Evaluation and Control of the Risk of Foodborne Pathogens and Spoilage Bacteria Present in Awa-Uirou, a Sticky Rice Cake Containing Sweet Red Bean Paste
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FOODBORNE PATHOGENS AND DISEASE Volume 5, Number 3, 2008 © Mary Ann Liebert, Inc. DOl: 10.1 089/fpd.2007.0073 Evaluation and Control of the Risk of Foodborne Pathogens and Spoilage Bacteria Present in Awa-Uirou, a Sticky Rice Cake Containing Sweet Red Bean Paste 2 Naoki Okahisa, 1 Yasuhiro Inatsu, Vijay K. Juneja, 3 and Shinichi Kawamoto2 Abstract The risk of food poisoning and growth of spoilage bacteria in Awa-Uirou, a sticky rice cake containing sweet red bean paste, was evaluated. Toxin-producing bacteria such as Staphylococcus aureus and Bacillus cereus are the main causes of food poisoning linked to this kind of food. The water activity in this product is in the range suitable for growth of S. aureus, B. cereus, and B. subtilis. The viable count of S. aureus or B. cereus spore cocktail was significantly reduced to 2.3 log colony-forming units (CFU)/g after 70 minutes steaming treatment at 100 - C. However, the heat-resistant endospores of B. subtilis germinated during storage at 30C to cause appreciable syneresis of the starch gel matrix in 4 days. The addition of 0.5% glycine before steaming treatment was found to effectively suppress the growth of B. cereus but was not effective in controlling S. aureus throughout the 7 days incubation period at 30 C. On the other hand, S. aureus and B. cereus could grow > 5.0 log CFU/g in an inoculated sample without glycine within 3 days when stored at 30C. Moreover, addition of 0.5% glycine before the steaming process did not have any significant effect on color, texture, or taste of sticky rice cake. Therefore, results of this study demonstrated that the addition of 05% glycine before the steaming process could inhibit B. cereus and B. subti/is multi- plication in the steamed rice confection which in turn may help reduce the risk of food poisoning or quality loss. Introduction cassava, and tapioca and ingredients such as fruits, sweet bean paste, or pandan leaves are TICKY STEAMED RICE CAKES such as Khanom used to enhance the texture, taste, and flavor of S Chan in Thailand, Banh da ion in Vietnam, the confections. Awa-Uirou, is a kind of Uirou Nian Gao in China, and Tteok in Korea are traditional ly produced in Tokushima prefecture, popular confections in East to Southeast Asia Japan, and is characterized by mixing sweet red and consumed widely. These confections are bean paste (Sarasi-An) with sticky rice flour and basically a mixture of sticky or ordinary rice sugar before steaming at 100 C C for 70 minutes. flours and sugar steamed to make a sticky starch The risk of food poisoning from raw sticky gel. Japanese raw confections such as Dango rice cake has been recognized because many of (rice dumpling) or Mochi and Uirou (rice cake) these steamed rice cakes are made in small are included in this food category. In addition to household enterprises and typically vended on rice flour, other flours made from mung bean, streets in the different countries (Notermans Tokushima Prefectural Industrial Technology Center, Tokushima City, Tokushima, Japan. 2National Food Research Institute, Tsukuha City, Ibaraki, Japan. Eastern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Wvndmoor Pennsylvania. 351 352 OKAHISA El AL. and Batt, 1998; Henson, 2003). Enterotoxin- starch–sugar gel matrix in bean jam jelly (Mizu- producing Staphylococcus aureus, Bacillus cereus, Youkan) or sticky rice cake (Uirou) resulting in and B. suhtilis are the major microbiological quality loss (Nanba and Itho, 1997; Naito and hazards of these foods (FDA, 2001). Matsunaga, 2002). S. aurcus is a gram-positive non–spore- Glycine is the simplest amino acid, and it forming bacterium. Around 25% of these has been used as an antibacterial agent in foods bacteria isolated from food samples produced due to its low toxicity in animals. Although heat-resistant enterotoxin (Le Loir ci al., 2003). glycine is toxic to humans when it is given in Several outbreaks due to S. aureus have oc- large amounts, it also has antibacterial potential curred due to steamed rice cake in Japan; for (Hammes et al., 1973). For example, Lactococcits example, Kashiwa-Mochi (Kaneko et al., 1982), lactis subspecies failed to grow in medium Tukimi-Dango (Ishii et al., 1988; Uematsu and containing > 2% glycine (Helge and Ingoif, Kaneko, 1989; Tsuji et al., 2003), Mitu-Dango, 1989). Furthermore, glycine concentrations of An-Dango, Dathuku (Houjyo et al., 2001), and 1.5-6% resulted in 70-90% reductions in growth Zunda-Mochi (Saito et al., 2001) outbreaks. All of Entcrococcusfaecalis (Gary et al., 1991). Glycine of these outbreaks involved sticky rice cakes is known to inhibit the synthesis of a peptido- except for Tsukimi- Dango and Mitsu-Dango, glycan component of the bacterial cell wall which contained sweet bean paste. (Hishinuma et al., 1969). Although the bacterial B. cereus, a gram-positive spore-forming bac- cell wall is thinner in gram-negative bacteria teria, produces either an emetic (vomit-inducing) than in gram-positive bacteria, it is thought toxin in foods or diarrheal toxins (enterotoxins) that the amount of glycine required to suppress in the intestine that induce acute symptoms bacillus proliferation is lower than that required between 8 and 16 hours after ingestion of the to suppress gram-positive bacteria (Schwartz contaminated foods (Drobniewski, 1993; Noter- et al., 1979). Hops extracts possess antimicro- mans and Batt, 1998; McKillip, 2000). B. cereus is bial activities against Streptococcus mutans and ubiquitous in nature and frequently isolated other streptococci (Bhattacharya et al., 2003), from soil. As a common inhabitant of soil, this and salmon roe protein has been shown to bacterium can be easily transmitted into vege- have a certain degree of antimicrobial activity tables or crops and hence into foods. B. cereus against gram-negative and gram-positive bac- grows well at room temperature in starchy foods teria (Anonymous, 2008). to produce a heat-resistant emetic toxin (Agata In this study, we evaluated the growth char- and Mori, 1997; Agata et al. 2002). It is becom- acteristics of B. subtilis, B. cereus, and S. aureus in ing apparent that many incidents of B. cereus Awa-Uirou to evaluate food safety and quality food poisoning involve consumption of starchy risks in traditional rice–bean confections. Heat- foods such as cooked rice, noodles, or sticky rice stable antimicrobial food additives—glycine, cakes (Shinagawa, 1990). In December 2001, 346 hop extract, and salmon roe protein—were patients who ate sweet red bean paste covered evaluated for their ability to control the growth with sticky rice cake (An-Iri-Mochi) in Kuma- of B. cereus, S. aureus, and B. subtilis in artificially moto prefecture in Japan suffered from food contaminated Awa-Uirou. poisoning caused by cereulide, a cyclic dodeca- depsi peptide (Matsuoka et al., 2003; Huruse Materials and Methods et al., 2004). Measurement of physical parameters B. subtilis is also distributed widely in the environment and foods. Contamination with Twenty-nine samples of sticky rice cake made the organism and its rapid growth in starchy locally with sweet red bean paste (Awa-Uirou) foods is associated with spoilage or quality loss and destined for distribution in Tokushima pre- (Thomas and Masters, 1988; Sorokulova et al., fecture, Japan, were collected. All samples were 2003). Rice flour and beans can be contaminated analyzed 2 days after production. The water with Bacillus spores as a result of soil contami- activity (a) was measured with AW-CENTER nation (Watanabe and Hayano, 1993). B. subtilis (Novasina Co. Ltd., Pfaffikon, Switzerland). The from food ingredients can cause syneresis of the sugar content was measured by an Appe type PATHOGENS AND SPOILAGE BACTERIA IN AWA-UIROU 353 refractometer IN-2E (As One Co. Ltd., Tokyo, at 30C C for 4 days (150 rpm). Spores were liar- Japan) after reconstituting the samples in dis- vested by centrifugation after lysozyme treat- tilled water followed by centrifugation. The ment (in case of B. subtilis) or self-lysis by 1 day water content was measured by weighting at 4 ,C storage (in case of B. cereus) followed by the samples before and after drying at 105C repeated centrifugation and washing twice in until the value reached a constant (5 hours). The sterile distilled water. Early stationary phase rate of syneresis of uninoculated laboratory- cells of S. aureus prepared in brain heart infusion prepared Awa-Uirou was calculated from the broth (Nissui Co. Ltd., Tokyo) were used for the number of samples showing syneresis out of inoculation studies. A cocktail of spores or pure the total 60 samples. bacterial culture was used as inocula. Trypticase soy agar (Nissui Co. Ltd.) containing 50 pg/mL Inocula and enumeration rifampicin (Wako Pure Chemical. Co. Ltd., To- kyo) was used for the enumeration of inoculated The bacterial strains used for this study were: samples. A standard plate count agar (Nissui B. subtilis strains 740, U-i, U-18, S-26, and S-56 Co. Ltd.) was used for the determination of aer- isolated from Awa-Uirou samples; B. cereus obic bacterial counts of noninoculated samples. strains 1F03457 (origin unknown), S-8 (from Awa-Uirou), 734 (from rice flour), and 1DC22, Food additives 1DC23, and 1DC24 (all from the swab of a food factory); S. aureus strains IF013726 (from a Glycine (Wako Pure Chemical Co. Ltd.), the human lesion), JCM2179 (origin unknown), hop extract Hoprex (Mitubishi Chemical Foods JCM2874 (from a wound), and 727 (from Awa- Co. Ltd., Tokyo), and protein from salmon soft Uirou). A rifarn picin-resistant spontaneous roe (Asama Chemical Co. Ltd., Tokyo) were mutant of each strain was isolated by the en- used for experiments. All of these additives richment cultivation method (Inatsu et al., 2004).