Food Microbiology 38 (2014) 122e127
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Fate of Bacillus cereus and naturally occurring microbiota on milled rice as affected by temperature and relative humidity Seonyeong Choi a, Hoikyung Kim b, Yoonsook Kim c, Byeong-sam Kim c, Larry R. Beuchat d, Jee-Hoon Ryu a, * a
Department of Food Bioscience and Technology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea Korea Food Research Institute, Baekhyun-dong, Seongnam, Gyeonggi 463-746, Republic of Korea d Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2013 Received in revised form 15 August 2013 Accepted 29 August 2013 Available online 12 September 2013
We studied the survival and growth patterns of Bacillus cereus, mesophilic aerobic bacteria (MAB), and molds and yeasts (MY) on rough and milled brown and white Korean rice stored at 12 and 21 C and 43, 68, and 85% relative humidity (RH) for up to 24 wk. The initial populations of MAB present on rough rice, brown rice, and white rice were 7.7, 5.7, and 3.3 log CFU/g, respectively, and remained constant or decreased (P 0.05) by 0.7e1.8 log CFU/g during storage. The initial populations of B. cereus on the three types of laboratory-inoculated rice were 3.1e3.8 log CFU/g and remained constant (P > 0.05) during storage, regardless of degree of milling, storage temperature, and RH. The initial populations of MY on rough rice, brown rice, and white rice were 6.2, 4.2, and 2.1 log CFU/g, respectively. At 12 C and 85% RH, the MY increased significantly (P 0.05) only on brown rice; however, at 21 C and 85% RH, MY increased (P 0.05) on all types of rice during storage. These observations will be useful when assessing conditions affecting survival of B. cereus and determining environmental conditions necessary to prevent growth of potentially mycotoxigenic molds on various types of milled rice during storage. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Rice Bacillus cereus Mesophilic aerobic bacteria Molds and yeasts Degree of milling Relative humidity Temperature
1. Introduction Rice (Oryza sativa L.) is a staple food consumed by almost half of the world’s population (Cottyn et al., 2001; FAO, 2004), and about 90% of the rice produced is consumed in Asia (FAOSTAT, 2009). Rice can be classified as rough, brown, or white, based on the degree of milling. Rough rice refers to rice kernels that contain outer hull, bran, and endosperm (Skyrme et al., 1998). Brown rice is produced when the outer hull is removed from rough rice, and white rice is produced by removing bran layers from brown rice. It is recommended that Korean rice be stored at less than 15 C and at a relative humidity (RH) less than 70% to retain microbiological and sensorial qualities; the optimum composition of air for stored rice is 5e7% oxygen and 3e5% carbon dioxide (Korea Rural Development Administration, 2007). Failure to maintain appropriate storage conditions can lead to proliferation of microorganisms on rice, resulting in post-harvest losses and reduction in
* Corresponding author. Tel.: þ82 2 3290 3409; fax: þ82 2 3290 3918. E-mail address: [email protected] (J.-H. Ryu). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.08.016
quality (Manabe and Tsuruta, 1991). Survival and growth of spoilage, pathogenic, and mycotoxigenic microorganisms on cereals and other low-moisture foods and food ingredients are known to be affected by environmental factors such as storage temperature and RH (Beuchat et al., 2013). Bacteria frequently found on rice belong mainly to the Enterobacteriaceae family (Cottyn et al., 2001). Bacillus spp. are also frequently present at low levels (Cottyn et al., 2001; Sarrías et al., 2002; Tahir et al., 2012). Rice can become contaminated with microorganisms during production, harvesting, processing, and handling as a result of contact with soil, soil amendments, irrigation water, dust, animal feces, or insects (Haque and Russell, 2005; Laca et al., 2006). Foodborne illness caused by Bacillus cereus has been associated with consumption of cooked rice (Batt, 1999; Granum and Lindback, 2013; Indukuri and Molohon, 2006). Spores of B. cereus can survive cooking and, if the cooked rice is left at room temperature, may germinate, multiply, and result in illnesses. Molds such as potentially mycotoxigenic Aspergillus spp. (Sales and Yoshizawa, 2005a), Fusarium spp. (Pitt et al., 1994), and Penicillium spp. (Makun et al., 2007) are not uncommonly found in raw rice. The presence of mycotoxins formed by molds during growth in
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Fig. 1. Populations of mesophilic aerobic bacteria on rough rice, brown rice, and white rice inoculated with B. cereus and incubated at 12 or 21 C and a relative humidity of 43 (circles), 68 (squares), or 85% (triangles) for up to 24 wk. The detection limit was 1.0 log CFU/g rice (10 CFU/g rice).
rice is a serious concern. Improper postharvest handling of agricultural commodities such as rice can lead to aflatoxin contamination (Sales and Yoshizawa, 2005a). It is important to understand the behavior of hazardous microorganisms such as pathogenic and spoilage microorganisms on rice during controlled and abusive storage conditions in order to develop more effective intervention strategies to enhance food safety. Studies reporting the behavior of harmful and naturally occurring microorganisms on rice have largely been done using cooked white rice. The objective of this study was to investigate the survival and growth patterns of mesophilic aerobic bacteria (MAB), B. cereus, and molds and yeasts (MY) on raw milled rice as affected by degree of milling, temperature, and RH during storage. 2. Materials and methods 2.1. Bacterial strains and preparation of inoculum Five strains of B. cereus (C1 [isolated from pasta], C9 [source of isolate unknown], 038-2 [isolated from infant formula], and F4810/ 72 [isolated from cooked rice], and KCTC 1013 [isolated from soil]) were used. B. cereus strains C1, C9, 038-2, and F4810/72 were obtained from Center for Food Safety at the University of Georgia in USA and strain KCTC 1013 was obtained from Korean Collection for Type Cultures in Republic of Korea. Cryopreserved B. cereus cells (vegetative cells plus spores) of each strain were separately inoculated in 10 ml of tryptic soy broth (TSB; BBL/Difco, Sparks, MD, USA) and incubated at 30 C for 24 h. After three consecutive loop (ca. 10 ml) transfers at 24-h intervals, 48 ml of a five-strain mixture of B. cereus was prepared by combining 9.6 ml of each of the five cultures. The suspension was centrifuged at 2000 g for 15 min at room temperature (21 2 C), the supernatant was decanted, and cells were resuspended in 480 ml of sterile distilled water (DW) to give a population of approximately 7 log CFU/ml.
2.2. Inoculation of B. cereus on rice Three types of rice (Oryza sativa L. ssp. Japonica; produced in Jincheon, Republic of Korea) obtained from the Korea Food Research Institute (Seongnam, Gyeonggi, Republic of Korea) were used: rough rice (rice without milling), brown rice (hulled rice), and white rice (rice with the bran layer completely removed). Rough rice produced in Jincheon was collected in October and November 2011 and milled to produce brown rice and white rice at a rice-processing complex. To inoculate with B. cereus, rough rice, brown rice, or white rice (160 g) was immersed in an inoculum prepared as described above (480 ml, ca. 7 log CFU/ml) for 5 min at room temperature with intermittent swirling. The inoculum was decanted and the rice was placed in a sterile sieve (diameter 203 mm; depth 41 mm; pore size 600 mm), spread evenly with a sterile spoon, and dried for 2 h in a laminar flow hood at room temperature. After drying, 10 g of rough rice, brown rice, or white rice were placed in a sterile Petri dish (diameter 60 mm; depth 15 mm; SPL, Pocheon, Republic of Korea). 2.3. Establishment of RH conditions Inoculated rice was stored at optimum and abusive RHs. To create RHs of 43.0 0.5, 68.0 0.5, or 85.0 0.5%, 200 ml of saturated potassium carbonate (Daejung, Siheung, Republic of Korea), lithium acetate (Junsei, Tokyo, Japan), or potassium chloride (Daejung) solution, respectively, were deposited in an airtight container (1.2 L; 155 mm long by 155 mm wide by 87 mm high; Lock & Lock, Seoul, Republic of Korea). The containers were held at 12 and 21 C for at least 24 h to enable equilibration of RH before use in experiments. Six lidless sterile Petri dishes, each containing 10 g of inoculated, dried rough rice, brown rice, or white rice (two Petri dishes per type of rice), were placed above the surface of saturated salt solutions in a sealed container and stored at 12 or 21 C for up to 24 wk.
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Fig. 2. Populations of B. cereus (open symbols) and B. cereus spores (closed symbols) on rough rice, brown rice, and white rice after inoculation of B. cereus and incubation at 12 or 21 C and a relative humidity of 43 (circles), 68 (squares), or 85% (triangles) for up to 24 wk. The detection limit was 1.0 log CFU/g rice (10 CFU/g rice).
2.4. Microbiological analyses
2.5. Statistical analysis
Populations of MAB, B. cereus, and MY on rough rice, brown rice, and white rice were determined after storage for 0, 1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 wk. At each sampling time, 5 g of rice were placed in a stomacher bag (BA 6040 standard bag; Seward, West Sussex, UK) containing 50 ml of TSB and pummeled for 2 min in a stomacher (Interscience BagMixerÒ 400W; Interscience, Saint Nom, France). The TSB wash was serially diluted in sterile 0.1% peptone water, and duplicate 0.1-ml samples and quadruplicate 0.25-ml samples (undiluted homogenate) were spread-plated on tryptic soy agar (TSA; BBL/Difco), mannitol egg yolk polymyxin agar (MYPA; Hangang, Gunpo, Republic of Korea), and Dichloran Rose Bengal Chloramphenicol (DRBC) agar (BBL/Difco) to determine populations of MAB, B. cereus, and MY, respectively. TSA and MYPA plates were incubated at 30 C for 24 h and DRBC plates were incubated at 25 C for 5 days before counting colonies. The remaining mixtures of rice and TSB were enriched by incubating at 25 C for 5 days. When no colonies were formed on MYPA or DRBC, enriched homogenates were streaked on MYPA or DRBC and incubated at 30 C for 24 h or at 25 C for 5 days, respectively. To determine the number of B. cereus spores on rice, 5 ml of homogenate were deposited in a 15-ml conical tube, heated at 80 C for 15 min, and immediately cooled by immersing in ice water. The heated suspensions (quadruplicate 0.25-ml samples) were spread-plated on MYPA. Heated homogenate was incubated at 30 C for 24 h to enrich for B. cereus, followed by streaking on MYPA and incubating at 30 C for 24 h. Typical B. cereus colonies (pink-red in color with zones of precipitate) formed on MYPA were counted. All colonies formed on TSA and DRBC were counted. Recovery of B. cereus from heated and/or heated and enriched homogenates indicated the presence of spores on rice. The detection limit for the direct plating was 1.0 log CFU/g rice (10 CFU/g rice); the detection limit by enrichment was 1 CFU/5 g of rice.
All experiments were replicated three times. Data were analyzed using the general linear model of the Statistical Analysis System software (SAS 9.1; SAS Institute, Cary, NC, USA). Fisher’s least significant difference (LSD) test was used to determine if populations of MAB, B. cereus, and MY were significantly (P 0.05) affected by storage temperature, RH, and time. 3. Results and discussion 3.1. MAB on rice Fig. 1 shows populations of MAB on rough rice, brown rice, and white rice inoculated with B. cereus and stored at 12 or 21 C and 43, 68, or 85% RH for up to 24 wk. To simulate a situation in which a high population of B. cereus is present on rice, rough, brown, and white rice were inoculated before determining numbers of MAB and MY. This approach facilitated measurement of populations of B. cereus, MAB, and MY at the time rice was placed into storage. The initial populations of MAB on rough rice, brown rice, and white rice were 7.7, 5.7, and 3.3 log CFU/g, respectively. This indicates that MAB (>2.0 log CFU/g) were physically removed from rice as the milling progressed. Counts for white rice largely reflect those on rice inoculated with B. cereus (Fig. 2). Our observations on the three types of rice are in good agreement with those reported for mesophilic aerobic plate counts (APC) on rice seed (rough rice), brown rice, and milled rice (white rice) produced and processed in Japan (Bainotti and Parra, 2000). The APCs in that study were 8.2, 6.7, and 3.4 log CFU/g, respectively. Differences in APCs on the three types of rice were attributed to partial decontamination caused by the milling process. Sarrías et al. (2003) investigated populations of microorganisms (APCs, coliforms, Escherichia coli, B. cereus, sulfite-
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Fig. 3. Populations of molds and yeasts rough rice, brown rice, and white rice inoculated with B. cereus and incubated at 12 or 21 C and a relative humidity of 43 (circles), 68 (squares), or 85% (triangles) for up to 24 wk. The detection limit was 1.0 log CFU/g rice (10 CFU/g rice).
reducing clostridia, and MY) on unhusked rice (rough rice) and husked rice (brown rice). They reported that counts on husked rice were 1.4e2.7 log CFU/g less than those on unhusked rice. At 12 C, perhaps with the exception of brown rice stored at 68% RH, populations of MAB on all three types of rice generally did not change significantly (P 0.05) during storage for 24 wk, regardless of the RH (Fig. 1). Compared to the initial population, MAB on brown rice stored at 68% RH decreased significantly (P 0.05) during storage but only by 0.7 log CFU/g (Fig. 1). At 21 C, populations of MAB on rough rice stored at 43% RH remained constant for 24 wk. At 68 and 85% RH, MAB populations decreased by 0.8 and 1.1 log CFU/g, respectively, after 24 wk. Populations of MAB on brown rice decreased by 0.8, 1.8, and 0.9 log CFU/g after 24 wk at 21 C and RHs of 43, 68, and 85%, respectively. Populations of MAB on white rice remained constant, regardless of RH, during storage time and are attributed mostly to the B. cereus inoculum. Extended survival of bacteria under adverse conditions has been described by other researchers (Beuchat et al., 2013). Lin and Beuchat (2007) reported that populations of Enterobacter sakazakii (4.93e5.64 log CFU/g) in infant rice, barley, oatmeal, and mixed grain cereal (aw 0.63e0.83) stored for 24 wk at 4 C decreased by only 0.4e1.5 log CFU/g. Extended survival, in part, has been attributed to a long-term stationary phase of bacterial cells. Finkel (2006) observed that after the logarithmic death phase, E. coli remained viable for long periods without the addition of nutrients. Wen et al. (2009) observed the long-term stationary phase of Listeria monocytogenes in TSB supplemented with yeast extract (TSBYE). They reported that the population of L. monocytogenes in long-term stationary phase remained at 8 log CFU/ml of TSBYE for 718 h. Our observations on extended survival of MAB on rice suggests that indigenous microorganisms on rice may have been in a long-term stationary phase in a desiccated environment.
3.2. B. cereus on rice Fig. 2 shows B. cereus populations on inoculated rough rice, brown rice, and white rice stored at 12 or 21 C and 43, 68, or 85% RH for up to 24 wk. B. cereus spores were not detected on uninoculated rough rice and white rice. On uninoculated brown rice, B. cereus spores (0.5 log CFU/g) were detected (data not shown). Others have reported higher levels of B. cereus on rice. Sarrías et al. (2003) reported that the mean population of naturally occurring B. cereus on unhusked rice was 3.4 log CFU/g. To simulate a worsecase scenario and to be able to determine if viability of B. cereus spores decreased during storage, we inoculated rice at a B. cereus population >4.0 log CFU/g. Total populations of B. cereus (vegetative cells plus spores) and B. cereus spores did not change significantly, regardless of the degree of milling, storage temperature, or RH during the 24-wk storage period (Fig. 2). Immediately after inoculation with B. cereus (before drying), populations of B. cereus (vegetative cells plus spores) on rough rice, brown rice, and white rice were 4.8, 4.3, and 4.1 log CFU/g, respectively; populations of B. cereus spores were 2.9, 2.7, and 2.1 log CFU/g, respectively. This indicates that the inoculum contained 1e2% B. cereus spores. After drying for 2 h in a laminar flow hood at room temperature, populations of total B. cereus (vegetative cells plus spores) decreased significantly by 1.0 log CFU/ g on all three types of rice. However, the number of B. cereus spores did not decrease. Consequently, 5e20% of the cells (vegetative and spores) that survived drying were spores, confirming their higher level of resistance to desiccation. Bacillus spp. spores are well known for resistance to environmental stresses such as desiccation, UV, and g-radiation (Setlow, 1995; Venkateswaran et al., 2003; Moeller et al., 2007). Death of B. cereus spores in dry infant rice cereal stored at 45 C for 48 wk is enhanced at aw 0.75e0.78 but unaffected by pH; loss of viability at 5, 25, and 35 C is largely
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unaffected by aw 0.27e0.28, 0.52e0.55, and 0.75e0.78 (Jaquette and Beuchat, 1998). There has been no report on the effect of temperature (12 and 21 C) and RH (43e85%) on survival patterns of B. cereus on rice as affected by degree of milling. However, there is a report describing the behavior of B. cereus in dry infant rice cereal as affected by temperature and aw. Jaquette and Beuchat (1998) studied the behavior of B. cereus in dry infant rice cereal as affected by temperature (5, 25, 35, and 45 C), pH (5.6 and 6.7), and aw (0.27e0.28, 0.52e0.55, and 0.75e0.78). Cereal was inoculated with vegetative cells and spores of B. cereus at populations of 5.0e6.5 log CFU/g and stored for 36e48 wk before analyzing for survivors. Vegetative cells decreased by 2.4e4.3 log CFU/g after 1 wk and remained constant for an additional 35 wk at 5 and 25 C, regardless of aw. Populations of B. cereus spores remained constant during storage for 48 wk at all test conditions, which is consistent with our findings on rough, brown, and white rice stored for 24 wk. 3.3. MY on rice Fig. 3 shows MY populations on rough rice, brown rice, and white rice inoculated with B. cereus and stored at 12 or 21 C and 43, 68, or 85% RH for up to 24 wk. The initial populations of MY on rough rice, brown rice, and white rice were 6.4, 4.2, and 2.1 log CFU/g, respectively. As with MAB counts, MY were significantly reduced by milling. When RH was maintained at 43 or 68%, populations of MY decreased significantly, regardless of the degree of milling and the storage temperature. After 24 wk, populations of MY on rough rice decreased by 0.5e1.4 log CFU/g at 12 and 21 C, respectively, compared to initial populations. Populations of MY on brown rice and white rice decreased by 1.1e2.6 log CFU/g and 1.0e1.8 log CFU/g, respectively. Notably, when white rice was stored at 21 C and 43 or 68% RH, MY decreased to levels below the detection limit (1.0 log CFU/g) after 24 wk but were not eliminated. At 85% RH, MY increased significantly or remained constant on rice for 24 wk. At 12 C and 85% RH, MY populations on rough rice remained constant for 24 wk; however, on brown rice MY increased gradually after 12 wk, from an initial population of 4.2 log CFU/g to a population of 6.9 log CFU/g after 24 wk. Populations of MY on white rice did not change significantly at 12 C after 24 wk, compared to the initial population. When rice was stored at 21 C and 85% RH, MY increased significantly, regardless of the degree of milling. On rough rice, the MY population was 7.3 log CFU/g after 24 wk, an increase of 1.1 log CFU/g compared to the initial population. On brown rice, MY increased significantly from 4.2 log CFU/g (initial population) to 8.8 log CFU/g after 16 wk and remained constant for the remaining storage period. Populations of MY on white rice increased by 2.3 log CFU/g after 24 wk compared to the initial population. MY increased to much higher populations on brown rice than on rough rice or white rice stored at 85% RH, suggesting that nutrients on the surface of brown rice that are removed during subsequent milling contribute significantly to supporting growth. Compared to the surface of white rice or the hull surrounding rough rice, the bran layer (shell surrounding brown rice) has a greater abundance of albumin, phosphorus, magnesium, and calcium (Tanaka et al., 1973; Dikeman et al., 1981; Juliano and Bechtel, 1985). Soluble protein and phosphorus can stimulate the growth of MY. Ravnskov et al. (1999) studied the effect of baker’s dry yeast, bovine serum albumin, starch, and cellulose on hyphal growth of Glomus intraradices and concluded that the growth was stimulated by albumin. Dorn and Rivera (1966) observed that the dry weight of Aspergillus nidulans increased more in high-phosphate medium than it did in a low-phosphate medium. We observed that, unlike MAB and B. cereus, populations of MY increased in rice held at 21 C and 85% RH. This was not unexpected
and indicates that if control of RH fails during storage of rice, potentially mycotoxigenic molds may grow. Aspergillus spp. and Penicillium spp. can grow at 85% RH (aw 0.85) (Magan and Lacey, 1984; Manabe and Tsuruta, 1991) and produce various mycotoxins. Aflatoxins and ochratoxin A (OTA) are the most extensively studied mycotoxins produced by these molds and have been detected on rice in many countries around the world. Park et al. (2005) studied the incidence of fungal infections and natural occurrence of mycotoxins in Korean polished rice. Aspergillus spp., Penicillium spp., and Fusarium spp. were isolated from 7e26%, 11e 27%, and 7e16% of 88 of the samples tested. They detected aflatoxin B1 in 5 of 88 polished rice samples with a mean concentration of 4.3 ng/g; OTA was detected in 8 of 88 samples with a mean concentration of 3.9 ng/g. They also detected fumonisin B1, deoxynivalenol, nivalenol, and zearalenone in Korean polished rice. Sales and Yoshizawa (2005b) reported that the mean concentrations of aflatoxin in brown rice and polished rice produced in the Philippines were 2.7 and 0.4 mg/kg, respectively. An investigation of rice produced in Nigeria showed that aflatoxin B1 was present in 97 of 196 samples and OTA was present in 56 of 140 samples (Makun et al., 2007). The average concentrations of aflatoxin B1 and OTA detected were 200 and 156 mg/kg, respectively. Our study showing that milled rice, particularly brown rice, stored at 12 or 21 C and 85% RH can support the growth of molds emphasizes the importance of storing milled rice at temperatures and RHs that will prevent mycotoxin production. We investigated the growth and survival of MAB, B. cereus, and MY separately. It would be interesting to study the influence riceborne microbiota on survival and growth of B. cereus. The presence of some microorganisms may influence the survival and growth of others. Cuero et al. (1987), for example, demonstrated that the growth of Aspergillus flavus on rice (aw 0.95 and 0.98) at 16 C is stimulated by the presence of Hyphopichia burtonii and Bacillus amyloliquefaciens. Tandon et al. (1979) showed Curvularia lunata and Chaetomium indicum suppressed the growth of seedborne microbiota. In summary, populations of MAB and MY present on rice decreased by ca. 2 log CFU/g during the milling process. Populations of MAB and B. cereus on rice either remained constant for 24 wk or decreased slightly, regardless of the degree of milling, temperature or RH. The MY population on rice generally increased at 85% RH, regardless of the degree of milling and storage temperature. MY grew on brown rice at 12 C and 85% RH, and on all types of rice at 21 C and 85% RH. Further investigation is needed to define conditions affecting the growth of mycotoxigenic molds and their mycotoxin production on rice during storage as affected by degree of milling, temperature, and RH. Acknowledgments This study was conducted with the support of the Korea Food Research Institute (project no. E132501). We thank the Korea University Food Safety Center and the Institute of Food and Biomedicine Safety for providing resources and facilities. References Bainotti, A.E., Parra, E.S.P., 2000. Microbiological evaluation of processed rice consumed in Japan. World J. Microbiol. Biotechnol. 16, 77e79. Batt, C.A., 1999. BACILLUS/Bacillus cereus. In: Batt, C.A., Patel, P., Robinson, R.K. (Eds.), Encyclopedia of Food Microbiology. Academic Press, San Diego, pp. 119e124. Beuchat, L.R., Komitopoulou, E., Beckers, H., Betts, R.P., Bourdichon, F., Fanning, S., Joosten, H.M., Ter Kuile, B.H., 2013. Low-water activity foods: increased concern as vehicles of foodborne pathogens. J. Food Prot. 76, 150e172. Cottyn, B., Regalado, E., Lanoot, B., Cleene, M.D., Mew, T.W., Swings, J., 2001. Bacterial populations associated with rice seed in the tropical environment. Phytopathology 91, 282e292.
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Moeller, R., Stackebrandt, E., Reitz, G., Berger, T., Rettberg, P., Doherty, A.J., Horneck, G., Nicholson, W.L., 2007. Role of DNA repair by nonhomologous-end joining in Bacillus subtilis spore resistance to extreme dryness, mono- and polychromatic UV, and ionizing radiation. J. Bacteriol. 189, 3306e3311. Park, J.W., Choi, S.-Y., Hwang, H.-J., Kim, Y.-B., 2005. Fungal mycoflora and mycotoxins in Korean polished rice destined for humans. Int. J. Food Microbiol. 103, 305e314. Pitt, J.I., Hocking, A.D., Bhudhasamai, K., Miscamble, B.F., Wheeler, K.A., TanboonEk, P., 1994. The normal mycoflora of commodities from Thailand. 2. Beans, rice, small grains and other commodities. Int. J. Food Microbiol. 23, 35e53. Ravnskov, S., Larsen, J., Olsson, P.A., Jakobsen, I., 1999. Effects of various organic compounds on growth and phosphorus uptake of an arbuscular mycorrhizal fungus. New Phytol. 141, 517e524. Sales, A.C., Yoshizawa, T., 2005a. Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines. J. Food Prot. 68, 120e 125. Sales, A.C., Yoshizawa, T., 2005b. Updated profile of aflatoxin and Aspergillus section Flavi contamination in rice and its byproducts from the Philippines. Food Addit. Cont. 22, 429e436. Sarrías, J.A., Valero, M., Salmerón, M.C., 2002. Enumeration, isolation and characterization of Bacillus cereus strains from Spanish raw rice. Food Microbiol. 19, 589e595. Sarrías, J.A., Valero, M., Salmerón, M.C., 2003. Elimination of Bacillus cereus contamination in raw rice by electron beam irradiation. Food Microbiol. 20, 327e332. Setlow, P., 1995. Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Ann. Rev. Microbiol. 49, 29e54. Skyrme, D.S., Marks, B.P., Johnson, M.G., Siebenmorgen, T.J., 1998. Distribution of total aerobic and coliform bacterial counts among rice kernel components. J. Food Sci. 63, 154e156. Tahir, A., Hameed, I., Aftab, M., Mateen, B., 2012. Microbial assessment of uncooked and cooked rice samples available in local markets of Lahore. Pak. J. Bot. 44, 267e270. Tanaka, K., Yoshida, T., Asada, K., Kasai, Z., 1973. Subcellular particles isolated from aleurone layer of rice seeds. Arch. Biochem. Biophys. 155, 136e143. Tandon, R.N., Gupta, R.C., Shukla, A.N., Arora, D.K., 1979. Antagonistic interactions among seedborne microflora and dynamics of microbial incidence. Arch. Microbiol. 120, 77e80. Venkateswaran, K., Kempf, M., Chen, F., Satomi, M., Nicholson, W., Kern, R., 2003. Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are g-radiation resistant. Int. J. Syst. Evol. Microbiol. 53, 165e172. Wen, J., Anantheswaran, R.C., Knabel, S.J., 2009. Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes. Appl. Environ. Microbiol. 75, 1581e1588.
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Fate of Bacillus cereus and naturally occurring microbiota on milled rice as affected by temperature and relative humidity Seonyeong Choi a, Hoikyung Kim b, Yoonsook Kim c, Byeong-sam Kim c, Larry R. Beuchat d, Jee-Hoon Ryu a, * a
Department of Food Bioscience and Technology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea Korea Food Research Institute, Baekhyun-dong, Seongnam, Gyeonggi 463-746, Republic of Korea d Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2013 Received in revised form 15 August 2013 Accepted 29 August 2013 Available online 12 September 2013
We studied the survival and growth patterns of Bacillus cereus, mesophilic aerobic bacteria (MAB), and molds and yeasts (MY) on rough and milled brown and white Korean rice stored at 12 and 21 C and 43, 68, and 85% relative humidity (RH) for up to 24 wk. The initial populations of MAB present on rough rice, brown rice, and white rice were 7.7, 5.7, and 3.3 log CFU/g, respectively, and remained constant or decreased (P 0.05) by 0.7e1.8 log CFU/g during storage. The initial populations of B. cereus on the three types of laboratory-inoculated rice were 3.1e3.8 log CFU/g and remained constant (P > 0.05) during storage, regardless of degree of milling, storage temperature, and RH. The initial populations of MY on rough rice, brown rice, and white rice were 6.2, 4.2, and 2.1 log CFU/g, respectively. At 12 C and 85% RH, the MY increased significantly (P 0.05) only on brown rice; however, at 21 C and 85% RH, MY increased (P 0.05) on all types of rice during storage. These observations will be useful when assessing conditions affecting survival of B. cereus and determining environmental conditions necessary to prevent growth of potentially mycotoxigenic molds on various types of milled rice during storage. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Rice Bacillus cereus Mesophilic aerobic bacteria Molds and yeasts Degree of milling Relative humidity Temperature
1. Introduction Rice (Oryza sativa L.) is a staple food consumed by almost half of the world’s population (Cottyn et al., 2001; FAO, 2004), and about 90% of the rice produced is consumed in Asia (FAOSTAT, 2009). Rice can be classified as rough, brown, or white, based on the degree of milling. Rough rice refers to rice kernels that contain outer hull, bran, and endosperm (Skyrme et al., 1998). Brown rice is produced when the outer hull is removed from rough rice, and white rice is produced by removing bran layers from brown rice. It is recommended that Korean rice be stored at less than 15 C and at a relative humidity (RH) less than 70% to retain microbiological and sensorial qualities; the optimum composition of air for stored rice is 5e7% oxygen and 3e5% carbon dioxide (Korea Rural Development Administration, 2007). Failure to maintain appropriate storage conditions can lead to proliferation of microorganisms on rice, resulting in post-harvest losses and reduction in
* Corresponding author. Tel.: þ82 2 3290 3409; fax: þ82 2 3290 3918. E-mail address: [email protected] (J.-H. Ryu). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.08.016
quality (Manabe and Tsuruta, 1991). Survival and growth of spoilage, pathogenic, and mycotoxigenic microorganisms on cereals and other low-moisture foods and food ingredients are known to be affected by environmental factors such as storage temperature and RH (Beuchat et al., 2013). Bacteria frequently found on rice belong mainly to the Enterobacteriaceae family (Cottyn et al., 2001). Bacillus spp. are also frequently present at low levels (Cottyn et al., 2001; Sarrías et al., 2002; Tahir et al., 2012). Rice can become contaminated with microorganisms during production, harvesting, processing, and handling as a result of contact with soil, soil amendments, irrigation water, dust, animal feces, or insects (Haque and Russell, 2005; Laca et al., 2006). Foodborne illness caused by Bacillus cereus has been associated with consumption of cooked rice (Batt, 1999; Granum and Lindback, 2013; Indukuri and Molohon, 2006). Spores of B. cereus can survive cooking and, if the cooked rice is left at room temperature, may germinate, multiply, and result in illnesses. Molds such as potentially mycotoxigenic Aspergillus spp. (Sales and Yoshizawa, 2005a), Fusarium spp. (Pitt et al., 1994), and Penicillium spp. (Makun et al., 2007) are not uncommonly found in raw rice. The presence of mycotoxins formed by molds during growth in
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Fig. 1. Populations of mesophilic aerobic bacteria on rough rice, brown rice, and white rice inoculated with B. cereus and incubated at 12 or 21 C and a relative humidity of 43 (circles), 68 (squares), or 85% (triangles) for up to 24 wk. The detection limit was 1.0 log CFU/g rice (10 CFU/g rice).
rice is a serious concern. Improper postharvest handling of agricultural commodities such as rice can lead to aflatoxin contamination (Sales and Yoshizawa, 2005a). It is important to understand the behavior of hazardous microorganisms such as pathogenic and spoilage microorganisms on rice during controlled and abusive storage conditions in order to develop more effective intervention strategies to enhance food safety. Studies reporting the behavior of harmful and naturally occurring microorganisms on rice have largely been done using cooked white rice. The objective of this study was to investigate the survival and growth patterns of mesophilic aerobic bacteria (MAB), B. cereus, and molds and yeasts (MY) on raw milled rice as affected by degree of milling, temperature, and RH during storage. 2. Materials and methods 2.1. Bacterial strains and preparation of inoculum Five strains of B. cereus (C1 [isolated from pasta], C9 [source of isolate unknown], 038-2 [isolated from infant formula], and F4810/ 72 [isolated from cooked rice], and KCTC 1013 [isolated from soil]) were used. B. cereus strains C1, C9, 038-2, and F4810/72 were obtained from Center for Food Safety at the University of Georgia in USA and strain KCTC 1013 was obtained from Korean Collection for Type Cultures in Republic of Korea. Cryopreserved B. cereus cells (vegetative cells plus spores) of each strain were separately inoculated in 10 ml of tryptic soy broth (TSB; BBL/Difco, Sparks, MD, USA) and incubated at 30 C for 24 h. After three consecutive loop (ca. 10 ml) transfers at 24-h intervals, 48 ml of a five-strain mixture of B. cereus was prepared by combining 9.6 ml of each of the five cultures. The suspension was centrifuged at 2000 g for 15 min at room temperature (21 2 C), the supernatant was decanted, and cells were resuspended in 480 ml of sterile distilled water (DW) to give a population of approximately 7 log CFU/ml.
2.2. Inoculation of B. cereus on rice Three types of rice (Oryza sativa L. ssp. Japonica; produced in Jincheon, Republic of Korea) obtained from the Korea Food Research Institute (Seongnam, Gyeonggi, Republic of Korea) were used: rough rice (rice without milling), brown rice (hulled rice), and white rice (rice with the bran layer completely removed). Rough rice produced in Jincheon was collected in October and November 2011 and milled to produce brown rice and white rice at a rice-processing complex. To inoculate with B. cereus, rough rice, brown rice, or white rice (160 g) was immersed in an inoculum prepared as described above (480 ml, ca. 7 log CFU/ml) for 5 min at room temperature with intermittent swirling. The inoculum was decanted and the rice was placed in a sterile sieve (diameter 203 mm; depth 41 mm; pore size 600 mm), spread evenly with a sterile spoon, and dried for 2 h in a laminar flow hood at room temperature. After drying, 10 g of rough rice, brown rice, or white rice were placed in a sterile Petri dish (diameter 60 mm; depth 15 mm; SPL, Pocheon, Republic of Korea). 2.3. Establishment of RH conditions Inoculated rice was stored at optimum and abusive RHs. To create RHs of 43.0 0.5, 68.0 0.5, or 85.0 0.5%, 200 ml of saturated potassium carbonate (Daejung, Siheung, Republic of Korea), lithium acetate (Junsei, Tokyo, Japan), or potassium chloride (Daejung) solution, respectively, were deposited in an airtight container (1.2 L; 155 mm long by 155 mm wide by 87 mm high; Lock & Lock, Seoul, Republic of Korea). The containers were held at 12 and 21 C for at least 24 h to enable equilibration of RH before use in experiments. Six lidless sterile Petri dishes, each containing 10 g of inoculated, dried rough rice, brown rice, or white rice (two Petri dishes per type of rice), were placed above the surface of saturated salt solutions in a sealed container and stored at 12 or 21 C for up to 24 wk.
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Fig. 2. Populations of B. cereus (open symbols) and B. cereus spores (closed symbols) on rough rice, brown rice, and white rice after inoculation of B. cereus and incubation at 12 or 21 C and a relative humidity of 43 (circles), 68 (squares), or 85% (triangles) for up to 24 wk. The detection limit was 1.0 log CFU/g rice (10 CFU/g rice).
2.4. Microbiological analyses
2.5. Statistical analysis
Populations of MAB, B. cereus, and MY on rough rice, brown rice, and white rice were determined after storage for 0, 1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 wk. At each sampling time, 5 g of rice were placed in a stomacher bag (BA 6040 standard bag; Seward, West Sussex, UK) containing 50 ml of TSB and pummeled for 2 min in a stomacher (Interscience BagMixerÒ 400W; Interscience, Saint Nom, France). The TSB wash was serially diluted in sterile 0.1% peptone water, and duplicate 0.1-ml samples and quadruplicate 0.25-ml samples (undiluted homogenate) were spread-plated on tryptic soy agar (TSA; BBL/Difco), mannitol egg yolk polymyxin agar (MYPA; Hangang, Gunpo, Republic of Korea), and Dichloran Rose Bengal Chloramphenicol (DRBC) agar (BBL/Difco) to determine populations of MAB, B. cereus, and MY, respectively. TSA and MYPA plates were incubated at 30 C for 24 h and DRBC plates were incubated at 25 C for 5 days before counting colonies. The remaining mixtures of rice and TSB were enriched by incubating at 25 C for 5 days. When no colonies were formed on MYPA or DRBC, enriched homogenates were streaked on MYPA or DRBC and incubated at 30 C for 24 h or at 25 C for 5 days, respectively. To determine the number of B. cereus spores on rice, 5 ml of homogenate were deposited in a 15-ml conical tube, heated at 80 C for 15 min, and immediately cooled by immersing in ice water. The heated suspensions (quadruplicate 0.25-ml samples) were spread-plated on MYPA. Heated homogenate was incubated at 30 C for 24 h to enrich for B. cereus, followed by streaking on MYPA and incubating at 30 C for 24 h. Typical B. cereus colonies (pink-red in color with zones of precipitate) formed on MYPA were counted. All colonies formed on TSA and DRBC were counted. Recovery of B. cereus from heated and/or heated and enriched homogenates indicated the presence of spores on rice. The detection limit for the direct plating was 1.0 log CFU/g rice (10 CFU/g rice); the detection limit by enrichment was 1 CFU/5 g of rice.
All experiments were replicated three times. Data were analyzed using the general linear model of the Statistical Analysis System software (SAS 9.1; SAS Institute, Cary, NC, USA). Fisher’s least significant difference (LSD) test was used to determine if populations of MAB, B. cereus, and MY were significantly (P 0.05) affected by storage temperature, RH, and time. 3. Results and discussion 3.1. MAB on rice Fig. 1 shows populations of MAB on rough rice, brown rice, and white rice inoculated with B. cereus and stored at 12 or 21 C and 43, 68, or 85% RH for up to 24 wk. To simulate a situation in which a high population of B. cereus is present on rice, rough, brown, and white rice were inoculated before determining numbers of MAB and MY. This approach facilitated measurement of populations of B. cereus, MAB, and MY at the time rice was placed into storage. The initial populations of MAB on rough rice, brown rice, and white rice were 7.7, 5.7, and 3.3 log CFU/g, respectively. This indicates that MAB (>2.0 log CFU/g) were physically removed from rice as the milling progressed. Counts for white rice largely reflect those on rice inoculated with B. cereus (Fig. 2). Our observations on the three types of rice are in good agreement with those reported for mesophilic aerobic plate counts (APC) on rice seed (rough rice), brown rice, and milled rice (white rice) produced and processed in Japan (Bainotti and Parra, 2000). The APCs in that study were 8.2, 6.7, and 3.4 log CFU/g, respectively. Differences in APCs on the three types of rice were attributed to partial decontamination caused by the milling process. Sarrías et al. (2003) investigated populations of microorganisms (APCs, coliforms, Escherichia coli, B. cereus, sulfite-
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Fig. 3. Populations of molds and yeasts rough rice, brown rice, and white rice inoculated with B. cereus and incubated at 12 or 21 C and a relative humidity of 43 (circles), 68 (squares), or 85% (triangles) for up to 24 wk. The detection limit was 1.0 log CFU/g rice (10 CFU/g rice).
reducing clostridia, and MY) on unhusked rice (rough rice) and husked rice (brown rice). They reported that counts on husked rice were 1.4e2.7 log CFU/g less than those on unhusked rice. At 12 C, perhaps with the exception of brown rice stored at 68% RH, populations of MAB on all three types of rice generally did not change significantly (P 0.05) during storage for 24 wk, regardless of the RH (Fig. 1). Compared to the initial population, MAB on brown rice stored at 68% RH decreased significantly (P 0.05) during storage but only by 0.7 log CFU/g (Fig. 1). At 21 C, populations of MAB on rough rice stored at 43% RH remained constant for 24 wk. At 68 and 85% RH, MAB populations decreased by 0.8 and 1.1 log CFU/g, respectively, after 24 wk. Populations of MAB on brown rice decreased by 0.8, 1.8, and 0.9 log CFU/g after 24 wk at 21 C and RHs of 43, 68, and 85%, respectively. Populations of MAB on white rice remained constant, regardless of RH, during storage time and are attributed mostly to the B. cereus inoculum. Extended survival of bacteria under adverse conditions has been described by other researchers (Beuchat et al., 2013). Lin and Beuchat (2007) reported that populations of Enterobacter sakazakii (4.93e5.64 log CFU/g) in infant rice, barley, oatmeal, and mixed grain cereal (aw 0.63e0.83) stored for 24 wk at 4 C decreased by only 0.4e1.5 log CFU/g. Extended survival, in part, has been attributed to a long-term stationary phase of bacterial cells. Finkel (2006) observed that after the logarithmic death phase, E. coli remained viable for long periods without the addition of nutrients. Wen et al. (2009) observed the long-term stationary phase of Listeria monocytogenes in TSB supplemented with yeast extract (TSBYE). They reported that the population of L. monocytogenes in long-term stationary phase remained at 8 log CFU/ml of TSBYE for 718 h. Our observations on extended survival of MAB on rice suggests that indigenous microorganisms on rice may have been in a long-term stationary phase in a desiccated environment.
3.2. B. cereus on rice Fig. 2 shows B. cereus populations on inoculated rough rice, brown rice, and white rice stored at 12 or 21 C and 43, 68, or 85% RH for up to 24 wk. B. cereus spores were not detected on uninoculated rough rice and white rice. On uninoculated brown rice, B. cereus spores (0.5 log CFU/g) were detected (data not shown). Others have reported higher levels of B. cereus on rice. Sarrías et al. (2003) reported that the mean population of naturally occurring B. cereus on unhusked rice was 3.4 log CFU/g. To simulate a worsecase scenario and to be able to determine if viability of B. cereus spores decreased during storage, we inoculated rice at a B. cereus population >4.0 log CFU/g. Total populations of B. cereus (vegetative cells plus spores) and B. cereus spores did not change significantly, regardless of the degree of milling, storage temperature, or RH during the 24-wk storage period (Fig. 2). Immediately after inoculation with B. cereus (before drying), populations of B. cereus (vegetative cells plus spores) on rough rice, brown rice, and white rice were 4.8, 4.3, and 4.1 log CFU/g, respectively; populations of B. cereus spores were 2.9, 2.7, and 2.1 log CFU/g, respectively. This indicates that the inoculum contained 1e2% B. cereus spores. After drying for 2 h in a laminar flow hood at room temperature, populations of total B. cereus (vegetative cells plus spores) decreased significantly by 1.0 log CFU/ g on all three types of rice. However, the number of B. cereus spores did not decrease. Consequently, 5e20% of the cells (vegetative and spores) that survived drying were spores, confirming their higher level of resistance to desiccation. Bacillus spp. spores are well known for resistance to environmental stresses such as desiccation, UV, and g-radiation (Setlow, 1995; Venkateswaran et al., 2003; Moeller et al., 2007). Death of B. cereus spores in dry infant rice cereal stored at 45 C for 48 wk is enhanced at aw 0.75e0.78 but unaffected by pH; loss of viability at 5, 25, and 35 C is largely
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unaffected by aw 0.27e0.28, 0.52e0.55, and 0.75e0.78 (Jaquette and Beuchat, 1998). There has been no report on the effect of temperature (12 and 21 C) and RH (43e85%) on survival patterns of B. cereus on rice as affected by degree of milling. However, there is a report describing the behavior of B. cereus in dry infant rice cereal as affected by temperature and aw. Jaquette and Beuchat (1998) studied the behavior of B. cereus in dry infant rice cereal as affected by temperature (5, 25, 35, and 45 C), pH (5.6 and 6.7), and aw (0.27e0.28, 0.52e0.55, and 0.75e0.78). Cereal was inoculated with vegetative cells and spores of B. cereus at populations of 5.0e6.5 log CFU/g and stored for 36e48 wk before analyzing for survivors. Vegetative cells decreased by 2.4e4.3 log CFU/g after 1 wk and remained constant for an additional 35 wk at 5 and 25 C, regardless of aw. Populations of B. cereus spores remained constant during storage for 48 wk at all test conditions, which is consistent with our findings on rough, brown, and white rice stored for 24 wk. 3.3. MY on rice Fig. 3 shows MY populations on rough rice, brown rice, and white rice inoculated with B. cereus and stored at 12 or 21 C and 43, 68, or 85% RH for up to 24 wk. The initial populations of MY on rough rice, brown rice, and white rice were 6.4, 4.2, and 2.1 log CFU/g, respectively. As with MAB counts, MY were significantly reduced by milling. When RH was maintained at 43 or 68%, populations of MY decreased significantly, regardless of the degree of milling and the storage temperature. After 24 wk, populations of MY on rough rice decreased by 0.5e1.4 log CFU/g at 12 and 21 C, respectively, compared to initial populations. Populations of MY on brown rice and white rice decreased by 1.1e2.6 log CFU/g and 1.0e1.8 log CFU/g, respectively. Notably, when white rice was stored at 21 C and 43 or 68% RH, MY decreased to levels below the detection limit (1.0 log CFU/g) after 24 wk but were not eliminated. At 85% RH, MY increased significantly or remained constant on rice for 24 wk. At 12 C and 85% RH, MY populations on rough rice remained constant for 24 wk; however, on brown rice MY increased gradually after 12 wk, from an initial population of 4.2 log CFU/g to a population of 6.9 log CFU/g after 24 wk. Populations of MY on white rice did not change significantly at 12 C after 24 wk, compared to the initial population. When rice was stored at 21 C and 85% RH, MY increased significantly, regardless of the degree of milling. On rough rice, the MY population was 7.3 log CFU/g after 24 wk, an increase of 1.1 log CFU/g compared to the initial population. On brown rice, MY increased significantly from 4.2 log CFU/g (initial population) to 8.8 log CFU/g after 16 wk and remained constant for the remaining storage period. Populations of MY on white rice increased by 2.3 log CFU/g after 24 wk compared to the initial population. MY increased to much higher populations on brown rice than on rough rice or white rice stored at 85% RH, suggesting that nutrients on the surface of brown rice that are removed during subsequent milling contribute significantly to supporting growth. Compared to the surface of white rice or the hull surrounding rough rice, the bran layer (shell surrounding brown rice) has a greater abundance of albumin, phosphorus, magnesium, and calcium (Tanaka et al., 1973; Dikeman et al., 1981; Juliano and Bechtel, 1985). Soluble protein and phosphorus can stimulate the growth of MY. Ravnskov et al. (1999) studied the effect of baker’s dry yeast, bovine serum albumin, starch, and cellulose on hyphal growth of Glomus intraradices and concluded that the growth was stimulated by albumin. Dorn and Rivera (1966) observed that the dry weight of Aspergillus nidulans increased more in high-phosphate medium than it did in a low-phosphate medium. We observed that, unlike MAB and B. cereus, populations of MY increased in rice held at 21 C and 85% RH. This was not unexpected
and indicates that if control of RH fails during storage of rice, potentially mycotoxigenic molds may grow. Aspergillus spp. and Penicillium spp. can grow at 85% RH (aw 0.85) (Magan and Lacey, 1984; Manabe and Tsuruta, 1991) and produce various mycotoxins. Aflatoxins and ochratoxin A (OTA) are the most extensively studied mycotoxins produced by these molds and have been detected on rice in many countries around the world. Park et al. (2005) studied the incidence of fungal infections and natural occurrence of mycotoxins in Korean polished rice. Aspergillus spp., Penicillium spp., and Fusarium spp. were isolated from 7e26%, 11e 27%, and 7e16% of 88 of the samples tested. They detected aflatoxin B1 in 5 of 88 polished rice samples with a mean concentration of 4.3 ng/g; OTA was detected in 8 of 88 samples with a mean concentration of 3.9 ng/g. They also detected fumonisin B1, deoxynivalenol, nivalenol, and zearalenone in Korean polished rice. Sales and Yoshizawa (2005b) reported that the mean concentrations of aflatoxin in brown rice and polished rice produced in the Philippines were 2.7 and 0.4 mg/kg, respectively. An investigation of rice produced in Nigeria showed that aflatoxin B1 was present in 97 of 196 samples and OTA was present in 56 of 140 samples (Makun et al., 2007). The average concentrations of aflatoxin B1 and OTA detected were 200 and 156 mg/kg, respectively. Our study showing that milled rice, particularly brown rice, stored at 12 or 21 C and 85% RH can support the growth of molds emphasizes the importance of storing milled rice at temperatures and RHs that will prevent mycotoxin production. We investigated the growth and survival of MAB, B. cereus, and MY separately. It would be interesting to study the influence riceborne microbiota on survival and growth of B. cereus. The presence of some microorganisms may influence the survival and growth of others. Cuero et al. (1987), for example, demonstrated that the growth of Aspergillus flavus on rice (aw 0.95 and 0.98) at 16 C is stimulated by the presence of Hyphopichia burtonii and Bacillus amyloliquefaciens. Tandon et al. (1979) showed Curvularia lunata and Chaetomium indicum suppressed the growth of seedborne microbiota. In summary, populations of MAB and MY present on rice decreased by ca. 2 log CFU/g during the milling process. Populations of MAB and B. cereus on rice either remained constant for 24 wk or decreased slightly, regardless of the degree of milling, temperature or RH. The MY population on rice generally increased at 85% RH, regardless of the degree of milling and storage temperature. MY grew on brown rice at 12 C and 85% RH, and on all types of rice at 21 C and 85% RH. Further investigation is needed to define conditions affecting the growth of mycotoxigenic molds and their mycotoxin production on rice during storage as affected by degree of milling, temperature, and RH. Acknowledgments This study was conducted with the support of the Korea Food Research Institute (project no. E132501). We thank the Korea University Food Safety Center and the Institute of Food and Biomedicine Safety for providing resources and facilities. References Bainotti, A.E., Parra, E.S.P., 2000. Microbiological evaluation of processed rice consumed in Japan. World J. Microbiol. Biotechnol. 16, 77e79. Batt, C.A., 1999. BACILLUS/Bacillus cereus. In: Batt, C.A., Patel, P., Robinson, R.K. (Eds.), Encyclopedia of Food Microbiology. Academic Press, San Diego, pp. 119e124. Beuchat, L.R., Komitopoulou, E., Beckers, H., Betts, R.P., Bourdichon, F., Fanning, S., Joosten, H.M., Ter Kuile, B.H., 2013. Low-water activity foods: increased concern as vehicles of foodborne pathogens. J. Food Prot. 76, 150e172. Cottyn, B., Regalado, E., Lanoot, B., Cleene, M.D., Mew, T.W., Swings, J., 2001. Bacterial populations associated with rice seed in the tropical environment. Phytopathology 91, 282e292.
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