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 Containing Sweet

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 in China, and 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 rice cake has been recognized because many of (rice dumpling) or 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). were dissolved in water to make solutions of Spores of B. subtiiis and B. cereus were produced different concentrations as shown in Table 1. in Schaeffers sporulation medium (containing, per liter, 8g of Bacto-nutrient broth, 10 mL of Preparation of Awa-Uirou and inoculation tests 10% KC1, lOmL of 1.2% MgSO 4-7H20, 0.5OmL Awa-Uirou was prepared from commercial of I M NaOH, 1.0mL of 1 MCa(NO 3) 2, 1.0mL of rice flour, sugar, and red beans. Raw sweet red 0.010M MnC12, I.OmL of 1mM FeSO4) shaken bean paste (Nama-Ann) was prepared from red

TABLE 1. EFFECTIVENESS OF FOOD ADDITIVES FOR INHIBITING B. SUBT1LIS IN UTROU-TANE BEFORE THE STEAMING PROCESS

Storage (30"C)

Concentration (mg/g sample) 0 drn 3 day

Glycine 0 2.7+0.1 6.6 ± 0.2 2.5 2.7± 0.1 5.3 ± 0.2 5.0 2.9±0.0 2.8 ± 0.0 10 2.9 ± 0.0 2.7± 0.1 20 2.8 ± 0.1 2.7+ 0.1 Hop extract 0 2.9 ± 0.1 6.8tM.0 0.125 2.7 ± 0.1 6.6 ± 0.2 0.25 2.8 ± 0.0 6.8 ± 0.2 0.5 2.9 ± 0.0 6.3±0.0 1.0 2.7± 0.0 5.4 ± 0.1 Salmon roe protein 0 2.8 ± 0.1 7.2 ± 0.0 0.05 2.7± 0.0 7.0 ± 0.1 0.1 2.8 ± 0.0 7.2 ± 0.2 0.2 2.8 ± 0.0 6.9 ± 0.1 0.4 2.8 ± 0.0 7.1 ±0.2 Data represent mean log value of the viable cell counts from three independent experiments as described in Methods.

354 OKAHISA ET AL. beans as follows: 100 g of red beans was soaked method, were stored at 30G. The syneresis in 300 mL of tap water for 16 hours at 25C. The caused by natural bacterial spore contamination swollen beans were then boiled in 250 mL of tap was evaluated visually. This experiment was water for 1 hour and the excess water was repeated three times. The rate of syneresis was drained off, followed by a second wash with tap calculated as the percentage of samples showing water. The beans were boiled again with 500 mL signs of syneresis out of the total 60 samples of tap water for 1 hour until softened. examined. Softened beans were mashed by using a food processor and then pureed with 400 mL of wa- Sensory evaluation ter. The supernatant was discarded after pre- cipitation and the puree was resuspended with Several packs of 100 g of noninoculated Awa- 500 mL of water. This washing step was re- Uirou samples with glycine (2.5 or 5.0mg/g peated again to purify the bean paste. To reduce sample) or without glycine were prepared. The the water content to 65-68%, the bean paste was color (appearance), taste, and texture were eval- dew atered by wringing in a sterile closed vessel. uated by a visual analogue scale method (Har- Sarashi-An (100g) and 70g of granulated Zion et al., 2004). Testing was conducted in the sugar were mixed well, then 36g of rice flour sensory laboratory at the National Food Re- (Jo-Yo-Ko) was added. Finally, the sugar con- search Institute. Twenty-one trained panelists tent was adjusted to 45 degrees Brix by adding (12 men and 9 women) were informed about the water. Different concentrations of glycine, hop nature of the study. The test area was free of extract, and protein from salmon soft roe was extraneous odors and sound, and panelists were mixed with this Awa-Uirou paste (Uirou-Tane). instructed not to talk during testing. Panelists Ten grams of prepared Uirou-Tane paste was evaluated samples and marked score sheets in packed into a sterile plastic bag. The paste was individual booths. Panelists were instructed to inoculated with spores of B. cereus, B. subti/is, or evaluate samples in the order of presentation vegetative cells of S. aureus for "presteaming and to clear their palates between samples with inoculation" tests. The tightly sealed bags were the crackers and water. The presentation order steamed at 100C for 70 minutes. For "post- of the samples was balanced among panelists to steaming inoculation" tests, the bags were avoid a position error bias. Three 15-cm hori- opened after steam treatment, the inoculum was zontal lines with two anchors corresponding to added, and the bags were sealed again. The in- the opposite (worst and best) attributes for each oculated and noninoculated (control) samples quality at 1.5 cm from each end of the line were were stored at 30C for 3 days. shown to the panelists. The panelists marked Each bag containing Awa-Uirou was then points on the lines corresponding to the value sanitized with 70% ethanol and opened with of their evaluation. The values corresponding sterile scissors in a laminar airflow biological to each quality were calculated by using the cabinet. The Awa-Uirou was transferred into a formula: value = length between left side (cor- stomacher bag, 90 mL of phosphate-buffered responding to "worst") to the mark (cm)/15 saline was added, and the bag contents were (cm)xlO. The Tukey—Kramer test was used to homogenized for 1 minute in a stomacher. Ten- determine the statistical significance. fold serial dilutions were made and samples were plated on selective and nonselective agars Statistical analysis for bacterial enumeration. All the inoculation studies were repeated four times. The logarith- All experiments were done in triplicate with mic values of viable cell counts were used for duplicate samples being analyzed at each sam- statistical analysis. pling time. Data were subjected to analysis of variance (ANOVA) using the Microsoft (Red- mond, WA) Excel program. Significant differ- Rate of syneresis ences in plate count data were established by Twenty bags of Uirou-Tane without inocula- least significant difference at the 5% level of tion of any bacteria, prepared by the above significance.

PATHOGENS AND SPOILAGE BACTERIA IN AWA-UIROU 355

70 tionship was observed between shelf life and y = - 289 x + 324 sugar or water content (data not shown). Z .R2=076i co 0 60 Evaluation of the natural micro flora a, 0 00 in Awa-Uirou 0 50 The growth of bacteria naturally present in laboratory-prepared uninoculated Awa-Uirou C/) was evaluated. The heat-resistant bacteria grew 40 rapidly to reach 5.0 log colony-forming units 0.88 0.90 0.92 0.94 0.96 Water activity (CFU)/g within 3 days and reached maximum level (6.4 log CFU/g) at day 6 and remained FIG. 1. Relationship of water activity and sugar contents constant throughout the incubation period of Awa-Uirou (u = 29). (Fig. 2). The rate of syneresis increased propor- tionally to the growth of bacteria and reached Results 28% at day 7 at 30 C storage. Physical parameters of commercial A wa-Uirou Artificial inoculation before steaming The average values of the sugar content, operation, evaluation, and control water content, and water activity (a) of the 29 The survival and growth of cells or spores in locally made samples were 56.7±4.1 degrees Awa-Uirou artificially inoculated with S. aureus, Brix, 41.4±4.0%, and 0.924±0.010, respec- B. cereus, and B. subtilis before steaming opera- tively. The ranges of these values were 48.0 to tion were evaluated. Each of 3.5, 4.5, and 4.8 log 64.0 degrees Brix, 32.1-48.7%, and 0.900-0.949, CFU/g of B. subtilis, B. cerei.is, and S. aureus respectively. A highly positive relationship spores or cells, respectively, were inoculated (R2 = 0.84) between the value of water content into lOg of Uirou-Tane (paste) and steamed and water activity was observed (data not at 100CC for 70 minutes. The residual viable shown). A clear negative relationship (R2 = 0.91) S. aureus cell and B. cereus spores were less than between the value of water content and sugar 2.3 log CFU/g after the steaming operation and content was also exhibited (data not shown). subsequent incubation for 7 days at 30CC (data Therefore, the sugar content was inversely pro- not shown). However, the 2.9 log CFU/g of re- portional to the water activity (Fig. 1). The shelf sidual B. subtilis spore started to germinate rap- life on the samples set by manufacturers was idly and reached 6.6 log CFU/g within 3 days 7.3 ±4.6 days (range, 1-16 days). No clear rela- when stored at 30C (Table 1). The effectiveness of glycine, hop extract, and salmon soft roe protein in presteamed Uirou-Tane inoculated 30 with B. subtilis is shown on Table 1. The hop extract and salmon soft roe protein did not 25 show any antimicrobial effect against B. suhtilis 0) at the concentrations recommended by the 0U- 20 company (Mitsubishi Chemical Food Co. Ltd., 15 > Tokyo, Japan). However, the addition of U) > 2.5 mg/g glycine effectively suppressed the

.0 10 growth of inoculated B. subtilis. The growth 54 > patterns of B. subtilis in Awa-Uirou supple- mented with 2.5 and 5.0 mg/g of glycine and stored at 30 C are shown in Fig. 3. The addition BS of 2.5 mg/g of glycine was found to suppress the Storage (days) growth of inoculated bacteria by 2 days and FIG. 2. The growth of heat-resistant spores in non- thereafter, the bacteria grew rapidly and reached inoculated Awa-Uirou (•) and the rate of syncresis (A). 6.0 log CFU/g level within 5 days. However, the

356 OKAHISA El AL.

8

7

C) 0U- FIG. 3. The growth characteristic of heat- C) resistant B. subtilis spore inoculated into 0 Uirou-Tane before steaming. The change of viable cell counts in Uirou-Tane inoculated 0 with heat-resistant B. subti/is spores prior to w steaming followed by storage at 30C for up 53 to 7 days is depicted. The symbols •, •, and > A represent the samples containing 0, 2.5, 2 and 5.0 mg/g sample of glycine, respectively.

BS 0 1 2 3 4 5 6 7 Storage (days) addition of 5.0 mg/g of glycine suppressed the B. cereus was suppressed throughout the 7-day growth of B. subtilis throughout the incubation incubation period at 30 C. period at 30C. Sensory evaluation Artificial inoculation after the steaming operation, evaluation, and control The sensory quality of Awa-Uirou supple- The growth of bacteria in Awa-Uirou artifi- mented with glycine (2.5 and 5.0mg/g) or without glycine was evaluated by 21 trained cially inoculated with S. aureus and B. cereus after the steaming operation was evaluated (Fig. panelists according to a visual analogue scale method. As shown in Table 2, no significant 4). S. aureus grew rapidly to reach 7.0 log CFU/g influence on color (appearance), taste, or texture level in 2 days under 30 :1C storage condition. was observed after the addition of glycine up to However, B. cereus grew slowly and reached 4.0 log CFU/g within 3 days and remained 5.0 mg/g. constant and/or increased slightly throughout the incubation period at 30C. The addition of Discussion 5.0 mg/g of glycine did not affect the growth of Enterotoxin-producing B. cercus and S. aureus S. aureus throughout the 7-day incubation pe- are recognized as potential hazards from raw riod at 30C (data not shown), but the growth of confections (Anunciacao et al., 1995; Agata and

8

7

FIG. 4. The growth characteristic of S. nu- C, reus and B. cereus inoculated into Awa-Uirou U- after the steaming process. The change of C-) viable cell counts in Awa-Uirou inoculated o5C) with B. cLrdus and S. aureus after steaming followed by storage at 30 CC for up to 7 days 0 is depicted. Symbols • and A represent B. C, cereus samples containing 0 and 5.0 mg/g S sample of glycine, respectively. The symbol > • represents S. aureus samples containing no 2 glycine.

0 1 2 3 4 5 6 7 Storage (days)

PATHOGENS AND SPOILAGE BACTERIA IN AWA-UIROU 357

TABLE 2. SENSORY EVALUATION OF AwA-UIRoc initial level of contaminated cells or spores was Glycine (mg/g sample) <3.0 log CFU/g Uirou Tane (Fig. 2). From this viewpoint, 70 minutes of steaming operation at 0 2.5 5.0 100C was thought to be sufficient to kill S. Color 7.2±2.1 7.1±2.0 7.0±2.1 aureus cells and most of the B. cereus spores. Taste 7.8±2.1 7.3± 1.9 7.1 ±2.0 However, extremely heat-resistant B. cereus Texture 7.2±2.2 6.8±1.8 6.9+2.1 spores can survive the steaming operation. The Continuous value 0 to 10 corresponds to bad to good After steaming, contaminating S. aureus cells quality. and B. cereus spores are thought to grow rapidly at 30C (Fig. 4). Agata et al. (2002) reported that the accumulation of emetic toxin by B. cereus Mori, 1997; Agata et at., 2002). Sticky rice cakes reached <10 ng/g when the viable B. cereus cell commonly consumed in Japan and other Far counts were > 6.0 log CFU/g in boiled rice. The East and Southeast Asian countries have been average value of B. cereus viable cells in Awa- associated with several outbreaks of food poi- Uirou in most cases was found to be > 5.0 log soning (Kaneko et at., 1982; Ishii et al., 1988; CFU/g after 7 days of storage at 30 CC and Uematsu and Kaneko, 1989; Anunciacao et al., some samples exhibited close to 6.0 log CFU/g 1995; Houjyo et at., 2001; Saito et at., 2001; Mat- (Fig. 4). Interestingly, the addition of 5.0 mg/g suoka et al., 2003; Niwa et at., 2003; Huruse et at., of glycine could suppress the growth of B. cereus 2004). To evaluate or control the risk of food significantly, suggesting the usefulness of such poisoning from this kind of food, we chose antimicrobials (Fig. 4). Awa-Uirou and surveyed its physical parame- A starch matrix such as rice flour gel is a good ters relating to bacterial growth and investi- medium for the growth of S. aureus and en- gated the sterilization efficacy of the steaming terotoxin production (Shinagawa et al., 1982). operation, growth characteristics of the bacteria, According to several reports of outbreaks re- and effectiveness of antimicrobial compounds lated to sticky rice cakes in Japan (Ishii et at., to control the growth of bacteria. 1988; Houjyo etal., 2001; Tsuji et at., 2003), 7.0 log The water activity of commercial Awa-Uirou CFU/g S. aureus could produce 2.4 to 5.1 ng/g of was found to be 0.900 to 0.949 (Fig. 1) and the enterotoxins, which is thought to be sufficient to pH was between 6.0 and 7.0, which suggests cause foodborne disease. Based upon these ob- that these products could have a potential risk servations and our experimental results (Fig. 4), to cause food poisoning. According to the FDAs post-process contamination of Awa-Uirou over definition of potentially hazardous foods (FDA, 2 days at 30C without any bacterial control 2001), those foods which have pH and a over may contribute to the risk of food poisoning by 5.0 and 0.88, respectively, are potentially haz- S. aureus enterotoxins. ardous foods. The syneresis of starch gel matrix is a major There are two possibilities for bacterial con- cause of quality loss of Uirou. As shown in Fig. 2, tamination of Awa-Uirou: 1) cells or spores with natural contamination, bacterial spores that survive after steaming and 2) surface grew rapidly to cause syneresis of Awa-Uirou contamination of the product by hands, cook- samples when stored at 30C. With artificial ing instruments, or the environment. How- contamination, B. subtilis spores, added before ever, during our experiments, S. aureus cell and steaming, exhibited similar growth patterns B. CLTCUS spore contamination of Uirou-Tane (6.0 log CFU/g) (Fig. 3) and resulted in syneresis was not detected even after 7 days of storage at of the samples. B. subtilis is known to produce 30C (data not shown). The decimal reduction several kinds of extracellular enzymes. The de- time at 58C (D58) of S. aureus cells in buffer struction of the sugar chain of starch by amylase solution at pH 7.0 was reported as 0.32+0.33 or decomposition of sucrose by levan sucrase is (0.09-1.1) minutes (Thomas and Masters, 1988). thought to increase or decrease the water- Dufrenne et at. (1994, 1995) reported the D90 holding capacity of the starch gel matrix and value of B. cereus to be 23.0 ± 41.6 (2.2 to >200) consequently cause syneresis of Awa-Uirou minutes. In our experiment, we found that the (Naito and Matsunaga, 2002).

358 OKAHISA ET AL.

It is not feasible to store ordinary rice flour hop (Hwnulus lupulus L.) constituents. Economic Botany gel foods such as Uirou at low temperature or 2003;57:118-125. freezing to prevent the growth of bacteria be- Drohniewski FA. Bacillus cereus and related species. Clin. Microbiol. Rev. 1993;6:324-338. cause the product easily loses its texture during Dufrenne J, Bijwaard M, te Giffel M, Beumer R, and aging due to disruption of the starch gel matrix. Notermans S. Characteristics of some psychrotrophic pH control is not suitable because it changes Bacillus cereus isolates. Int. J. Food Microhiol. 1995; not only the taste and texture but also the color. 27:175-183. However, the addition of 5.0 mg/g of glycine DLlfrcnne J, Soentoro P. Tatini 5, Day T, and Notermans S. was found to effectively suppress the growth of Characteristics of Bacillus cereus related to safe food production. Int. J . Food Microbiol. 1994;23:99-109. B. cereiis (Figs. 3 and 4) without changing the [FDA] U.S. Food and Drug Administration. Evaluation and sensory characteristics of Awa-Uirou (Table 2). Definition of Potentially Hazardous Foods. Available These results suggested that glycine could be at http://www.cfsaii.fda.gov/-coniiii //ift4-toc.litin], ac- useful for reducing the risk of food poisoning by cessed July 16, 2007. Rockville, MD: FDA, 2001. (Online.) B. cereus enterotoxins and preventing quality Gary MD, Linda NL, and Donald JL. Improved electro- poration and cloning vector system for gram-positive B. subtilis. losses caused by bacteria. AppI. Environ. Microhiol. 1991;57:1194-1201. In conclusion, the results of this study dem- Hammes W, Schleifer KH, and Kandler 0. Mode of action onstrated that the addition of 0.5% glycine be- of glycine on the biosynthesis of peptidoglycan. J . Bac- fore the steaming process could reduce the terial. 1973;116:1029-1053. microbial risk of the steamed rice confection and Har-Zion G, Brin I, and Steiner J . Psychophysical testing of prevent the risk of food poisoning and quality taste and flavour reactivity in young patients undergo- loss. Therefore, addition of glycine along with ing treatment with removable orthodontic appliances. Fur. J. Orthodontics. 2004;26:73-78. good manufacturing and handling practices Helge H and Ingolf FN. High-frequency transformation, could be an effective means for reducing the risk by electroporation, of Lactococcus lactis subsp. cremoris of contamination of Awa-Uirou. grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 1989;55:3119-3123. Henson S. The economics of food safety in developing Acknowledgment countries. No. 03-19, Working Papers. Rome: Agri- This work was done as part of "Food Project" cultural and Development Economics Division of the Food and Agriculture Organization of the United Na- with financial support by the Ministry of Agri- tions (FAO - ESA), 2003. culture, Forestry and Fisheries. We would like Hishinuma F, Izaki K, and Takahashi H. Effects of glvcine to thank Mitsubishi Chemical Food Co. Ltd. and and u-amino acids on growth of various microorganism. Asama Chemical Co. Ltd. for kindly providing Agric. Biol. Chem. 1969;33:1577-1586. the food additives. We are grateful to Ms. Man Houjyo K, Shimizu K, Yamamoto K, Nagai H, Shibata Y, and Iwamoto S. S. aureus outbreaks caused by "Mitsu- Mochida and Ms. Tomoko Kitagawa for their dango", "An-dango" and "Daihuku" in Sizuoka prefec- technical assistance and support. ture [in Japanese]. Infect Agents Surv. Rep. 2001;22: 192-193. References Huruse A, Ushigome T, Tsuruta M, Mata yoshi Y, and Ootsuka H. An outbreak of Bacillus cereus [in Japanese]. Agata N and Mori M. Growth and emetic toxin production Infect. immun. Childhood 2004;16:151-155. of Bacillus cereus in various food (in Japanese). Jpn. Inatsu Y, Bari ML, Kawasaki S. and lsshiki K. Survival of J. Food Microbiol. 1997;14:145-148. Escherichia co/i 0157:H7, Salmonella Enteritidis, Stap/u- Agata N, Ohta M, and Yokoyama K. Production of Bacillus lococcus aureus, and Lisferia mowci/toçenes in Kimchi. cereus emetic toxin (cereulide) in various foods. Int. J. Food Prot. 2004;67:1497-1500. J. Food Microbiol. 2002;73:23-27. Ishii T, Mazaki M, Takebayashi I, Nagao 5, Morioka M, Anonymous. 2008. Asama Chemical Co. Ltd. homepage. Ishikawa H, and Yano H. An outbreak of poisoning due Available at http://www.asama-chemical.co.jp/, ac- to Staphyloccocus aureus producing enterotoxin origi- cessed February 2, 2008. Tokyo: Asarna Chemical Co. nated to Tsukimi dumpling [in Japanese]. Rep. Kouchi Ltd., 2008. (Online.) Res. Inst. Hygiene 1988;34:99-103. Anunciacao LL, Linardi WR, do Carmo LS, and Bergdoll Kaneko T, Kanamaru Y, and Kasuga T. An outbreak of MS. Production of staphylococcal enterotoxin A in S. aureus caused by "Kash iwa-mochi" [in Japanese]. cream-filled cake. Int. J . Food Microbiol. 1995;26:259- Rep. Yamanashi Pref. Inst. Environ. Hygiene 1982;25:51- 263. - 55. Bhattacharya S. Virani 5, Zavro M, and Haas GJ. Inhibition Le Loir Y, Baron F, and Gautier M. Staphylococcus aureus of Streptococcus mu tans and other oral streptococci by and food poisoning. Genet. Mol. Re. 2003;2:63-76.

PATHOGENS AND SPOILAGE BACTERIA IN AWA-UIROU 359

Matsuoka Y, Arava I, and Hujii K. A case of B. cLrLus food Shinagawa K, Kunita N, Matuzaka N, and Sakaguchi C. poisoning caused by rice dumpling with sweet bean Experimental production of Staphylococcal enterotoxins paste [in Japanese]. Rep. Kumamoto Pref. Res. Tnstit. A, B, and C on rice flour gel medium. Jap. J . Med. Biol. Envron. Sd. 2003;9:33-38. Sci. 1982;35:1-8. McKillip JL. Prevalence and expression of enterotoxins in Sorokulova lB. Reva ON, Smirnov VV, Pinchuk IV, Lapa Bacillus cereus and other Bacillus spp., a literature review. SV, and Urdaci MC. Genetic diversity and involvement Ant Van Leeuwenhoek 2000;77:393-399. in bread spoilage of Bacillus strains isolated from flour Naito S and Matsunaga D. Syneresis of Mizu-yokan (soft and ropy bread. Lett. AppI. Microbiol. 2003;37:169-173. bean jam jelly) by Bacillus spp. Jpn. J. Food Microbiol. Thomas CJ and Masters F. Microbial spoilage of pre- 2002;19:119-125. cooked potato-topped pies. J. AppI. Bacteriol. 1988;64: Nanba A and Itho M. Combinated effect of heat treatment 227-233. and various chemical on sterilization of Uirou [in Japa- Tsuji H, Oshibe T, Ikeno M, Yamamoto A, Matuda K, nese]. Ann. Rep. Food Res. Cent., AlTEC 1997;38:13-17. Totsuka A, Ooya M, Uchigoe S, Tsuruhayashi I, Segou E, Niwa A, Kato T, Yamamoto K, Toni T, Ito A, Ishikawa K, Enornoto M, and Flujimoto T. A case of food poisoning and Naito S. 2003. Application of biopreservation on caused by Tsukimi dango in Hyogo prefecture [in Jap- Azuki bean paste and its product [in Japanese]. Ann. anese]. Infect. Agents Surv. Rep. 2003;24:11-12. Rep. Food Res. Cent., AlTEC 2003;44:20-22. Uematsu II and Kaneko T. Food poisoning caused by Notermans S and Batt CA. A risk assessment approach for Staphylococci in Tsukimi-dango [in Japanese]. Rep. Ya- food-borne Bacillus cereus and its toxins. Symp. Ser. Soc. manashi Pref. Res. Inst. Environ. lolutry. 1989;33:30-33. Appl. Microbiol. 1998;27:51S-61S. Watanabe K and Hayano K. Distribution and identification Saito N, Sasaki M, Yamaguchi I, Hatayama K, Siraishi H, of proteolytic Bacillus spp. in paddy field soil under rice T, Hino K, Uziie Y, Kobayasi T, Oikawa T, Ohba 0, cultivation. Can. J. Microbiol. 1993;39:674-680. Itho Z, Nakajima H, Shisido Y, Kato E, Takauchi J, and Suzuki K. An outbreak case of S. aureus caused by Address reprint requests to: "Zunda machi" in Miyagi prefecture [in Japanese]. Infect Agents Surv. Rep. 2001;22:191-192. Yasuhiro Inatsu, Ph.D. Schwartz AC, Quecke W, and Brenschede C. Inhibition by National Food Research Ins fit i.te glycine of the catabolic reduction of proline in Cbs- 2-1-12, Kannondai tridiurn sticklaudii: evidence on the regulation of amino Tsukuba City acid reduction. Z. AlIg. Mikrohiol. 1979;19:211-220. Ibaraki, 305-8642 Shinagawa K. Analytical method for Bacillus cereus and Japan other Bacillus species. Int. J. Food Microhiol. 1990;10: 125-142. E-mail: [email protected], [email protected]