International Journal of Pure and Applied Sciences and Technology

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1 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp International Journal of Pure and Applied Sciences and Technology ISSN Available online at Research Paper Isolation and Quantification of Aflatoxin from Aspergillus Flavus Infected Rice G. Ravi Babu 2, M. Guru Prasad 1 and T.N.V.K.V. Prasad 1,* 1 Institute of Frontier Technology, Regional Agricultural Research Station, Acharya NG Ranga Agricultural University, Tirupati , A.P., India 2 Department of Veterinary Pathology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati , A.P., India (All authors contributed equally to this work) * Corresponding author, (tnvkvprasad@gmail.com) (Received: ; Accepted: ) Abstract: Isolation of Apergillus flavus from groundnut and production of aflatoxin on rice. a method has been developed for the production of aflatoxin by growing Aspergillus flavus strain AJ on the solid substrate rice. Optimal yields, more than 1mg of aflatoxin B1 per gram of starting material, were obtained in 5 days at 28C 0.A crude product containing aflatoxin was isolated by HPLC (High Performance Liquid Chromatography). The cruded product consists of 55% aflatoxin in the following ratio:b1-b2-g1- G2,100:0.15:0.22:0.02.Aflatoxin B1 was separated from all the impurities and from the other aflatoxins by chromatograph on silica gel with 1% ethyl alcohol in chloroform. Keywords: Rice, Aspergillus flavus, Aflatoxin, Metabolite, HPLC Introduction Aflatoxins are considered to be one of the most dangerous contaminants in food and feeds. Aflotoxins are a special group of naturally occurring metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus [7,12]. The primary metabolites of A.flavus are

2 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp aflotoxins B1, B2, G1 and G2 which contaminate a variety of agricultural food and feed products [17]. A number of investigators have found that aflatoxins are acutely toxic, carcinogenic, teratogenic and mutagenic compounds [2,5,11,15,18]. Aflatoxin B1 is the most potential toxic and carcinogenic metabolite and found naturally in large amounts in agricultural crops. Since the discovery of deaths of a hundred thousand poults in 1960 [12] and the hepatomas in trout [1] were caused by feeds containing aflatoxins, there has been much effort to determine the effect of aflatoxin on other animals [8,10]. Feeding chickens with aflatoxin contamination cause growth retardation and increase in liver/body weight ratio, biochemical alterations and pathological changes. To conduct the necessary feed trails, large quantities of toxin are necessary [16]. Aflatoxin has produced on groundnut [13], crushed wheat and corn meal. A method for producing the substance in submerged culture has b e e n developed [ 14]. Production of aflatoxins on the agricultural commodities like groundnut, rice, wheat, corn, soybeans, cotton and sorghum was done and found that rice was the best substrate [6]. This paper describes a method for production of toxin on rice with the adaptation of the technique fermentation. Materials and Method Culture and inoculums: A.flavus strain AJ was used to produce aflatoxins because it was identified asone of the best found in a survey of A.flavus strains (Regional Agricultural Research Station, Tirupati, India). This strain was isolated from groundnut in This strain is very stable and consistently yields higher levels of aflatoxin, e s p e c i a l l y B1, even after man transfers. Inoculums were prepared by inoculating tubes (1.5 by 15 cm) of potato dextrose-agar with spores of AJ 2010.The potato-dextrose- agar was prepared as follows. The flask contained distilled water 100ml and dextrose, 20g; NaCl-20mg and 1g agar. Then the p H is adjusted to the 7.0.The contents of flask were brought momentarily to C in an autoclave and then platted in laminar flow. Inoculated slants were incubated for 7 to 21 days at 28 0 C. By 7 days, the cultures had a heavy crop of green conidia, spores were scraped loose with a loop,the slants was shaken to give a uniform suspension of spores,and the spore suspension (0.5ml) was used to inoculate each 50g of substrate. Fermentation: Fermentations were carried out in 500ml Erlenmeyer flasks containing 100g of polished rice or 50ml water Erlenmeyer flask containing 300g of rice. Indented Erlenmeyer flask

3 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp did not increase yields of aflatoxin. After distilled water (25ml) was added to the rice (50g) in the Erlenmeyer flask, the mixture was allowed to stand for 1hr with frequent shaking. The flasks were autoclaved at 15 psi for 15 min and cooled. They were then inoculated and placed on a Erlenmeyer flask at 28C 0 and 188 rev/min. If the did pack in clumps, the flasks were removed from the shaker and the materials were loosened by shaking each flask vigorously. It is extremely important that the rice does not remain as a compact mass with the mold mycelium binding the rice. The technique was adapted from the fermentation, which involves the growth of Aspergillus flavus on rice at moisture levels to produce yellow rice. At 48 hr after inoculation, the rice should show small white areas at the sites where the molds has begun to grow. Shortly after, the rice assumes a bright yellowish colour. In all successful fermentation, these colour changes occur. Sporulation does not occur except perhaps on the walls of the flasks above the fermentation rice. If rice is inoculated in flasks and allowed to stand with daily shaking b hand, some aflatoxin is formed but not nearly as much as with continuous shaking. When rice is allowed to stand, it does not go through the colour changes described, but the mould sporulation to give a large crop of green conidia (FIG.1). When such a flask was opened, a cloud of spores was released. At the end of the fermentation (6 to 8 days), the flasks were briefly steamed to destroy the fungus. FIG. 1. (left) Flask of uninoculated rice ; (centre) flask of fermented rice allowed to stand for 8 days with shaking by hand once a day; and (right) flask of fermented rice incubated for 8 days on a rotary shaker with continuous agitation. High Performance Liquid chromatography: The most wide spread methods for quantitative determination of aflatoxin content in different samples are thin-layer chromatography (TLC) [3] and high performance liquid chromatography (HPLC) [4]. Aflatoxins can be separated and detected using

4 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp either normal-or reversed-phase HPLC methods mainly with fluorescence spectrometric detection. Preparation of sample: Weigh 20gms of seed samples into a blender jar and add 100ml of 80% methanol then blend at high speed. Filter blender contents through a filter paper and add 25ml of filtrate and filter these contents. This filtrate is passed through aflatest-p column and sample is now ready for injection into HPLC. HPLC instrument set up protocol for estimation of aflatoxins:- Columns for chromatography were prepared by symmetry C-18, mobile phase is isocratic and flow rate is 1.0ml/min. Now the sample is injected volume is 20 µl.columns and to elute product. Absorption was detected by fluorescence detection with excitation λ 365nm and emission λ 464nm. As eluting solvent was added, 20-ml fractions were collected and were chromatographed on thin-layer plates to determine their aflatoxin composition. Appropriate fractions were combined, concentrated, and precipitated in hexane. Precipitates were dried in vacuo and analysed by thin-layer chromatography. Results and Discussion: A typical data on fermentation of rice in 500-ml flasks with A. flavus strain AJ is shown in Table 1. Table 1:Production of aflatoxin on rice by Aspergillus flavus AJ in 300ml Erlenmeyer flask Aflatoxin (µ g/g of substrate) Das B1 B2 G1 G2 Total , ,113 1,511 (Incubate on shaker for 188 rev/min) Many of the fluctuations reported in amounts of aflatoxins on rice samples are caused by expected variations in the assay rather than in actual quantities present. Peak yields were obtained in 8 days. Aflatoxin B1 predominated over other aflatoxins in the product. Data obtained with indented flasks containing 100 g of rice and incubated on a rotar shaker at 28 0 C are shown in Table 2.

5 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp Table 2: Production of aflatoxin B1 on rice by A.flavus in Erlenmeyer flasks No.of Days Aflatoxin b1 (µ g/g of Wt of rice at time of substrate) harvest (Incubate on shaker, 200rev/min). Eight fermentations on rice in 500-ml Erlenmeyer flasks gave the following yields in milligrams per gram of substrate: 1.00, 0.16, 0.80, 1.11, 1.10, 0.74, 1.19, and A biological assay with ducklings of the moldy rice, conducted at the Regional Research Laboratory, confirmed the results of our chemical analysis. The crude isolated product was about 50% pure in terms of total aflatoxins with a distribution of individual factors as indicated by the ratio BI-B2-G--G2; 1.00:0.15:0.22:0.02. The yield in flasks was lower (0.68 mg of aflatoxin B1 per g of substrate). To save time, moldy rice can be used in animal feeding tests without extraction. It should be easier to prepare a feed homogeneously contaminated with ground rice containing aflatoxin than with the extracted product. Aflatoxins are produced when A.flavus is grown on a number of agricultural substrates [6], any one of which could be used to prepare feeds containing levels of toxin desired for animal trials. A

6 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp B C

7 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp D Fig.2. Quantification of aflatoxin A)G2, B)G1, C)B1 and D)B1 with High Performance Liquid Chromatography (HPLC) produced by Aspergillus flavus infected rice The crude product precipitated from extracts was filter on filter paper purified on aflatest-p columns to yield several fractions containing about one-half of the total aflatoxin B1 uncontaminated by other aflatoxins. Fractions that followed contained mixtures of B1, B2, GI, and G2. Composition of products obtained from a typical column was as follows: product from (table 2) Fractions 8 to 10, 100% aflatoxin, 334 mg of aflatoxin B1; product from fractions 11 and 12, 100% aflatoxin, 209 mg of B1, 60 mg of B2, and 5 mg of G1; product from fractions 13 to 22, 75% aflatoxin, 66 mg of B1, 51 mg of B2, 71 mg of G1, and 13 mg of G2. Recoveries of 80% can be expected from aflatest-p column. Rechromatography with the same conditions of fractions containing mixtures of aflatoxins yielded more aflatoxin B1. A convenient method for the production of quantities of moldy rice containing aflatoxins, crude aflatoxins, or pure aflatoxin B1 have been developed for animal feeding trails. Similar results on production of aflatoxin using rice have been reported with the rice variety NRRL2999 [9]. Conclusion The results of our present study clearly indicated that rice is one of the best substrates to produce aflatoxin by A.flavus. Throughout the incubation period, different levels of toxin yields were recorded. Higher percent of B1 toxin was produced which is of great interest in respect to conduct trails on animal feeding.

8 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp Acknowledgements The authors are thankful to Acharya N G Ranga Agricultural University for providing research facilities at Institute of Frontier Technology, Regional Agricultural Research Station, Tirupati to carry out this work. References [1] Anonymous, Hepatomas in trout, Nutr. Rev, 22(1964), [2] A.C.Keyl and A.N.Booth, Aflatoxin effects in liver stock, J.Amer.Oilchem.Soc, 48(1971), [3] AOAC Official Method (1988). [4] AOAC Official Method A (1994). [5] C.Gopalan, P.G.Tulpule, D.Krishnamurthy, Introduction of hepatic carcinoma with aflatoxin in Rhesus monkey, Fd. Cosmet.Toxicol, 10(1972), [6] C.W.Hesseltine, A millennium of fungi, food and fermentation, Mycologia, 57(1965) [7] K. Sargeant, A.Sheridan, J.O Kelly and R.B.A.Carnaghan,Toxicity associated with certain samples of groundnuts, Nature, 192 (1961), [8] N.G.Wogan and P.M. Newberne, Dose response characteristics of aflatoxin B1 carcinogenesis in the rat, Cancer Res., 27 (1967), [9] O.L.Shotwell, C.W.Hesseltine, R.D.Stubblefield and W.G.Sorenson, Production of aflatoxin in rice, Appl.Microbiol., 14(3)(1966), [10] P.M.Scott, Mycotoxins in feeds and ingredients and their origin, J. Food. Prot., 41, 5 (1978), [11] P.M.Newberne and W.H.Butler, Acute and chromic effect of aflatoxin on the liver of domestic and laboratory animals, Cancer Res, 29(1969), [12] R.Allcroft and R.B.A.Carnaghan, Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal, Vet.Res., 75 (1963), [13] R.C.Codner, K.Sargeant and R.Yeo, Production of aflatoxin by the culture of strains of Aspergillus flavus-oryzae on sterilized peanuts, Biotechnol.Bioeng, 5(1963), [14] R.I.Mateles and J.C.Adye, Production of aflatoxins in submerged culture, Appl.Microbiol., 13(1965), [15] R.H.Adamson, P.Correa and D.W.Dalgard, Brief communication occurance of primary liver carcinoma in a Rhesus monkey fed aflatoxin B1, J. Antn. Cancer Inst., 50 (1973), [16] R.B.A. Carnaghan, G.Levis, D.S.P. Paterson and R.Allcroft, Biochemical and pathological aspects of groundnut poisoning in chickens, Pathol.Vet., 3(1966),

9 Int. J. Pure Appl. Sci. Technol., 5(1) (2011), pp [17] U.L.Diener and N.D.Davis, Aflatoxin formation by Aspergillus flavus, In: L.A.Gold Blatt (Ed), Aflatoxin (Ed) NY, Academic press, [18] W.H.Butler and J.M.Barnes, Carcinogenic action of groundnut meal containing aflatoxin in rats, Fd. Cosmet.Toxicol, 6(1968),

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