each strain were spread plated onto brain heart infusion (BHI) agar and incubated at 20C for about 3 weeks until 85 to 100% of the bacteria had sporulated. Spores of each strain were individually harvested by depositing 1 to 2 ml of sterile water onto the surface of BHI culture plates and gently rubbing with a sterile swab. Pooled suspensions were transferred to centrifuge tubes containing 20 ml of sterile water and centrifuged at 5,900 g for 15 min at 4C. Spore pellets were washed three times with sterile distilled water and resuspended in a small volume of sterile water. The final pellet was resuspended in sterile phosphate buffer (ph 7.0), corresponding to approximately 10 8 to 10 9 spores per ml, and stored in 1.8-ml cryogenic tubes (Fisher Scientific, Pittsburgh, Pa.) at 20C until use. Spore concentrations were determined after a 100-l sample of spore concentrate diluted in 900 l of sterile water was heat shocked at 80C for 20 min, chilled in ice water, serially diluted 10-fold in peptone water, and plated on tryptic soy agar. Colony counts were made after incubation at 37C for 24 h. Effect of storage temperature on the growth of B. cereus in rice cakes: enumeration of B. cereus. Four strains of spore suspensions were combined to generate a spore cocktail. Five milliliters of the spore cocktail was inoculated into gof rice flour or mung bean flour at 10 6 to 10 7 CFU/g and mixed thoroughly for 5 min with gloved hands. Inoculated flours were placed in a sieve lined with cheesecloth over a pot containing boiling water and steam cooked for 30 min. cooking, rice cakes were cooled to room temperature and divided into g slices with a flame-sterilized knife. The rice cakes were stored at room temperature or under refrigeration for 3 days. Samples were taken at six points (before and after cooking and at 12 h and 1, 2, and 3 days of storage), and the B. cereus populations were determined as described for isolation of B. cereus. The effect of storage temperature on the growth of B. cereus spores was investigated in five types of rice cakes at two storage temperatures (4 and 22C). Two inoculation studies were designed. First, inoculation with low concentrations of B. cereus spores (10 to 100 CFU/g) was used to simulate as closely as possible the natural contamination of B. cereus spores in rice cakes, and the growth of surviving and germinating B. cereus spores was studied. Second, a higher concentration of inoculum (10 3 to 10 4 CFU/g) was used to represent a worst-case scenario to help ensure a margin of safety. Effect of storage temperature on the growth of B. cereus in rice cakes: enterotoxin production. B. cereus enterotoxin produced during storage of rice cakes was detected using the B. cereus enterotoxin reverse passive latex agglutination test (BCET- RPLA; Oxoid) following the manufacturer s instructions. Statistical analysis. All experiments were repeated three times, and dilutions were plated in duplicate. Data were processed with the Statistical Analysis System (SAS Institute, Cary, N.C.) for analysis of variance and Duncan s multiple range tests to determine whether significant differences (P 0.05) existed between mean values of treatment groups. RESULTS AND DISCUSSION Total mesophilic plate counts and concentrations of B. cereus in rice cake flours. Samples of each type of flour (RR, SR, WR, TP, and MB) collected from 10 different sources were evaluated for total mesophilic microorganisms TABLE 1. Cooked rice cake water activity and ph measured at room temperature Rice cake ingredient a a w ph RR SR WR TP MB a RR, regular rice flour; SR, sweet rice flour; WR, white rice flour; TP, tapioca flour; MB, mung bean flour. and for the presence of B. cereus spores. Average concentrations of total mesophilic microorganisms were 3.0 to 3.6 log CFU/g in RR, SR, WR, and TP in each of the 10 samples. In MB flour, an average of 4.7 log CFU/g total mesophilic microorganisms was detected in only 3 of 10 samples. Contamination with B. cereus spores in raw ingredients was detected with or without enrichment. Most of the raw ingredients contained B. cereus spores below the detection limit (0.6 log CFU/g) of direct plating with the exception of TP, which contained an average of 53 CFU/g. However, with enrichment, WR, TP, and MB tested positive for B. cereus (data not shown). All presumptively positive colonies isolated from direct plating and enrichment procedures were confirmed as B. cereus following the staining method of Holbrook and Andersson (12) (data not shown). Effect of steam cooking on foodborne pathogens in rice cakes. All rice cakes were evaluated for water activity (a w ) and ph. The a w ranged from 0.92 in SR to 0.99 in MB, and ph ranged from 5.4 for RR to 6.9 for MB. All samples had a w and ph values favorable for growth of microorganisms (Table 1). E. coli O157:H7 and Salmonella Typhimurium were very sensitive to steam cooking. More than 6-log reductions were obtained for both pathogens, and no survivors were detected in rice cakes made from all five ingredients (Table 2). For L. monocytogenes and S. aureus, at least 5-log reductions were observed in all rice cakes, but small populations of both pathogens were detected in cakes with RR and WR and in cakes with SR and WR, respectively (Table 2). Overall, steam cooking effectively reduced inoculated foodborne pathogens in rice cakes by more than 6 log CFU/ g. These results verify that steam cooking for 30 min is a very effective method for eliminating foodborne pathogens in manufactured rice cakes. Effect of storage temperature on the growth of B. cereus spores. Steam cooking for 30 min resulted in a 1- to 2-log reduction of B. cereus spores inoculated into raw ingredients. There was no significant difference among ingredients (P 0.05, data not shown). When cooked rice cakes were stored at room temperature (22C), the concentration of B. cereus spores increased by more than 5 log CFU/g in MB rice cakes during the first day of storage. For other rice cakes (RR, SR, WR, and TR), populations of B. cereus increased during the first

4 3040 LEE ET AL. J. Food Prot., Vol. 69, No. 12 TABLE 2. Rice cake populations of inoculated Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus before and after steam cooking for 30 min a Rice cake type b E.coli O157:H7 Salmonella Typhimurium L. monocytogenes S. aureus RR SR WR TP MB a Values are presented in log CFU per gram. Detection limit was 0.78 log CFU/g. b RR, regular rice flour; SR, sweet rice flour; WR, white rice flour; TP, tapioca flour; MB, mung bean flour. day of storage and remained below 10 6 CFU/g (the critical point associated with illness) (15, 16, 21) until day 3. The least growth of B. cereus spores was observed in SR rice cakes followed by RR rice cakes (Fig. 1A). This difference may be due to different a w and ph values of rice cakes made from different types of flours; low a w and low ph have an adverse effect on B. cereus growth during storage. The a w and ph values of rice cakes made from MB, RR, FIGURE 1. Growth of surviving Bacillus cereus in steam-cooked rice cakes stored at room temperature (22 2C) (A) and refrigeration temperature (4 1C) (B). cooking, the initial concentration of B. cereus was about 1 to 2 log CFU/g. and SR were 0.99 and 6.9, 0.96 and 5.4, and 0.93 and 5.5, respectively (Table 1). The minimum ph and a w for B. cereus growth are 4.35 and 0.912, respectively (4, 18). Gonzalez et al. (9) investigated the effect of ph on a medium used for recovery of heat-injured B. cereus spores. When the heat-treated spores (96 to 104C for 3 min) were transferred to a recovery medium with ph values of 5.4 to 7.6, higher recovery of heat-injured spores was observed at near neutral ph (ph 6.8 and 7.6), whereas spore counts decreased markedly as ph decreased below 6. Under refrigeration (4C), there was no apparent growth of B. cereus in any of the rice cakes. The number of surviving spores was maintained in rice cakes made from MB and WR during refrigerated storage, whereas there was a slight decrease in B. cereus populations in TP, RR, and SR cakes (Fig. 1B). Our observations were in agreement with the results of Guinebretiere et al. (10) and Valero et al. (22). Guinebretiere et al. (10) studied the effect of storage temperature (4, 10, and 20 to 25C) on the survival of B. cereus spores in zucchini purée following heat treatment at 80C for 10 min. pasteurization, B. cereus concentrations were less than 0.2 log CFU/g, and remained below the detection limit (1.7 log CFU/g) at 4C for 21 days, whereas counts of B. cereus increased to 4.6 log CFU/g in purée stored at 10C for 21 days and reached 6.4 log CFU/ gat20to25c for 5 days, respectively. Valero et al. (22) studied growth of B. cereus in zucchini broth (ph 5.0 and 6.5) and carrot purée at low temperatures (5 to 16C). Six B. cereus strains were able to grow at 12C in vegetable broth (ph 6.5), but no growth was observed at ph 5.0 under the storage temperatures. As the ph decreased, an elevated temperature (16C) was required for the growth of B. cereus. Some strains showed growth at ph 5.0 and 16C, but there was a significantly extended lag phase. Under the same storage conditions, mesophilic microflora showed dramatic growth during the first day and reached about 10 5 to 10 6 CFU/g at room temperature. In MB rice cakes, growth of more than 7 log CFU/g was observed after 1 day (Fig. 2A). Refrigeration suppressed the growth of total mesophilic microorganisms during 3 days of storage (Fig. 2B). With high inoculum concentrations of B. cereus spores in rice cakes, growth of B. cereus spores was greater yet similar to the results obtained with low inoculum concentrations at both storage temperatures (data

5 J. Food Prot., Vol. 69, No. 12 MICROBIAL STUDIES OF ORIENTAL-STYLE RICE CAKES 3041 TABLE 3. Growth of surviving Bacillus cereus cells and enterotoxin production during storage of rice cakes Storage time (days) Mung bean flour cakes B. cereus (log CFU/g) Toxin a White rice flour cakes B. cereus (log CFU/g) Toxin a Detected using BCET-RPLA. Agglutination: +++, heavy; ++, moderate; +, slight;, none. FIGURE 2. Growth of total mesophilic microorganisms in steamcooked rice cakes stored at room temperature (22 2C) (A) and refrigeration temperature (4 1C) (B). storage, B. cereus was added to ingredients at 1 to 2 log CFU/g. not shown). However, rice cakes stored at low temperatures were of unacceptable quality because of starch retrogradation. For this reason, although refrigerated storage is a good intervention method for inhibiting B. cereus growth, it is not recommended because of degradation of product quality. B. cereus produces toxin in foods when it reaches 10 5 to 10 6 CFU/g, making foods hazardous for human consumption (15, 16, 21). Because produced higher concentrations of B. cereus were found in cakes made with MB and WR than in cakes made with other ingredients, we investigated enterotoxin production of B. cereus using BCET-RPLA in cooked MB and WR cakes during 7 days of storage at room temperature (Table 3). In MB cakes, enterotoxin was detected from the second day of storage when B. cereus reached about 6.9 log CFU/g. Toxin production increased with storage time. In WR cakes, growth of B. cereus was slower than that in MB cakes. Toxin production was detected on day 7 when B. cereus reached about 6.6 log CFU/g (Table 3). Finlay et al. (8) examined toxin production by B. cereus in skim milk medium at incubation temperatures ranging from 10 to 50C. Seven strains of B. cereus produced significantly higher concentrations of toxin at 12 or 15C than at 30C, even though longer incubation periods were required. This finding suggests that toxin production by B. cereus is dependent on storage conditions such as ph, a w, and temperature and that the risk of food poisoning by B. cereus can be reduced even at room temperature provided other conditions are properly controlled to keep B. cereus concentrations below 10 5 to 10 6 CFU/g. In summary, steam cooking is a critical control point in manufacturing rice cakes for controlling foodborne pathogens. Our data indicated that steam cooking resulted in more than 6-log reductions of foodborne pathogenic bacteria, effectively eliminating them from the rice cakes. Steam cooking reduced B. cereus spores by 1 to 2 log CFU/ g. Spores of B. cereus survived steam cooking and germinated during 3 days of storage at room temperature. However, concentrations were below 10 6 CFU/g in most rice cakes. Although rice cakes made from MB supported full growth and germination of B. cereus spores above that critical level, faster growth of total mesophilic microorganisms to more than 7 to 8 log CFU/g during the first day of storage resulted in noticeable spoilage of rice cakes and offflavors even before B. cereus grew and produced toxins. This study was conducted with higher concentrations of pathogenic bacteria and B. cereus than would be found naturally to ensure a worst-case scenario. Therefore, steamcooked rice cakes made from a variety of flours, including MB, can be considered safe for sale after up to 1 day of storage at room temperature and should be free from B. cereus toxin. REFERENCES 1. Anonymous California Uniform Food Facilities Law. California Department of Health Services. Available at: Accessed 3 January Bean, N. H., J. S. Goulding, C. Lao, and F. J. Angulo Surveillance for foodborne-disease outbreaks United States, Morb. Mortal. Wkly. Rep. 45(SS-5): Brown, K. L Control of bacterial spores. Br. Med. Bull. 56: Bryan, F. L., C. A. Bartleson, and N. Christopherson Hazard analysis in references to Bacillus cereus, of boiled and fried rice in Cantonese-style restaurants. J. Food Prot. 44:

6 3042 LEE ET AL. J. Food Prot., Vol. 69, No Centers for Disease Control Bacillus cereus Maine. Morb. Mortal. Wkly. Rep. 35(25): Centers for Disease Control and Prevention Epidemiologic notes and reports: Bacillus cereus food poisoning associated with fried rice at two child day care centers Virginia, Morb. Mortal. Wkly. Rep. 43(10): Drobnicwski, F. A Bacillus cereus and related species. Clin. Microbiol. Rev. 6: Finlay, W. J., N. A. Logan, and A. D. Sutherland Bacillus cereus produces most emetic toxin at lower temperatures. Lett. Appl. Microbiol. 31: Gonzalez, I., M. Lopez, M. Mazas, A. Bernardo, and R. Martin Effect of ph of the recovery medium on the apparent heat resistance of three strains of Bacillus cereus. Int. J. Food Microbiol. 31: Guinebretiere, M. H., H. Girardin, C. Dargaignaratz, F. Carlin, and C. Nguyen-The Contamination flows of Bacillus cereus and spore-forming aerobic bacteria in a cooked, pasteurized and chilled zucchini puree processing line. Int. J. Food Microbiol. 82: Hebeda, R. E., and H. F. Zobel Baked goods freshness. Marcel Dekker, New York. 12. Holbrook, R., and J. M. Andersson An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Can. J. Microbiol. 26: Kim, S. S Scientific study about the traditional Korean food: a review, p Publishing department at Sook-Myung Women s University, Seoul, Korea. 14. Morris, V. J Starch gelation and retrogradation. Trends Food Sci. Technol. 1: Notermans, S., and C. A. Batt A risk assessment approach for food-borne Bacillus cereus and its toxins. J. Appl. Microbiol. Symp. Suppl. 84:51S 61S. 16. Notermans, S., J. Dufrenne, P. Teunis, R. Beumer, M. te Giffel, and P. P. Weem A risk assessment study of Bacillus cereus present in pasteurised milk. Food Microbiol. 14: Olsen, S. J., L. C. MacKinon, J. S. Goulding, N. H. Bean, and L. Slutsker Surveillance for foodborne-disease outbreaks United States, Morb. Mortal. Wkly. Rep. 49(SS01): Raevuori, M., and C. Genigeorgis Effect of ph and sodium chloride on growth of Bacillus cereus in laboratory media and certain foods. Appl. Microbiol. 29: Schiraldi, A., and D. Fessas Mechanism of staling: an overview, p In P. Chinachoti and Y. Vodovotz (ed.), Bread staling. CRC Press, Boca Raton, Fla. 20. Todd, E. C. D Worldwide surveillance of foodborne disease: the need to improve. J. Food Prot. 59: U.S. Food and Drug Administration The Bad Bug Book : foodborne pathogenic microorganisms and natural toxins handbook. U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition. Available at: intro.html. Accessed 3 January Valero, M., P. S. Fernández, and M. C. Salmerón Influence of ph and temperature on growth of Bacillus cereus in vegetable substrates. Int. J. Food Microbiol. 82: Yoon, S, S Korean food (history and cooking). Su-Hak Publisher, Seoul, Korea.