Abstract
Rice (Oryza sativa Linn) is one of the basic diets in the north of Iran. The aim of present study was to detect total aflatoxin (AFT) in domestic and imported rice in Amol (in the north of Iran) and to evaluate the effect of different cooking methods on the levels of the toxin. For this purpose, 42 rice samples were collected from retail stores. The raw samples were analysed by enzyme-linked immunosorbent assay (ELISA) technique for toxin assessment and then submitted to two different cooking methods including traditional local method and in rice cooker. After treatment, AFT was determined. Results show that the average concentration of AFT in domestic and imported samples was 1.08 ± 0.02 and 1.89 ± 0.87 ppb, respectively, which is lower than national and European Union standards. The highest AFT reduction (24.8%) was observed when rice samples were cooked by rice cooker but the difference with local method was not statistically significant (p > 0.05).
Introduction
Mycotoxins are secondary metabolites of moulds, which are associated with certain disorders in animals and humans. In addition to being acutely toxic, some mycotoxins are now linked with the incidence of certain types of cancer, and it is this aspect which has evoked global concern over feed and food safety (Castegnaro and Mcgregor, 1998). Aflatoxins (AFTs) are naturally occurring toxins produced by many ubiquitous fungi, mainly Aspergillus flavus (which produces AFT B1 and AFT B2) and Aspergillus parasiticus (which produces AFT B1, B2, G1 and G2). These fungi can grow on foods such as cereals, dried fruits and spices under certain environmental conditions (Moss, 1998; Shephard, 2009). Mycotoxin contamination in rice is usually lower as in wheat or corn. However, there are some reports that rice has been contaminated with mycotoxins such as AFT B1, B2, G1, G2 (AFT), citrinin, deoxynivalenol (DON), fumonisin B1, B2, B3 (FMs), fusarenon-X (Fus.-X), nivalenol (NIV), ochratoxin A (OTA), sterigmatocystin (STE), and zearalenone (Tanaka et al., 2007). As a result, European Union (EU) food safety legislation on AFT levels is becoming increasingly strict and has established maximum residue limits (MRLs) for these compounds by means of the Regulation (EU) No. 165/2010 of 26 February 2010 (Regulation). For instance, these MRLs are 5 ppb for B1 and 10 ppb for the sum of B1, B2, G2 and G1 for rice. United States has regulated the AFT level in their foods to lower than 20 ppb, and in Korea and Japan the maximum allowed level is 10 ppb (Chiavaro et al., 2001). Currently, the worldwide range of limits for AFB1 and AFT are 1–20 ppb and 1–35 ppb, respectively (FAO, 2004). The MRL for AFT in cereals according to national standard is currently set by Institute of Standard and Industrial Research of I.R. Iran at 30 ppb (ISIRI, 2002).
It is very difficult to remove AFT since they are heat resistant and soluble in intermediate polar solvents. So far, detoxification methods are classified as physical, chemical and biological ones. Although AFT are highly stable to dry heat up to temperatures below their thermal decomposition temperature (Betina, 1989), use of heat to inactivate AFT in contaminated food has been attempted. Some studies have indicated that AFT in contaminated food can be degraded by heat treatment. The extent of destruction achieved depends on the initial level of contamination, heating temperature, moisture content and duration of heating (Rustom, 1997).
The objective of our study was to investigate the presence of AFTs in imported and domestic rice samples collected from retail stores of Amol city in north Iran and to evaluate the effect of different cooking methods including in rice cooker and traditional local method on the levels of the toxin.
Materials and methods
Materials
A total of 42 rice samples included imported (21 samples from Pakistan) and domestic (21) rice samples from open packages (20 kg each package), each sample weighing 500 g, were obtained from retail stores in Amol in the winter of 2011 (Figure 1). Samples were transported to the laboratory under controlled conditions (4–6°C) and analysed within 4–6 h.
Calibration curve of AFT represents absorbance at 450 nm for the sample, or the standard divided by absorbance at the same wavelength for the control. AFT: aflatoxin.
Methods and analysis
The method used in this study was enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of AFT residues in grains, cereals, nuts, animal feeds and other commodities. Commercial ELISA kits were purchased from Romer Labs Singapore Pte Ltd.: Agraquant® Total AF test (COKAQ1000). According to manufacturer’s description, the detection limit, quantification limit and quantitation range for AFT were 1, 1, and 1–20 ppb.
Sample preparation
Samples preparation and test method were conducted according to the instructions outlined in the Romer Labs ELISA kits (Romer Labs, 2007) as is described below. All rice samples were ground by a hammer mill (Pars-technique, Iran) to a fine powder such that over 75% of the material passed through a 20-mesh sieve (Zarrin-khooshe, Iran).
Briefly, a 20-g of ground rice was weighed into a clean jar; 100 mL of 70/30 (v/v) methanol/water was added and blended for 3 min at 25°Ċ. The top layer of the extract was filtered through filter paper (Whatman no. 1) and the filtrate was collected.
Analysis of mycotoxins in samples by ELISA
Prior to analysis of the samples, the ELISA method was validated to ensure data quality. Validation of ELISA was carried out by determination of recoveries for rice samples spiked with different concentrations of AFM1 (0, 1, 2, 4, 10 and 20 ppb). According to the manufacturer’s instructions, a sufficient number of microtiter wells were inserted into the microwell holder for all standards (at the concentrations 0, 1, 2, 4, 10 and 20 ppb supplied with the KIT) and samples (100 μL/well/sample) to be run in duplicate. Briefly, standard solutions and prepared samples (100 μL) were mixed with 200 μL of conjugate in individual dilution wells, and then 100 μL from each dilution well were transferred to a respective antibody-coated well. After 15 min of incubation at room temperature, the wells were washed five times with 250 μL distilled water. Substrate (100 μL) was added to each well and incubated for 5 min at room temperature. Following the addition of stop solution (100 μL) to each well, the intensity of the resulting yellow colour was measured at a wavelength of 450 nm using ELISA 96-well plate reader. The absorbance values obtained for standards and the samples were divided by the absorbance value of the first standard (zero standard) and multiplied by 100 (percentage of maximum absorbance). The absorption intensity was found to be inversely proportional to mycotoxin concentration in the samples.
Rice cooking methods
Rice cooker method
Twenty-one rice samples (250 g) were washed using 500 mL drinking water and soaked in 500 mL water for 1 h. The water was drained completely. Then 250 mL water was poured on rice in electric rice cooker (Toshiba-Japan) and then the lid was closed. Cooking was done in reduced flame for 30 min.
Local cooking method
Twenty-one rice samples (250 g) were washed four times using 500 mL drinking water until the water looks clear. This is a very important step in local cooking method of rice. Essentially, this step is to remove the starch from the rice. Then the rice was soaked for 2 h in 500 mL drinking water. Once the water started boiling, the rice was placed in the water and cooked for about 10 min. During the cooking time, the rice was stirred a couple of times from bottom to up. After cooking, rice was drained in a colander with a quick rinse with cold water (10–15°C) to stop additional cooking. The cooked rice was poured in a pot and then 1 spoon of oil (about 10 g) was added onto the rice. After that, a towel was placed over the lid of the pot and covered the pot. Finally, cooking was done on medium low flame for 1 h. Then the cooked rice samples were dried and ground according to the method noted in “sample preparation” section.
Statistical analysis
Statistical analysis was performed using SPSS version 11.5. Data were analysed by analysis of variance (ANOVA) and reported as mean ± SD. The levels were considered significantly different at p < 0.05.
Results and discussion
AFT occurrence in investigated samples
The standard curve obtained in the present study for AFT detection using the competitive ELISA test is depicted in Figure 1. The resolution of the curve within the range 0–1 ppb was insufficient to enable reliable interpolation of AFT concentrations based on their corresponding absorbance values. Therefore, concentration values of AFT obtained from the curve within this range cannot be considered reliable. However, the standard curve for the range 1–20 ppb was reliable, and within this range the inverse correlation between AFT concentrations in the standard samples and their corresponding absorbance values at 450 nm was good (Figure 1). The ELISA validation results are expressed in Table 1, showing excellent recovery for the method.
Validation data of the competitive ELISA for AFT.
ELISA: enzyme-linked immunosorbent sassy; AFT: aflatoxin.
The levels of mycotoxin contamination in Iranian rice samples examined in our study are shown in Table 2. Of the 42 samples, 45% were found to contain total AF levels ranging from 1.03 to 3.7 ppb. Most importantly, none of the samples exceeded the maximum tolerable limit for total AF as stated in the EU Regulation (10 ppb), FAO Regulation (35 ppb) and national standard (30 ppb). The highest levels of total AF in the raw imported and domestic samples were 3.7 and 1.10 ppb, respectively.). In a study reported by Bandara et al. (1991), AFT levels in parboiled rice were found to be significantly higher than in raw milled rice, with the highest levels of AFB1 and AFG1 being 185 and 963 ppb, respectively. Toteja et al. (2006) examined parboiled rice collected from India and found 38.5% of the samples to be positive for AFB1. Reddy et al. (2009) analysed 1200 rice samples by ELISA and 67.8% of the samples were positive to AFB1. In another study, Sugita-Konishi et al. (2006) reported natural contamination of AFTs and ochratoxin A (OTA) in retail rice in 2004 and 2005. AFTs were not detected in 53 samples. The limit of quantification was 0.1 ppb. No OTA was detected in 50 retail rice samples. The limit of quantification was 0.1 μg/kg. Zuoxin et al. (2006) analysed AFTs in 37 rice samples. The results showed that almost all samples collected contained AFTs. The average contents in whole grain rice and brown rice were found to be 3.87 and 0.88 ppb, respectively.
Levels of AFT in rice samples.
AFT: aflatoxin; Nd: not detectable (below the detection limit).
According to Manabe and Tsuruta (1991), the majority of fungal species in rice are mesophylls (optimal growth temperature: 22–35°C) that are capable of growing at temperatures between 5 and 45°C, and therefore, in the temperate climate of Amol, the prevailing temperature satisfies the growth conditions for a wide variety of mycelium fungi. According to them, the required level of water activity in the commercial distribution of harvested grains is considered to be 0.65–0.70 or below. The average relative humidity in Amol is high, between 65 and 98%. AFT contamination and A. flavus infection are often associated with drought stress and high temperature in pre-harvest and high humidity and high temperature in post-harvest (Moreno and Kang, 1999). Poor sanitation and handling conditions during the harvest, drying, transport and storage stages of cereal production can result in fungal contamination and subsequent formation of mycotoxins (Aydin et al., 2007). Iran has encountered AFT contamination in various foods including cereals, nuts, feed and dairy products (Mohamadi Sani et al., 2010). The low level of AFT contamination in rice grains might be due, in particular, to sufficient ventilation to reduce moisture and reduce the risk of mould and mycotoxin contamination. Another reason for this low AFT content might be the storage period of rice; domestic rice samples were selected from the batches that were stored less than 1 year while the imported rice samples usually have storage time of more than 1 year.
Effect of cooking method on AFT
Table 3 shows the effect of cooking method on the level of AFT content in rice samples. Results show the average AFT reduction in rice cooker and local methods, respectively, 24.8 and 17.5% and the difference is not statistically significant (p > 0.05). The relationship between moisture content in foods and destruction of AFT has been already reported several times (Hwang & Lee, 2006; Mendez-Albores et al., 2002; Torres et al., 2001). According to these reports, the increased moisture content enhances the destruction of AFT during cooking or baking. Cooking rice with both rice cooker and local methods involve steam-based cooking methods that can reduce the AFT; but the difference is that in local method, semi-cooked rice is drained compared to rice cooker method. This may led to extraction of AFT to the boiling and washing water. However, in rice cooker method, the excess water is removed in the form of vapour during cooking period (after closing the lid), which can destroy the toxins. According to the results, there is no significant difference between the effectiveness of the two methods in destroying the AFT.
Effect of cooking method on reduction of AFT in rice.
AFT: aflatoxin.
Conclusion
The low level of AFT contamination in rice grains might be due, in particular, to the fact that in Amol, the conditions of storage vary but the local storehouses use sufficient ventilation to reduce moisture and reduce the risk of mould and mycotoxin contamination. None of the samples tested had higher AFT content than the regulated maximum amount allowed. Since rice is an important part of the main diet of the local population and is consumed in large quantities, AFT contamination could be a serious problem even at low levels (Paranagama et al., 2003). Improvement in storage conditions to prevent grain spoilage and reduce AFT contamination is recommended.
Footnotes
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.