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Behaviour of Aspergillus flavus and Fusarium graminearum on rice as affected by degree of milling, temperature, and relative humidity during storage
Food Microbiology 46 (2015) 307e313
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Behaviour of Aspergillus flavus and Fusarium graminearum on rice as affected by degree of milling, temperature, and relative humidity during storage Seonyeong Choi a, Hyejung Jun a, Jihyun Bang a, Soo-Hyun Chung b, Yoonsook Kim c, Byeong-sam Kim c, Hoikyung Kim d, Larry R. Beuchat e, Jee-Hoon Ryu a, * a
Department of Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Department of Food and Nutrition, Korea University, Jeongneung-dong, Sungbuk-ku, Seoul 136-703, Republic of Korea Neo Food Resources Research Group, Korea Food Research Institute, Baekhyun-dong, Seongnam, Gyeonggi 463-746, Republic of Korea d Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea e 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 1 April 2014 Received in revised form 30 July 2014 Accepted 23 August 2014 Available online 2 September 2014
We investigated the survival and growth patterns of Aspergillus flavus and Fusarium graminearum, as well as mycotoxin production, on Korean rice as affected by the degree of milling (rough, brown, and white rice) and storage conditions (21 C/85% relative humidity [RH], 21 C/97% RH, and 30 C/85% RH). When rice was stored at 21 C/85% RH, the population of A. flavus remained constant and aflatoxin was not produced, regardless of the degree of milling. At 21 C/97% RH and 30 C/85% RH, the populations of A. flavus increased significantly (P 0.05) and aflatoxins were produced. The highest population of A. flavus and highest amount of aflatoxin B1 were observed on brown rice stored at 21 C/97% RH. For F. graminearum, when stored at 85% RH, the populations were reduced to less than a detectable level (5 CFU/g of rice) within 120 days and no deoxynivalenol (DON) was produced, regardless of the degree of milling and storage temperature. However, at 21 C/97% RH, the population of F. graminearum increased significantly (P 0.05) and DON was produced on all types of rice. Findings from this study provide insights concerning storage conditions necessary to prevent growth and mycotoxin production by A. flavus and F. graminearum on Korean rice with different degrees of milling. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Rice Aspergillus flavus Fusarium graminearum Milling Relative humidity Temperature
1. Introduction Rice (Oryza sativa L.) is a staple food for more than half of the global population (FAO, 2004a) and provides 20% of the dietary energy supply in the world (FAO, 2004b). According to the Food and Agriculture Organization of the United Nations, world rice production has increased steadily in recent years. Production increased from ca. 518 million tons in 1990 to ca. 599 million tons in 2000 and ca. 701 million tons in 2010 (FAOSTAT, 2013). Rice is stored for several months or even years as rough rice after harvesting, and brown rice or white rice after milling. Rough rice consists of the hull, bran, and endosperm. Rough rice is dehulled to produce brown rice and the bran layer of brown rice is removed to produce white rice (Skyrme et al., 1998). Rice is usually consumed in the
* Corresponding author. Tel.: þ82 2 3290 3409; fax: þ 82 2 3290 3918. E-mail address: [email protected] (J.-H. Ryu). http://dx.doi.org/10.1016/j.fm.2014.08.019 0740-0020/© 2014 Elsevier Ltd. All rights reserved.
latter form, but demands for brown rice have increased because of its high nutritional value (FAO, 2004b; Heinemann et al., 2005). As rice is an essential part of the human diet of many people, it is important to maintain its sensorial, nutritional, and microbiological qualities. The growth of some fungal species on rice, with consequent mycotoxin production, results in a microbiological safety concern (Kumar et al., 2008; Reddy et al., 2009). The major genera of fungi found on rice are Aspergillus, Penicillium, Fusarium, Alternaria, Mucor, Rhizopus, Trichoderma, Curvularia, Helminthosporium, and Cladosporium (Makun et al., 2007). Among them, Aspergillus flavus and Fusarium graminearum raise particular concern because some strains can produce mycotoxins. Contamination of rice with mycotoxigenic fungi and the presence of mycotoxins have been reported in several countries (Desjardins et al., 1997; Fredlund et al., 2009; Makun et al., 2007; Ok et al., 2009; Tanaka et al., 1988). Fredlund et al. (2009) reported that 21% of rice samples collected from Swedish retail markets were contaminated with A. flavus. Total aflatoxin in contaminated rice was as high as 50.7 mg/kg Park
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et al. (2005) reported that 17% and 10% of polished rice samples obtained from grain wholesale markets in the Republic of Korea were contaminated with A. flavus and F. graminearum, respectively; the mean aflatoxin B1 (AFB1) and deoxynivalenol (DON) concentrations in rice were 4.3 and 139 ng/g, respectively. A. flavus is a mycotoxigenic fungus that produces AFB1 and aflatoxin B2 (AFB2). AFB1 is classified as an International Agency for Research on Cancer (IARC) Group 1 human carcinogen (IARC, 1993). The optimum temperatures for growth and aflatoxin production are ca. 33 C and 16e31 C, respectively, and the optimum water activities (aw) for growth and aflatoxin production are 0.98 and 0.95e0.99, respectively (ICMSF, 1996). An outbreak of 397 cases of hepatitis with 106 deaths occurred in India in 1974. It was concluded that this outbreak was associated with the consumption of maize heavily contaminated with A. flavus and containing aflatoxin of concentrations up to 15.6 mg/g (Krishnamachari et al., 1975). In Kenya, 317 people became ill after eating maize contaminated with aflatoxin; 125 deaths occurred in this outbreak (CDC, 2004). The government of the Republic of Korea has set limits of 15 ng/g and 10 ng/g for total aflatoxins and AFB1, respectively, in cereal grains (Korean Ministry of Food and Drug Safety, 2013). In the USA, the limit for total aflatoxins in food is 20 ng/g, but no limit has been specifically set for AFB1 (FAO, 2003). In the EU, maximum concentrations of 10 ng/g for total aflatoxins and 5 ng/g for AFB1 have been established in rice (European Commission, 2010). F. graminearum is a plant pathogen that can produce DON, nivalenol, and zearalenone (Pitt and Hocking, 2009). Growth of F. graminearum is most rapid at 25 C and aw 0.95e0.995, and DON production is highest at 25 C and aw 0.995 (Ramirez et al., 2006). DON is classified by the IARC as a Group 3 agent (not classifiable as to carcinogenicity in humans) (IARC, 1993). However, consumption of food contaminated with DON may result in nausea, vomiting, gastrointestinal upset, dizziness, diarrhoea, and headache (JECFA, 2002). An outbreak has been reported in India in 1987, in which 97 people became sick after eating wheat bread contaminated with DON and other trichothecene mycotoxins (Bhat et al., 1989). The limit for DON in grains in the Republic of Korea is 1000 ng/g (Korean Ministry of Food and Drug Safety, 2013). In the USA, the limit of DON in finished wheat products for human consumption is 1000 ng/g (FDA, 2010). The EU has set limits for DON in unprocessed cereal (1250 ng/g) and in cereals for direct human consumption (750 ng/g) (European Commission, 2006). Although there have been no documented outbreaks of foodborne intoxication associated with the consumption of rice containing aflatoxin or DON, there is a potential risk of mycotoxin production in rice contaminated with A. flavus or F. graminearum and stored at abusive temperature and relative humidity (RH) conditions. In countries such as the Republic of Korea, where rice is a staple food and the climate is changing from temperate to subtropical due to global warming, potential risks of human illness associated with consumption of rice and other grains containing mycotoxins are likely to increase. Mycotoxins are difficult to eliminate in foods without compromising sensorial and nutritional quality because they are heat-resistant and do not decompose readily (Korzun, 2002; Shapira and Paster, 2004). It is important to prevent contamination of rice with mycotoxigenic fungi and store rice under conditions that will prevent growth mycotoxin production. The growth of fungi and production of mycotoxins in rice and other grains are affected by the availability of nutrients and environmental conditions, such as temperature, aw, and pH (Holmquist et al., 1983; Llorens et al., 2004; Mylona et al., 2012; Ramirez et al., 2006; Sweeney and Dobson, 1998). There have been a number of reports describing conditions affecting growth of fungi and production of mycotoxins affected by storage temperature and aw. However, none has addressed the
patterns of fungal growth and mycotoxin production in rice as affected by different degrees of milling and long-term storage under various temperatures and RH. The study reported here was done to determine the patterns of growth and survival of A. flavus and F. graminearum, as well as mycotoxin production, on rice as affected by the degree of milling, temperature, RH, and storage time. 2. Materials and methods 2.1. A. flavus strains and preparation of inocula Five strains of A. flavus known to produce AFB1 and AFB2 were used in this study: A. flavus strain ATCC 22546 (isolated from mouldy corn), CN 008 (isolated from malted wheat), CN 028 (isolated from malted wheat [nuruk]), CN 029 (isolated from malted wheat [nuruk]), and M 2034 (isolated from fermented soybeans [meju]). These strains were chosen because they had been isolated from grain-based products and their abilities to produce mycotoxin had been confirmed. A. flavus strain ATCC 22546 was obtained from Korean Culture Center of Microorganisms in Seoul, Republic of Korea, and strains CN 008, CN 028, CN 029, and M 2034 were supplied by Dr. Soo-Hyun Chung (Department of Food and Nutrition, Korea University, Seoul, Republic of Korea). Each strain was streaked separately onto potato dextrose agar (PDA; BBL/Difco, Sparks, MD, USA) supplemented with 10% tartaric acid (PDAT; pH 3.5) slants and incubated at 28 C for 7e10 days. Spores were harvested by depositing 10 mL of distilled water (DW) containing 0.02% Tween 80 (Junsei, Tokyo, Japan) on the mat surface, gently rubbing with a sterile loop, and filtering the suspension through sterile cheesecloth. Spore counts (spores/mL) were determined using a haemocytometer. More than 98% of the propagules were spores. A five-strain cocktail of A. flavus spores was prepared by combining suspensions of each of the five strains (2 106 spore per strain). The suspension was centrifuged at 20,000 g for 15 min. The supernatant was decanted and spores were resuspended in sterile DW to give ca. 6.3 log spores/mL. 2.2. F. graminearum strains and preparation of inocula Five strains of F. graminearum known to produce DON were used in this study: F. graminearum strains KACC 46434 (isolated from rice), KACC 46437 (isolated from barley), KACC 46438 (isolated from barley), KACC 46439 (isolated from barley), and KACC 46441 (isolated from rice). These strains were chosen because they had been isolated from grain and their abilities to produce DON had been confirmed. All strains were obtained from the Rural Development Administration-Genebank Information Center, Suwon, Republic of Korea. Each strain was inoculated separately on the PDAT plates and incubated at 28 C for 7 days. Five agar plugs (ca. 6 mm diam.) from the edges of colonies of each strain were deposited in 100 mL of carboxymethyl cellulose (CMC) broth (ammonium nitrate [NH4NO3], 1 g; monopotassium phosphate [KH2PO4], 1 g; magnesium sulphate heptahydrate [MgSO4∙7H2O], 0.5 g; yeast extract, 1 g; carboxylmethyl cellulose, 15 g; DW, 1 L) and incubated on a rotary shaker at 200 rpm for 5 days at 25 C. The CMC culture was filtered through sterile cheesecloth and spore counts for each strain were determined with a haemocytometer. More than 98% of the propagules were spores. A five-strain cocktail of F. graminearum spores was prepared by combining suspensions of each of the five strains (2 106 spore per strain). The suspension was centrifuged at 20,000 g for 15 min. The supernatant was decanted and the pellet was resuspended in sterile DW to give ca. 6.3 log spores/mL.
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2.3. Creation of relative humidity To create atmospheres with 85% or 97% RH, 200 mL of saturated potassium chloride (Daejung, Siheung, Republic of Korea) or potassium sulphate (Daejung) solution, respectively, was added to an airtight container (1.2 L, 155 mm long 155 mm wide 87 mm high; Lock & Lock, Seoul, Republic of Korea) and stored at 21 or 30 C for at least 24 h before use in the experiments. 2.4. Inoculation and storage Three types of rice (O. sativa L. ssp. japonica; produced in Jincheon, Republic of Korea) were provided by the Korea Food Research Institute (Seongnam, Gyeonggi, Republic of Korea): rough rice, brown rice, and white rice. Rough rice was collected in October and November 2011 and milled at a rice processing complex to obtain brown rice and white rice. Rice was subjected to gamma irradiation (15 kGy; Greenpia Technology, Yeoju, Republic of Korea) before use in the experiments. It was confirmed that there were no viable cells of bacteria or moulds in the irradiated rice samples by spread-plating and enriching the samples on PDA and in tryptic soy broth (TSB; BBL/Difco), respectively. Gamma-irradiated rough rice, brown rice, or white rice (10 g) was placed in a sterile Petri dish (60mm diameter 15-mm depth; SPL, Pocheon, Republic of Korea) and 50 mL of inoculum (A. flavus or F. graminearum cocktail) prepared as described above was spot-inoculated at five randomly selected locations on the rice. Open Petri dishes containing 10 g of inoculated rough rice, brown rice, or white rice (two Petri dishes per type of rice in each of three replicate trials) were placed above the surface of saturated potassium chloride or potassium sulphate solutions in an airtight container and incubated at 21 C/85% RH, 21 C/97% RH, or 30 C/ 85% RH for up to 120 days. In total, more than 180 airtight containers were prepared. 2.5. Microbiological analysis After storing inoculated rice for 0, 10, 20, 30, 40, 50, 60, 80, 100, and 120 days, populations of A. flavus or F. graminearum were determined. At each sampling time, 10 g of rice were placed in a stomacher bag (BA 6040 standard bags; Seward, West Sussex, UK) containing 50 mL of potato dextrose broth (PDB; BBL/Difco) and pummelled for 2 min in a stomacher (Interscience BagMixer® 400W; Interscience, St. Nom, France). The homogenate was serially diluted in 0.1% peptone water. Duplicate 0.1-mL samples (diluted or undiluted homogenate) or quadruplicate 0.25-mL samples (undiluted homogenate) were spread plated on PDAT. The PDAT plates for enumerating A. flavus were incubated at 28 C for 2 days, while those for F. graminearum were incubated at 25 C for 2 days before counting colonies. The remaining F. graminearum homogenate was incubated at 25 C for up to 5 days to enrich viable cells of F. graminearum. When colonies of F. graminearum did not form on PDAT, the enriched homogenates (1 mL) were spread on PDAT and incubated at 25 C for 2 days. The limits of detection were 0.7 log CFU/g of rice (5 CFU/g of rice) by direct plating, and 1.0 log CFU/g (1 CFU/10 g) of rice by enrichment. 2.6. Aflatoxin analysis The amounts of AFB1 and AFB2 produced in rough rice, brown rice, and white rice stored at various temperatures and RHs for 0, 10, 20, 30, 40, 50, 60, 80, 100, and 120 were determined. Aflatoxin content was determined by a method described in the Korean Food Standards Codex (Korean Ministry of Food and Drug Safety, 2013), with a slight modification. Briefly, rough rice, brown rice, or white
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rice (10 g) was ground in a blender, an extractant (40 mL: 1 g of sodium chloride, 30 mL of DW, and 70 mL of methanol) was added, and the mixture was blended at high speed for 1 min. The homogenate was filtered through Whatman grade no. 1 filter paper and 10 mL of filtrate were mixed with 30 mL of 1% Tween 20 (Junsei) in DW. The mixture was filtered through Whatman grade GF/A. Filtered extract (10 mL) was cleaned up using an immunoaffinity column (IAC) (AflaTest; VICAM, Watertown, MA, USA) at a rate of approximately 1 drop/s until air came through the column. Then, 10 mL of DW were passed through the column at the same flow rate until air came through the column. Aflatoxins were eluted by passing 1.5 mL of high-performance liquid chromatography (HPLC)egrade methanol through the column at a rate of ca. 1 drop/s and collected in a glass vial. The eluate (1.5 mL) was evaporated to dryness under a stream of nitrogen gas at 50 C using a nitrogen concentrator (EYELA MGS-2200; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The residue was dissolved in 0.5 mL of derivatisation solution consisting of 9 mL of 10% acetonitrile (ACN) and 1 mL of trifluoroacetic acid (CHROMASOLV®; SigmaeAldrich, St. Louis, MO, USA) and kept in the dark for at least 3 h. Derivatised aflatoxins were filtered through 0.45-mm syringe filters. Quantification of aflatoxins was performed by HPLC (Waters, Milford, MA, USA). Aflatoxins were separated using Nova-Pak C18 columns (3.9 150 mm, 4-mm particle size; Waters) and detected using a fluorescence detector (Waters 2475; Waters) at an excitation wavelength of 360 nm and emission wavelength of 430 nm. Samples (50 mL) were injected and eluted with a mobile phase (DW:methanol:ACN [660:170:170, v/v/v]) at a flow rate of 0.5 mL/min. The limits of detection of AFB1 and AFB2 were 0.1 and 0.03 ng/g, respectively. 2.7. Deoxynivalenol analysis The amounts of DON produced in rough rice, brown rice, and white rice inoculated with F. graminearum and stored at various temperatures and RHs for 0, 10, 20, 30, 40, 50, 60, 80, 100, and 120 days were determined. DON content was determined using a modification of the method described in the VICAM DONtest HPLC Instruction Manual (Vicam, 2005). Rough rice, brown rice, or white rice (10 g) were ground in a blender, 2 g of polyethylene glycol 8000 (Yakuri Pure Chemicals, Kyoto, Japan) and 40 mL of DW were added, and the mixture was blended at high speed for 1 min. The homogenate was filtered sequentially through a Whatman grade no. 4 filter and a Whatman grade GF/A filter to extract DON. The filtered extract (1 mL) was cleaned up by passing through IAC (DONtest; VICAM) at a rate of approximately 1 drop/s until air came through the column. Then, 5 mL of DW were passed through the column at the same flow rate until air came through the column. DON was eluted by passing 1.5 mL of HPLC-grade methanol through the column at a rate of about 1 drop/s and collected in a glass vial. The eluate (1.5 mL) was evaporated to dryness under a stream of nitrogen gas at 45 C using a nitrogen concentrator (EYELA MGS2200; Tokyo Rikakikai Co., Ltd.). The residue was dissolved in 0.5 mL of 10% ACN and filtered through a 0.45-mm syringe filter. Quantification of DON was performed by HPLC. DON was separated using a Nova-Pak C18 column (3.9 150 mm, 4-mm particle size; Waters) and detected using a photodiode array detector (Waters 2996; Waters) at 220 nm. Samples (50 mL) were injected and eluted with a mobile phase consisting of 10% ACN at a flow rate of 0.5 mL/ min. The detection limit of DON was 30 ng/g. 2.8. Statistical analysis All experiments were performed in triplicate. Data were analysed using the general linear model of the Statistical Analysis System procedure (SAS 9.3; SAS Institute, Cary, NC, USA). The
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effects of the degree of milling of rice and storage time on populations of A. flavus and F. graminearum as well as the amounts of mycotoxins produced on rice were analysed using Fisher's least significant difference test. In all analyses, P 0.05 was taken to indicate statistical significance. 3. Results 3.1. Growth of A. flavus and production of aflatoxin in rice as affected by the degree of milling, temperature, and RH Fig. 1 shows populations of A. flavus in inoculated rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. The initial populations of A. flavus in rough, brown, and white rice samples were 3.3e3.8 log CFU/g of rice. At 21 C/85% RH, populations in rough rice and brown rice did not change significantly (P > 0.05) during storage for 120 days. The number of A. flavus significantly decreased by 0.6 log CFU/g in white rice stored for 120 days. When rice was stored at 21 C/97% RH, A. flavus increased in rough rice, brown rice, and white rice to 5.9, 7.3, and 6.7 log CFU/g, respectively, within 20 days. Populations remained constant for an additional 100 days, regardless of the degree of milling. The number of A. flavus detected in brown rice was significantly higher than that in rough rice and white rice by 0.8e1.0 log CFU/g after 120 days (P 0.05). At 30 C/85% RH, A. flavus increased significantly (P 0.05), regardless of the degree of milling during storage. After 120 days, populations in rough rice, brown rice, and white rice increased by 2.6, 3.2, and 2.9 log CFU/g, respectively, compared to the initial populations. Table 1 shows the concentrations of AFB1 detected in rough rice, brown rice, and white rice inoculated with A. flavus and stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. In non-inoculated rough rice, brown rice, and white rice, 0.27, 0.13, and 0.30 ng/g of AFB1 were detected, respectively (data not shown). When rice was stored at 21 C/85% RH for 0 and 10 days, trace
▫
amounts of AFB1 were detected, regardless of the degree of milling; AFB1 was not detected in rice stored for more than 10 days. At 21 C/ 97% RH, AFB1 was detected in all three types of rice stored for 20e120 days. The maximum levels of AFB1 detected in rough rice, brown rice, and white rice were 140.9, 491.0, and 144.9 ng/g, respectively. At 30 C/85% RH, AFB1 was detected in rough rice and brown rice after storage for 40 days and reached levels up to 96.7 and 203.2 ng/g, respectively, after 120 days. On white rice, however, AFB1 was detected after storage for 60 days and reached 1.8 ng/g after 120 days. Table 2 shows the concentrations of AFB2 detected in rough rice, brown rice, and white rice inoculated with A. flavus when stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. In non-inoculated rough rice, brown rice, and white rice, AFB2 was not detected (data not shown). At 21 C/85% RH, AFB2 was not detected, regardless of the degree of milling. At 21 C/97% RH, AFB2 was detected in rough, brown, and white rice stored for 30 days or longer, and reached levels up to 7.4, 2.6, and 2.4 ng/g, respectively, within 120 days. AFB2 was detected in rough rice and brown rice stored at 30 C/85% RH for 40 days, reaching levels of 3.0 and 1.9 ng/ g after 50 and 120 days, respectively. In white rice, a trace amount (0.5 ng/g) of AFB2 was detected after 120 days. 3.2. Growth of F. graminearum and DON production in rice as affected by the degree of milling, temperature, and RH Fig. 2 shows populations of F. graminearum in inoculated rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. The initial population of F. graminearum in rice was 2.3e3.8 log CFU/g. When rice was stored at 21 C/85% RH, F. graminearum decreased to an undetectable level by enrichment (<1 CFU/10 g) in all types of rice over time during the 120-day storage period. At 21 C/97% RH, populations in rough rice, brown rice, and white rice increased significantly (P 0.05) to 4.4, 4.2, and 4.0 log CFU/g after 120 days. At 30 C/85% RH,
Fig. 1. Numbers of viable cells of A. flavus in rough rice (B), brown rice ( ) and white rice (D) stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Error bars indicate standard deviations across replicated experiments. The detection limit ($$$$$$) by direct plating was 0.7 log CFU/g rice (5 CFU/g rice).
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Table 1 Concentrations of aflatoxin B1 (ng/g rice) in rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Storage conditions
Type of rice
Mean concentrations of aflatoxin B1 (ng/g rice) after the indicated storage time (days)a 0
21 C/85% RH
21 C/97% RH
30 C/85% RH
Rough Brown White Rough Brown White Rough Brown White
10
A 0.9 a A 0.6 a A 0.6 a ND ND ND ND ND ND
20
A 0.1 b A 0.1 a A 0.1 a ND ND ND ND ND ND
30 b
ND ND ND C 4.5 a A 318.3 c B 144.9 a ND ND ND
c
e e e A 24.4 a A 183.2 d A 56.5 b ND ND ND
40
50
60
80
100
120
e e e B 128.4 a A 445.6 ab B 14.8 bc A 18.5 a A 18.7 b ND
ND ND ND B 83.1 a A 466.3 a B 1.7 c A 18.4 a A 107.2 ab ND
e e e B 9.0 a A 100.0 d B 6.4 bc A 1.5 a A 14.2 b A 1.1 ab
ND ND ND B 140.9 a A 491.0 a B 5.2 bc A 1.3 a A 47.2 b A 0.2 b
e e e AB 115.3 a A 350.0 c B 5.2 bc A 0.1 a A 58.1 ab A 0.2 b
ND ND ND B 7.5 a A 361.8 bc B 4.8 bc A 96.7 a A 203.2 a A 1.8 a
a Comparison of the effects of storage time: values in the same row that are not followed by the same lowercase letter are significantly different (P 0.05). Comparison of the effects of type of rice: under the same storage conditions, values in the same column that are not preceded by the same uppercase letter are significantly different (P 0.05). b ND, not detected (<0.1 ng/g). c e, not determined.
F. graminearum decreased to undetectable levels in rough, brown, and white rice within 50, 80, and 50 days, respectively. Table 3 shows the concentrations of DON detected in rough rice, brown rice, and white rice inoculated with F. graminearum and stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. DON was not detected in all types of non-inoculated rice (data not shown). At 21 C/85% RH, DON was not detected in brown rice or white rice throughout storage, but was detected at 76.3 and 134.6 ng/g in rough rice after 80 and 120 days, respectively. DON was detected on all three types of rice stored at 21 C/97% RH. In rough rice, 154.3 ng/g were detected after storage for 50 days and a maximum level (307.6 ng/g) was detected after 60 days. In brown rice, DON was detected after storage for 40 days, and reached 954.4 ng/g after 50 days. DON was not detected in white rice stored for less than 80 days, and reached 174.7 ng/g at 100 days. DON was not detected in rice stored at 30 C/85% RH for up to 120 days. 4. Discussion As expected, growth of A. flavus and production of aflatoxins were affected by storage temperature and RH. These observations are in agreement with findings of Niles et al. (1985), who reported that A. flavus did not grow on wheat during storage at 20 C and 0.85 aw for 30 days. However, Mousa et al. (2013) reported that A. flavus could grow in rice held under similar conditions. They reported that the A. flavus showed radial growth (0.1e0.2 mm/day) in brown and polished rice (aw 0.84 and 0.86) stored at 20 C with for 5 weeks but did not produce aflatoxins. Growth of A. flavus and production of aflatoxins at 21 C/97% RH were affected markedly by the degree of milling of rice. Higher populations and AFB1 concentrations were observed in brown rice,
compared to rough rice and white rice. These results imply that AFB1 may be produced in greater amounts in naturally contaminated brown rice than in rough or white rice. The higher levels of aflatoxins in brown rice than in rough rice probably result in part from physical protection by the hull surrounding the rice against invasion of A. flavus, whereas brown rice with hull removed can be easily colonized (Juliano and Bechtel, 1985). Other researchers have reported that greater amounts of AFB1 were found in brown rice than in white rice. Sales and Yoshizawa (2005) investigated the level of aflatoxins in naturally contaminated rice. They found that brown rice (2.7 mg/kg) had significantly higher amounts of aflatoxins than did polished rice (0.37 mg/kg). Takahashi et al. (1989) examined the location of aflatoxin in brown rice artificially inoculated with A. flavus and stored at about 85e90% RH and 28 C for up to 15 days. They observed that considerably higher concentrations of AFB1 were detected on bran layer (8930 ng/g) than in the starchy endosperm portion (250 ng/g). Aflatoxin was present near the invading A. flavus mycelia, which were present mainly in the caryopsis coat, aleurone layer, germ, and outer starchy endosperm. In our study, AFB1 was produced at much higher levels than AFB2. This observation is in agreement with studies of Shotwell et al. (1966) and Taber and Schroeder (1967). Shotwell et al. (1966) reported that the ratio of AFB1 and AFB2 produced on polished rice was approximately 6:1, while Taber and Schroeder (1967) reported a ratio of AFB1 to AFB2 of about 10:1 on rough rice. In our study, approximate ratios of the amounts of AFB1 and AFB2 produced in rough, brown, and white rice after 120 days were 8:1, 170:1, and 9:1, respectively, when rice was stored at 21 C/97% RH. Growth of F. graminearum (Fig. 2) and production of DON (Table 3) were unaffected by the degree of milling of rice but were
Table 2 Concentrations of aflatoxin B2 (ng/g rice) in rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Storage conditions
Type of rice
Mean concentrations of aflatoxin B2 (ng/g rice) after the indicated storage time (days)a 0
10
20
30
40
50
60
80
100
120
21 C/85% RH
Rough Brown White Rough Brown White Rough Brown White
NDb ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
ec e e A 1.1 b A 1.8 b A 2.4 a ND ND ND
e e e A 1.0 A 1.9 A 1.4 A 2.5 A 1.8 ND
ND ND ND A 5.8 ab AB 1.9 b B 0.2 a A 3.0 a A 0.7 a ND
e e e A 1.4 A 2.2 A 1.0 A 0.1 A 1.3 ND
ND ND ND A 7.4 A 2.6 A 0.6 A 0.3 A 0.4 ND
e e e A 1.6 ab A 2.3 ab A 0.8 a ND 0.4 a ND
ND ND ND B 0.9 b A 2.1 ab B 0.5 a A 1.3 a A 1.9 a A 0.5
21 C/97% RH
30 C/85% RH
b b a a a
b ab a a a
a a a a a
a Comparison of the effects of storage time: values in the same row that are not followed by the same lowercase letter are significantly different (P 0.05). Comparison of the effects of type of rice: under the same storage conditions, values in the same column that are not preceded by the same uppercase letter are significantly different (P 0.05). b ND, not detected (<0.03 ng/g). c e, not determined.
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▫
Fig. 2. Numbers of viable cells of F. graminearum in rough rice (B), brown rice ( ) and white rice (D) stored at 21 C/85% RH, 21 C/97% RH and 30 C/85% RH for up to 120 days. Error bars indicate standard deviations across replicated experiments. The detection limit ($$$$$$) by direct plating was 0.7 log CFU/g rice (5 CFU/g rice). The detection limit by enrichment was 1.0 log CFU/g (1 CFU/10 g). To plot the numbers of F. graminearum on rice, the number of cells was considered as 0.7 log CFU/g when cells were not detected on agar plates but detected after enrichment. If cells were not detected after enrichment, it was considered as 1.0 log CFU/g.
Table 3 Concentrations of deoxynivalenol (ng/g rice) in rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Storage conditions
Type of rice
Mean concentrations of deoxynivalenol (ng/g rice) after indicated storage time (days)a 0
21 C/85% RH
21 C/97% RH
30 C/85% RH
Rough Brown White Rough Brown White Rough Brown White
10 b
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
20 ND ND ND ND ND ND ND ND ND
30 c
e e e ND ND ND e e e
40
50
60
80
100
120
e e e ND 244.5 b ND e e e
ND ND ND A 154.3 a A 954.4 a ND ND ND ND
e e e A 307.6 a A 92.2 b ND e e e
76.3 a ND ND A 54.0 a A 551.7 ab A 164.3 a ND ND ND
e e e A 141.8 a A 37.9 b A 174.7 a e e e
134.6 a ND ND A 11.2 a A 107.3 b A 51.9 a ND ND ND
a Comparison of the effects of storage time: values in the same row that are not followed by the same lowercase letter are significantly different (P 0.05). Comparison of the effects of type of rice: under the same storage conditions, values in the same column that are not preceded by the same uppercase letter are significantly different (P 0.05). b ND, not detected (<30 ng/g). c e, not determined.
enhanced by increased RH. Hope et al. (2005) determined the effects of aw (0.99e0.85) and temperature (15 and 25 C) on growth and production of DON by F. graminearum on wheat grain. They reported that mycelial growth did not occur at aw 0.85, regardless of temperature, and DON was not produced at aw 0.93. Sanchis and Magan (2004) reported that F. graminearum did not produce DON on wheat at aw 0.93. Cuero et al. (1987) reported that F. graminearum could not grow on cracked irradiated rice at aw less than 0.90. These studies indicate that growth of F. graminearum and production of DON can be effectively suppressed by lowering RH below 85% during storage. In our study, F. graminearum was inactivated at 85% RH more rapidly at 30 C than at 21 C. The amount of DON produced on rice was less than the maximum level of 1000 ng/ g set for grains by the Korean Ministry of Food and Drug Safety and for wheat set by the FDA. In summary, we observed that A. flavus cannot grow or produce aflatoxins in rice at 21 C/85% RH, regardless of the degree of
milling. At 21 C/97% RH and 30 C/85% RH, however, A. flavus was able to grow and produce aflatoxins. The population of A. flavus and the AFB1 concentration in brown rice was significantly (P 0.05) greater than that in rough rice or white rice stored at 21 C/97% RH. F. graminearum did not survive or produce DON on rice stored at 21 C/85% RH or 30 C/85%, regardless of the degree of milling. At 21 C/97% RH, F. graminearum was able to grow and produce DON. Findings from this study provide information useful when developing interventions to enhance the microbiological safety of rough, brown, and white rice by controlling temperature and RH during storage, thereby preventing growth of A. flavus and F. graminearum and production of mycotoxins. Acknowledgements This study was conducted with the support of the Korea Food Research Institute (project no. E132501) and a Korea University
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Grant. We thank the Institute of Control Agents for Microorganisms at Korea University for providing resources and facilities. References Bhat, R.V., Beedu, S.R., Ramakrishna, Y., Munshi, K.L., 1989. Outbreak of trichothecene mycotoxicosis associated with consumption of mould-damaged wheat products in Kashmir Valley, India. Lancet 333, 35e37. CDC (Centers for Disease Control and Prevention), 2004. Outbreak of aflatoxin poisoning e eastern and central provinces, Kenya, January e July 2004. Morb. Mortal. Wkly. Rep. 53, 790e793. Cuero, R.G., Smith, J.E., Lacey, J., 1987. Interaction of water activity, temperature and substrate on mycotoxin production by Aspergillus flavus, Penicillium viridicatum and Fusarium greaminearum in irradiated grains. Trans. Br. Mycol. Soc. 89, 221e226. Desjardins, A.E., Plattner, R.D., Nelson, P.E., 1997. Production of fumonisin B1 and moniliformin by Gibberella fujikuroi from rice from various geographic areas. Appl. Environ. Microbiol. 63, 1838e1842. European Commission, 2006. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. Union L345, 5e24. European Commission, 2010. Commission Regulation (EU) No 165/2010 of 26 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins. Off. J. Eur. Union L50, 8e12. FAO (Food and Agriculture Organization of the United Nations), 2003. Worldwide Regulations for Mycotoxins in Food and Feed in 2003. Retrieved from. ftp://ftp. fao.org/docrep/fao/007/y5499e/y5499e00.pdf. FAO (Food and Agriculture Organization of the United Nations), 2004a. International Year of Rice 2004: Rice and Us. Retrieved from. http://www.fao.org/ rice2004/en/rice-us.htm. FAO (Food and Agriculture Organization of the United Nations), 2004b. Rice and Human Nutrition. Retrieved from. http://www.fao.org/rice2004/en/f-sheet/ factsheet3.pdf. FAOSTAT (Food and Agriculture Organization of the United Nations Statistics), 2013. Retrieved from. http://faostat.fao.org/site/291/default.aspx. FDA (Food and Drug Administration), 2010. Guidance for Industry and FDA: Advisory Levels for Deoxynivalenol (DON) in Finished Wheat Products for Human Consumption and Grains and Grain By-products Used for Animal Feed. Retrieved from. http://www.fda.gov/downloads/Food/GuidanceRegulation/ UCM217558.pdf. Fredlund, E., Thim, A.-M., Gidlund, A., Brostedt, S., Nyberg, M., Olsen, M., 2009. Moulds and mycotoxins in rice from the Swedish retail market. Food Addit. Contam. 26, 527e533. Heinemann, R.J.B., Fagundes, P.L., Pinto, E.A., Penteado, M.V.C., LanferMarquez, U.M., 2005. Comparative study of nutrient composition of commercial brown, parboiled and milled rice from Brazil. J. Food Compos. Anal. 18, 287e296. Holmquist, G.U., Walker, H.W., Stahr, H.M., 1983. Influence of temperature, pH, water activity and antifungal agents on growth of Aspergillus flavus and A. parasiticus. J. Food Sci. 48, 778e782. Hope, R., Aldred, D., Magan, N., 2005. Comparison of environmental profiles for growth and deoxynivalenol production by Fusarium culmorum and F. graminearum on wheat grain. Lett. Appl. Microbiol. 40, 295e300. IARC (International Agency for Research on Cancer), 1993. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Human, vol. 52. IARC Press, Lyon, pp. 245e251. ICMSF (International Commission on Microbiological Specifications for Foods), 1996. Toxigenic fungi: Aspergillus. In: ICMSF (Ed.), Microorganisms in Foods 5. Characteristics of Microbial Pathogens. Blackie Academic and Professional, London, pp. 347e381. JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2002. Evaluation of Certain Mycotoxins in Food: Fifty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Tech. Rep. Ser. 906. Juliano, B.O., Bechtel, D.B., 1985. The rice grain and its gross composition. In: Juliano, B.O. (Ed.), Rice: Chemistry and Technology. The American Association of Cereal Chemists, St. Paul, pp. 17e57.
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Food Microbiology journal homepage: www.elsevier.com/locate/fm
Behaviour of Aspergillus flavus and Fusarium graminearum on rice as affected by degree of milling, temperature, and relative humidity during storage Seonyeong Choi a, Hyejung Jun a, Jihyun Bang a, Soo-Hyun Chung b, Yoonsook Kim c, Byeong-sam Kim c, Hoikyung Kim d, Larry R. Beuchat e, Jee-Hoon Ryu a, * a
Department of Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Department of Food and Nutrition, Korea University, Jeongneung-dong, Sungbuk-ku, Seoul 136-703, Republic of Korea Neo Food Resources Research Group, Korea Food Research Institute, Baekhyun-dong, Seongnam, Gyeonggi 463-746, Republic of Korea d Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea e 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 1 April 2014 Received in revised form 30 July 2014 Accepted 23 August 2014 Available online 2 September 2014
We investigated the survival and growth patterns of Aspergillus flavus and Fusarium graminearum, as well as mycotoxin production, on Korean rice as affected by the degree of milling (rough, brown, and white rice) and storage conditions (21 C/85% relative humidity [RH], 21 C/97% RH, and 30 C/85% RH). When rice was stored at 21 C/85% RH, the population of A. flavus remained constant and aflatoxin was not produced, regardless of the degree of milling. At 21 C/97% RH and 30 C/85% RH, the populations of A. flavus increased significantly (P 0.05) and aflatoxins were produced. The highest population of A. flavus and highest amount of aflatoxin B1 were observed on brown rice stored at 21 C/97% RH. For F. graminearum, when stored at 85% RH, the populations were reduced to less than a detectable level (5 CFU/g of rice) within 120 days and no deoxynivalenol (DON) was produced, regardless of the degree of milling and storage temperature. However, at 21 C/97% RH, the population of F. graminearum increased significantly (P 0.05) and DON was produced on all types of rice. Findings from this study provide insights concerning storage conditions necessary to prevent growth and mycotoxin production by A. flavus and F. graminearum on Korean rice with different degrees of milling. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Rice Aspergillus flavus Fusarium graminearum Milling Relative humidity Temperature
1. Introduction Rice (Oryza sativa L.) is a staple food for more than half of the global population (FAO, 2004a) and provides 20% of the dietary energy supply in the world (FAO, 2004b). According to the Food and Agriculture Organization of the United Nations, world rice production has increased steadily in recent years. Production increased from ca. 518 million tons in 1990 to ca. 599 million tons in 2000 and ca. 701 million tons in 2010 (FAOSTAT, 2013). Rice is stored for several months or even years as rough rice after harvesting, and brown rice or white rice after milling. Rough rice consists of the hull, bran, and endosperm. Rough rice is dehulled to produce brown rice and the bran layer of brown rice is removed to produce white rice (Skyrme et al., 1998). Rice is usually consumed in the
* Corresponding author. Tel.: þ82 2 3290 3409; fax: þ 82 2 3290 3918. E-mail address: [email protected] (J.-H. Ryu). http://dx.doi.org/10.1016/j.fm.2014.08.019 0740-0020/© 2014 Elsevier Ltd. All rights reserved.
latter form, but demands for brown rice have increased because of its high nutritional value (FAO, 2004b; Heinemann et al., 2005). As rice is an essential part of the human diet of many people, it is important to maintain its sensorial, nutritional, and microbiological qualities. The growth of some fungal species on rice, with consequent mycotoxin production, results in a microbiological safety concern (Kumar et al., 2008; Reddy et al., 2009). The major genera of fungi found on rice are Aspergillus, Penicillium, Fusarium, Alternaria, Mucor, Rhizopus, Trichoderma, Curvularia, Helminthosporium, and Cladosporium (Makun et al., 2007). Among them, Aspergillus flavus and Fusarium graminearum raise particular concern because some strains can produce mycotoxins. Contamination of rice with mycotoxigenic fungi and the presence of mycotoxins have been reported in several countries (Desjardins et al., 1997; Fredlund et al., 2009; Makun et al., 2007; Ok et al., 2009; Tanaka et al., 1988). Fredlund et al. (2009) reported that 21% of rice samples collected from Swedish retail markets were contaminated with A. flavus. Total aflatoxin in contaminated rice was as high as 50.7 mg/kg Park
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et al. (2005) reported that 17% and 10% of polished rice samples obtained from grain wholesale markets in the Republic of Korea were contaminated with A. flavus and F. graminearum, respectively; the mean aflatoxin B1 (AFB1) and deoxynivalenol (DON) concentrations in rice were 4.3 and 139 ng/g, respectively. A. flavus is a mycotoxigenic fungus that produces AFB1 and aflatoxin B2 (AFB2). AFB1 is classified as an International Agency for Research on Cancer (IARC) Group 1 human carcinogen (IARC, 1993). The optimum temperatures for growth and aflatoxin production are ca. 33 C and 16e31 C, respectively, and the optimum water activities (aw) for growth and aflatoxin production are 0.98 and 0.95e0.99, respectively (ICMSF, 1996). An outbreak of 397 cases of hepatitis with 106 deaths occurred in India in 1974. It was concluded that this outbreak was associated with the consumption of maize heavily contaminated with A. flavus and containing aflatoxin of concentrations up to 15.6 mg/g (Krishnamachari et al., 1975). In Kenya, 317 people became ill after eating maize contaminated with aflatoxin; 125 deaths occurred in this outbreak (CDC, 2004). The government of the Republic of Korea has set limits of 15 ng/g and 10 ng/g for total aflatoxins and AFB1, respectively, in cereal grains (Korean Ministry of Food and Drug Safety, 2013). In the USA, the limit for total aflatoxins in food is 20 ng/g, but no limit has been specifically set for AFB1 (FAO, 2003). In the EU, maximum concentrations of 10 ng/g for total aflatoxins and 5 ng/g for AFB1 have been established in rice (European Commission, 2010). F. graminearum is a plant pathogen that can produce DON, nivalenol, and zearalenone (Pitt and Hocking, 2009). Growth of F. graminearum is most rapid at 25 C and aw 0.95e0.995, and DON production is highest at 25 C and aw 0.995 (Ramirez et al., 2006). DON is classified by the IARC as a Group 3 agent (not classifiable as to carcinogenicity in humans) (IARC, 1993). However, consumption of food contaminated with DON may result in nausea, vomiting, gastrointestinal upset, dizziness, diarrhoea, and headache (JECFA, 2002). An outbreak has been reported in India in 1987, in which 97 people became sick after eating wheat bread contaminated with DON and other trichothecene mycotoxins (Bhat et al., 1989). The limit for DON in grains in the Republic of Korea is 1000 ng/g (Korean Ministry of Food and Drug Safety, 2013). In the USA, the limit of DON in finished wheat products for human consumption is 1000 ng/g (FDA, 2010). The EU has set limits for DON in unprocessed cereal (1250 ng/g) and in cereals for direct human consumption (750 ng/g) (European Commission, 2006). Although there have been no documented outbreaks of foodborne intoxication associated with the consumption of rice containing aflatoxin or DON, there is a potential risk of mycotoxin production in rice contaminated with A. flavus or F. graminearum and stored at abusive temperature and relative humidity (RH) conditions. In countries such as the Republic of Korea, where rice is a staple food and the climate is changing from temperate to subtropical due to global warming, potential risks of human illness associated with consumption of rice and other grains containing mycotoxins are likely to increase. Mycotoxins are difficult to eliminate in foods without compromising sensorial and nutritional quality because they are heat-resistant and do not decompose readily (Korzun, 2002; Shapira and Paster, 2004). It is important to prevent contamination of rice with mycotoxigenic fungi and store rice under conditions that will prevent growth mycotoxin production. The growth of fungi and production of mycotoxins in rice and other grains are affected by the availability of nutrients and environmental conditions, such as temperature, aw, and pH (Holmquist et al., 1983; Llorens et al., 2004; Mylona et al., 2012; Ramirez et al., 2006; Sweeney and Dobson, 1998). There have been a number of reports describing conditions affecting growth of fungi and production of mycotoxins affected by storage temperature and aw. However, none has addressed the
patterns of fungal growth and mycotoxin production in rice as affected by different degrees of milling and long-term storage under various temperatures and RH. The study reported here was done to determine the patterns of growth and survival of A. flavus and F. graminearum, as well as mycotoxin production, on rice as affected by the degree of milling, temperature, RH, and storage time. 2. Materials and methods 2.1. A. flavus strains and preparation of inocula Five strains of A. flavus known to produce AFB1 and AFB2 were used in this study: A. flavus strain ATCC 22546 (isolated from mouldy corn), CN 008 (isolated from malted wheat), CN 028 (isolated from malted wheat [nuruk]), CN 029 (isolated from malted wheat [nuruk]), and M 2034 (isolated from fermented soybeans [meju]). These strains were chosen because they had been isolated from grain-based products and their abilities to produce mycotoxin had been confirmed. A. flavus strain ATCC 22546 was obtained from Korean Culture Center of Microorganisms in Seoul, Republic of Korea, and strains CN 008, CN 028, CN 029, and M 2034 were supplied by Dr. Soo-Hyun Chung (Department of Food and Nutrition, Korea University, Seoul, Republic of Korea). Each strain was streaked separately onto potato dextrose agar (PDA; BBL/Difco, Sparks, MD, USA) supplemented with 10% tartaric acid (PDAT; pH 3.5) slants and incubated at 28 C for 7e10 days. Spores were harvested by depositing 10 mL of distilled water (DW) containing 0.02% Tween 80 (Junsei, Tokyo, Japan) on the mat surface, gently rubbing with a sterile loop, and filtering the suspension through sterile cheesecloth. Spore counts (spores/mL) were determined using a haemocytometer. More than 98% of the propagules were spores. A five-strain cocktail of A. flavus spores was prepared by combining suspensions of each of the five strains (2 106 spore per strain). The suspension was centrifuged at 20,000 g for 15 min. The supernatant was decanted and spores were resuspended in sterile DW to give ca. 6.3 log spores/mL. 2.2. F. graminearum strains and preparation of inocula Five strains of F. graminearum known to produce DON were used in this study: F. graminearum strains KACC 46434 (isolated from rice), KACC 46437 (isolated from barley), KACC 46438 (isolated from barley), KACC 46439 (isolated from barley), and KACC 46441 (isolated from rice). These strains were chosen because they had been isolated from grain and their abilities to produce DON had been confirmed. All strains were obtained from the Rural Development Administration-Genebank Information Center, Suwon, Republic of Korea. Each strain was inoculated separately on the PDAT plates and incubated at 28 C for 7 days. Five agar plugs (ca. 6 mm diam.) from the edges of colonies of each strain were deposited in 100 mL of carboxymethyl cellulose (CMC) broth (ammonium nitrate [NH4NO3], 1 g; monopotassium phosphate [KH2PO4], 1 g; magnesium sulphate heptahydrate [MgSO4∙7H2O], 0.5 g; yeast extract, 1 g; carboxylmethyl cellulose, 15 g; DW, 1 L) and incubated on a rotary shaker at 200 rpm for 5 days at 25 C. The CMC culture was filtered through sterile cheesecloth and spore counts for each strain were determined with a haemocytometer. More than 98% of the propagules were spores. A five-strain cocktail of F. graminearum spores was prepared by combining suspensions of each of the five strains (2 106 spore per strain). The suspension was centrifuged at 20,000 g for 15 min. The supernatant was decanted and the pellet was resuspended in sterile DW to give ca. 6.3 log spores/mL.
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2.3. Creation of relative humidity To create atmospheres with 85% or 97% RH, 200 mL of saturated potassium chloride (Daejung, Siheung, Republic of Korea) or potassium sulphate (Daejung) solution, respectively, was added to an airtight container (1.2 L, 155 mm long 155 mm wide 87 mm high; Lock & Lock, Seoul, Republic of Korea) and stored at 21 or 30 C for at least 24 h before use in the experiments. 2.4. Inoculation and storage Three types of rice (O. sativa L. ssp. japonica; produced in Jincheon, Republic of Korea) were provided by the Korea Food Research Institute (Seongnam, Gyeonggi, Republic of Korea): rough rice, brown rice, and white rice. Rough rice was collected in October and November 2011 and milled at a rice processing complex to obtain brown rice and white rice. Rice was subjected to gamma irradiation (15 kGy; Greenpia Technology, Yeoju, Republic of Korea) before use in the experiments. It was confirmed that there were no viable cells of bacteria or moulds in the irradiated rice samples by spread-plating and enriching the samples on PDA and in tryptic soy broth (TSB; BBL/Difco), respectively. Gamma-irradiated rough rice, brown rice, or white rice (10 g) was placed in a sterile Petri dish (60mm diameter 15-mm depth; SPL, Pocheon, Republic of Korea) and 50 mL of inoculum (A. flavus or F. graminearum cocktail) prepared as described above was spot-inoculated at five randomly selected locations on the rice. Open Petri dishes containing 10 g of inoculated rough rice, brown rice, or white rice (two Petri dishes per type of rice in each of three replicate trials) were placed above the surface of saturated potassium chloride or potassium sulphate solutions in an airtight container and incubated at 21 C/85% RH, 21 C/97% RH, or 30 C/ 85% RH for up to 120 days. In total, more than 180 airtight containers were prepared. 2.5. Microbiological analysis After storing inoculated rice for 0, 10, 20, 30, 40, 50, 60, 80, 100, and 120 days, populations of A. flavus or F. graminearum were determined. At each sampling time, 10 g of rice were placed in a stomacher bag (BA 6040 standard bags; Seward, West Sussex, UK) containing 50 mL of potato dextrose broth (PDB; BBL/Difco) and pummelled for 2 min in a stomacher (Interscience BagMixer® 400W; Interscience, St. Nom, France). The homogenate was serially diluted in 0.1% peptone water. Duplicate 0.1-mL samples (diluted or undiluted homogenate) or quadruplicate 0.25-mL samples (undiluted homogenate) were spread plated on PDAT. The PDAT plates for enumerating A. flavus were incubated at 28 C for 2 days, while those for F. graminearum were incubated at 25 C for 2 days before counting colonies. The remaining F. graminearum homogenate was incubated at 25 C for up to 5 days to enrich viable cells of F. graminearum. When colonies of F. graminearum did not form on PDAT, the enriched homogenates (1 mL) were spread on PDAT and incubated at 25 C for 2 days. The limits of detection were 0.7 log CFU/g of rice (5 CFU/g of rice) by direct plating, and 1.0 log CFU/g (1 CFU/10 g) of rice by enrichment. 2.6. Aflatoxin analysis The amounts of AFB1 and AFB2 produced in rough rice, brown rice, and white rice stored at various temperatures and RHs for 0, 10, 20, 30, 40, 50, 60, 80, 100, and 120 were determined. Aflatoxin content was determined by a method described in the Korean Food Standards Codex (Korean Ministry of Food and Drug Safety, 2013), with a slight modification. Briefly, rough rice, brown rice, or white
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rice (10 g) was ground in a blender, an extractant (40 mL: 1 g of sodium chloride, 30 mL of DW, and 70 mL of methanol) was added, and the mixture was blended at high speed for 1 min. The homogenate was filtered through Whatman grade no. 1 filter paper and 10 mL of filtrate were mixed with 30 mL of 1% Tween 20 (Junsei) in DW. The mixture was filtered through Whatman grade GF/A. Filtered extract (10 mL) was cleaned up using an immunoaffinity column (IAC) (AflaTest; VICAM, Watertown, MA, USA) at a rate of approximately 1 drop/s until air came through the column. Then, 10 mL of DW were passed through the column at the same flow rate until air came through the column. Aflatoxins were eluted by passing 1.5 mL of high-performance liquid chromatography (HPLC)egrade methanol through the column at a rate of ca. 1 drop/s and collected in a glass vial. The eluate (1.5 mL) was evaporated to dryness under a stream of nitrogen gas at 50 C using a nitrogen concentrator (EYELA MGS-2200; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The residue was dissolved in 0.5 mL of derivatisation solution consisting of 9 mL of 10% acetonitrile (ACN) and 1 mL of trifluoroacetic acid (CHROMASOLV®; SigmaeAldrich, St. Louis, MO, USA) and kept in the dark for at least 3 h. Derivatised aflatoxins were filtered through 0.45-mm syringe filters. Quantification of aflatoxins was performed by HPLC (Waters, Milford, MA, USA). Aflatoxins were separated using Nova-Pak C18 columns (3.9 150 mm, 4-mm particle size; Waters) and detected using a fluorescence detector (Waters 2475; Waters) at an excitation wavelength of 360 nm and emission wavelength of 430 nm. Samples (50 mL) were injected and eluted with a mobile phase (DW:methanol:ACN [660:170:170, v/v/v]) at a flow rate of 0.5 mL/min. The limits of detection of AFB1 and AFB2 were 0.1 and 0.03 ng/g, respectively. 2.7. Deoxynivalenol analysis The amounts of DON produced in rough rice, brown rice, and white rice inoculated with F. graminearum and stored at various temperatures and RHs for 0, 10, 20, 30, 40, 50, 60, 80, 100, and 120 days were determined. DON content was determined using a modification of the method described in the VICAM DONtest HPLC Instruction Manual (Vicam, 2005). Rough rice, brown rice, or white rice (10 g) were ground in a blender, 2 g of polyethylene glycol 8000 (Yakuri Pure Chemicals, Kyoto, Japan) and 40 mL of DW were added, and the mixture was blended at high speed for 1 min. The homogenate was filtered sequentially through a Whatman grade no. 4 filter and a Whatman grade GF/A filter to extract DON. The filtered extract (1 mL) was cleaned up by passing through IAC (DONtest; VICAM) at a rate of approximately 1 drop/s until air came through the column. Then, 5 mL of DW were passed through the column at the same flow rate until air came through the column. DON was eluted by passing 1.5 mL of HPLC-grade methanol through the column at a rate of about 1 drop/s and collected in a glass vial. The eluate (1.5 mL) was evaporated to dryness under a stream of nitrogen gas at 45 C using a nitrogen concentrator (EYELA MGS2200; Tokyo Rikakikai Co., Ltd.). The residue was dissolved in 0.5 mL of 10% ACN and filtered through a 0.45-mm syringe filter. Quantification of DON was performed by HPLC. DON was separated using a Nova-Pak C18 column (3.9 150 mm, 4-mm particle size; Waters) and detected using a photodiode array detector (Waters 2996; Waters) at 220 nm. Samples (50 mL) were injected and eluted with a mobile phase consisting of 10% ACN at a flow rate of 0.5 mL/ min. The detection limit of DON was 30 ng/g. 2.8. Statistical analysis All experiments were performed in triplicate. Data were analysed using the general linear model of the Statistical Analysis System procedure (SAS 9.3; SAS Institute, Cary, NC, USA). The
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effects of the degree of milling of rice and storage time on populations of A. flavus and F. graminearum as well as the amounts of mycotoxins produced on rice were analysed using Fisher's least significant difference test. In all analyses, P 0.05 was taken to indicate statistical significance. 3. Results 3.1. Growth of A. flavus and production of aflatoxin in rice as affected by the degree of milling, temperature, and RH Fig. 1 shows populations of A. flavus in inoculated rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. The initial populations of A. flavus in rough, brown, and white rice samples were 3.3e3.8 log CFU/g of rice. At 21 C/85% RH, populations in rough rice and brown rice did not change significantly (P > 0.05) during storage for 120 days. The number of A. flavus significantly decreased by 0.6 log CFU/g in white rice stored for 120 days. When rice was stored at 21 C/97% RH, A. flavus increased in rough rice, brown rice, and white rice to 5.9, 7.3, and 6.7 log CFU/g, respectively, within 20 days. Populations remained constant for an additional 100 days, regardless of the degree of milling. The number of A. flavus detected in brown rice was significantly higher than that in rough rice and white rice by 0.8e1.0 log CFU/g after 120 days (P 0.05). At 30 C/85% RH, A. flavus increased significantly (P 0.05), regardless of the degree of milling during storage. After 120 days, populations in rough rice, brown rice, and white rice increased by 2.6, 3.2, and 2.9 log CFU/g, respectively, compared to the initial populations. Table 1 shows the concentrations of AFB1 detected in rough rice, brown rice, and white rice inoculated with A. flavus and stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. In non-inoculated rough rice, brown rice, and white rice, 0.27, 0.13, and 0.30 ng/g of AFB1 were detected, respectively (data not shown). When rice was stored at 21 C/85% RH for 0 and 10 days, trace
▫
amounts of AFB1 were detected, regardless of the degree of milling; AFB1 was not detected in rice stored for more than 10 days. At 21 C/ 97% RH, AFB1 was detected in all three types of rice stored for 20e120 days. The maximum levels of AFB1 detected in rough rice, brown rice, and white rice were 140.9, 491.0, and 144.9 ng/g, respectively. At 30 C/85% RH, AFB1 was detected in rough rice and brown rice after storage for 40 days and reached levels up to 96.7 and 203.2 ng/g, respectively, after 120 days. On white rice, however, AFB1 was detected after storage for 60 days and reached 1.8 ng/g after 120 days. Table 2 shows the concentrations of AFB2 detected in rough rice, brown rice, and white rice inoculated with A. flavus when stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. In non-inoculated rough rice, brown rice, and white rice, AFB2 was not detected (data not shown). At 21 C/85% RH, AFB2 was not detected, regardless of the degree of milling. At 21 C/97% RH, AFB2 was detected in rough, brown, and white rice stored for 30 days or longer, and reached levels up to 7.4, 2.6, and 2.4 ng/g, respectively, within 120 days. AFB2 was detected in rough rice and brown rice stored at 30 C/85% RH for 40 days, reaching levels of 3.0 and 1.9 ng/ g after 50 and 120 days, respectively. In white rice, a trace amount (0.5 ng/g) of AFB2 was detected after 120 days. 3.2. Growth of F. graminearum and DON production in rice as affected by the degree of milling, temperature, and RH Fig. 2 shows populations of F. graminearum in inoculated rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. The initial population of F. graminearum in rice was 2.3e3.8 log CFU/g. When rice was stored at 21 C/85% RH, F. graminearum decreased to an undetectable level by enrichment (<1 CFU/10 g) in all types of rice over time during the 120-day storage period. At 21 C/97% RH, populations in rough rice, brown rice, and white rice increased significantly (P 0.05) to 4.4, 4.2, and 4.0 log CFU/g after 120 days. At 30 C/85% RH,
Fig. 1. Numbers of viable cells of A. flavus in rough rice (B), brown rice ( ) and white rice (D) stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Error bars indicate standard deviations across replicated experiments. The detection limit ($$$$$$) by direct plating was 0.7 log CFU/g rice (5 CFU/g rice).
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Table 1 Concentrations of aflatoxin B1 (ng/g rice) in rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Storage conditions
Type of rice
Mean concentrations of aflatoxin B1 (ng/g rice) after the indicated storage time (days)a 0
21 C/85% RH
21 C/97% RH
30 C/85% RH
Rough Brown White Rough Brown White Rough Brown White
10
A 0.9 a A 0.6 a A 0.6 a ND ND ND ND ND ND
20
A 0.1 b A 0.1 a A 0.1 a ND ND ND ND ND ND
30 b
ND ND ND C 4.5 a A 318.3 c B 144.9 a ND ND ND
c
e e e A 24.4 a A 183.2 d A 56.5 b ND ND ND
40
50
60
80
100
120
e e e B 128.4 a A 445.6 ab B 14.8 bc A 18.5 a A 18.7 b ND
ND ND ND B 83.1 a A 466.3 a B 1.7 c A 18.4 a A 107.2 ab ND
e e e B 9.0 a A 100.0 d B 6.4 bc A 1.5 a A 14.2 b A 1.1 ab
ND ND ND B 140.9 a A 491.0 a B 5.2 bc A 1.3 a A 47.2 b A 0.2 b
e e e AB 115.3 a A 350.0 c B 5.2 bc A 0.1 a A 58.1 ab A 0.2 b
ND ND ND B 7.5 a A 361.8 bc B 4.8 bc A 96.7 a A 203.2 a A 1.8 a
a Comparison of the effects of storage time: values in the same row that are not followed by the same lowercase letter are significantly different (P 0.05). Comparison of the effects of type of rice: under the same storage conditions, values in the same column that are not preceded by the same uppercase letter are significantly different (P 0.05). b ND, not detected (<0.1 ng/g). c e, not determined.
F. graminearum decreased to undetectable levels in rough, brown, and white rice within 50, 80, and 50 days, respectively. Table 3 shows the concentrations of DON detected in rough rice, brown rice, and white rice inoculated with F. graminearum and stored at 21 C/85% RH, 21 C/97% RH, or 30 C/85% RH for up to 120 days. DON was not detected in all types of non-inoculated rice (data not shown). At 21 C/85% RH, DON was not detected in brown rice or white rice throughout storage, but was detected at 76.3 and 134.6 ng/g in rough rice after 80 and 120 days, respectively. DON was detected on all three types of rice stored at 21 C/97% RH. In rough rice, 154.3 ng/g were detected after storage for 50 days and a maximum level (307.6 ng/g) was detected after 60 days. In brown rice, DON was detected after storage for 40 days, and reached 954.4 ng/g after 50 days. DON was not detected in white rice stored for less than 80 days, and reached 174.7 ng/g at 100 days. DON was not detected in rice stored at 30 C/85% RH for up to 120 days. 4. Discussion As expected, growth of A. flavus and production of aflatoxins were affected by storage temperature and RH. These observations are in agreement with findings of Niles et al. (1985), who reported that A. flavus did not grow on wheat during storage at 20 C and 0.85 aw for 30 days. However, Mousa et al. (2013) reported that A. flavus could grow in rice held under similar conditions. They reported that the A. flavus showed radial growth (0.1e0.2 mm/day) in brown and polished rice (aw 0.84 and 0.86) stored at 20 C with for 5 weeks but did not produce aflatoxins. Growth of A. flavus and production of aflatoxins at 21 C/97% RH were affected markedly by the degree of milling of rice. Higher populations and AFB1 concentrations were observed in brown rice,
compared to rough rice and white rice. These results imply that AFB1 may be produced in greater amounts in naturally contaminated brown rice than in rough or white rice. The higher levels of aflatoxins in brown rice than in rough rice probably result in part from physical protection by the hull surrounding the rice against invasion of A. flavus, whereas brown rice with hull removed can be easily colonized (Juliano and Bechtel, 1985). Other researchers have reported that greater amounts of AFB1 were found in brown rice than in white rice. Sales and Yoshizawa (2005) investigated the level of aflatoxins in naturally contaminated rice. They found that brown rice (2.7 mg/kg) had significantly higher amounts of aflatoxins than did polished rice (0.37 mg/kg). Takahashi et al. (1989) examined the location of aflatoxin in brown rice artificially inoculated with A. flavus and stored at about 85e90% RH and 28 C for up to 15 days. They observed that considerably higher concentrations of AFB1 were detected on bran layer (8930 ng/g) than in the starchy endosperm portion (250 ng/g). Aflatoxin was present near the invading A. flavus mycelia, which were present mainly in the caryopsis coat, aleurone layer, germ, and outer starchy endosperm. In our study, AFB1 was produced at much higher levels than AFB2. This observation is in agreement with studies of Shotwell et al. (1966) and Taber and Schroeder (1967). Shotwell et al. (1966) reported that the ratio of AFB1 and AFB2 produced on polished rice was approximately 6:1, while Taber and Schroeder (1967) reported a ratio of AFB1 to AFB2 of about 10:1 on rough rice. In our study, approximate ratios of the amounts of AFB1 and AFB2 produced in rough, brown, and white rice after 120 days were 8:1, 170:1, and 9:1, respectively, when rice was stored at 21 C/97% RH. Growth of F. graminearum (Fig. 2) and production of DON (Table 3) were unaffected by the degree of milling of rice but were
Table 2 Concentrations of aflatoxin B2 (ng/g rice) in rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Storage conditions
Type of rice
Mean concentrations of aflatoxin B2 (ng/g rice) after the indicated storage time (days)a 0
10
20
30
40
50
60
80
100
120
21 C/85% RH
Rough Brown White Rough Brown White Rough Brown White
NDb ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
ec e e A 1.1 b A 1.8 b A 2.4 a ND ND ND
e e e A 1.0 A 1.9 A 1.4 A 2.5 A 1.8 ND
ND ND ND A 5.8 ab AB 1.9 b B 0.2 a A 3.0 a A 0.7 a ND
e e e A 1.4 A 2.2 A 1.0 A 0.1 A 1.3 ND
ND ND ND A 7.4 A 2.6 A 0.6 A 0.3 A 0.4 ND
e e e A 1.6 ab A 2.3 ab A 0.8 a ND 0.4 a ND
ND ND ND B 0.9 b A 2.1 ab B 0.5 a A 1.3 a A 1.9 a A 0.5
21 C/97% RH
30 C/85% RH
b b a a a
b ab a a a
a a a a a
a Comparison of the effects of storage time: values in the same row that are not followed by the same lowercase letter are significantly different (P 0.05). Comparison of the effects of type of rice: under the same storage conditions, values in the same column that are not preceded by the same uppercase letter are significantly different (P 0.05). b ND, not detected (<0.03 ng/g). c e, not determined.
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▫
Fig. 2. Numbers of viable cells of F. graminearum in rough rice (B), brown rice ( ) and white rice (D) stored at 21 C/85% RH, 21 C/97% RH and 30 C/85% RH for up to 120 days. Error bars indicate standard deviations across replicated experiments. The detection limit ($$$$$$) by direct plating was 0.7 log CFU/g rice (5 CFU/g rice). The detection limit by enrichment was 1.0 log CFU/g (1 CFU/10 g). To plot the numbers of F. graminearum on rice, the number of cells was considered as 0.7 log CFU/g when cells were not detected on agar plates but detected after enrichment. If cells were not detected after enrichment, it was considered as 1.0 log CFU/g.
Table 3 Concentrations of deoxynivalenol (ng/g rice) in rough rice, brown rice, and white rice stored at 21 C/85% RH, 21 C/97% RH, and 30 C/85% RH for up to 120 days. Storage conditions
Type of rice
Mean concentrations of deoxynivalenol (ng/g rice) after indicated storage time (days)a 0
21 C/85% RH
21 C/97% RH
30 C/85% RH
Rough Brown White Rough Brown White Rough Brown White
10 b
ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
20 ND ND ND ND ND ND ND ND ND
30 c
e e e ND ND ND e e e
40
50
60
80
100
120
e e e ND 244.5 b ND e e e
ND ND ND A 154.3 a A 954.4 a ND ND ND ND
e e e A 307.6 a A 92.2 b ND e e e
76.3 a ND ND A 54.0 a A 551.7 ab A 164.3 a ND ND ND
e e e A 141.8 a A 37.9 b A 174.7 a e e e
134.6 a ND ND A 11.2 a A 107.3 b A 51.9 a ND ND ND
a Comparison of the effects of storage time: values in the same row that are not followed by the same lowercase letter are significantly different (P 0.05). Comparison of the effects of type of rice: under the same storage conditions, values in the same column that are not preceded by the same uppercase letter are significantly different (P 0.05). b ND, not detected (<30 ng/g). c e, not determined.
enhanced by increased RH. Hope et al. (2005) determined the effects of aw (0.99e0.85) and temperature (15 and 25 C) on growth and production of DON by F. graminearum on wheat grain. They reported that mycelial growth did not occur at aw 0.85, regardless of temperature, and DON was not produced at aw 0.93. Sanchis and Magan (2004) reported that F. graminearum did not produce DON on wheat at aw 0.93. Cuero et al. (1987) reported that F. graminearum could not grow on cracked irradiated rice at aw less than 0.90. These studies indicate that growth of F. graminearum and production of DON can be effectively suppressed by lowering RH below 85% during storage. In our study, F. graminearum was inactivated at 85% RH more rapidly at 30 C than at 21 C. The amount of DON produced on rice was less than the maximum level of 1000 ng/ g set for grains by the Korean Ministry of Food and Drug Safety and for wheat set by the FDA. In summary, we observed that A. flavus cannot grow or produce aflatoxins in rice at 21 C/85% RH, regardless of the degree of
milling. At 21 C/97% RH and 30 C/85% RH, however, A. flavus was able to grow and produce aflatoxins. The population of A. flavus and the AFB1 concentration in brown rice was significantly (P 0.05) greater than that in rough rice or white rice stored at 21 C/97% RH. F. graminearum did not survive or produce DON on rice stored at 21 C/85% RH or 30 C/85%, regardless of the degree of milling. At 21 C/97% RH, F. graminearum was able to grow and produce DON. Findings from this study provide information useful when developing interventions to enhance the microbiological safety of rough, brown, and white rice by controlling temperature and RH during storage, thereby preventing growth of A. flavus and F. graminearum and production of mycotoxins. Acknowledgements This study was conducted with the support of the Korea Food Research Institute (project no. E132501) and a Korea University
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