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Indian Journal of Science and Technology http://www.indjst.org Vol.1 No 4 (Sep. 2008)

Department of Biotechnology, Vels college of Science, Pallavarm, Chennai-117, India 2 Centre for Advanced Studies in Botany, University of Madras, Chennai-25, India.
banunkl@yahoo.com

Screening of commercial feed samples for the presence of multimycotoxins Narasimhan Banu1 and Johnpaul Muthumary2

Abstract: Multimycotoxin screening methods for carcinogenic effect is of the most concern. simultaneously testing combinations of aflatoxins, Because of their presence in foods and feeds and ochratoxins, sterigmatocystin and T2-toxins have strong evidence of their association with human been carried out for 18 different commercial feed carcinogenesis, aflatoxins are still a serious threat samples collected from various local markets of to human health. New mycotoxins and simultaneous Tamil Nadu. Of these, 14 showed contamination with aflatoxin B1, aflatoxin B2 and Ochratoxin. But contamination of known mycotoxins are being all the 18 samples showed negative results to discovered at high rates. Thus, the mycotoxin risk aflatoxin G1, aflatoxin G2, sterigmatocystin and T2 management requires the development of toxin. Negative correlations were observed for feed multimycotoxin analysis methods to be able to identify and quantify simultaneously several samples with moisture content. Key words: Multimycotoxins, feed samples, mycotoxins of different structures or families. Materials and methods aflatoxins, moisture content, ochratoxin A. Eighteen different feed samples collected from Introduction Feed is a milled product of cereals or other the local markets of Tamil Nadu state and a milled grains combined with oilcakes of sample from Kerela state was also evaluated for groundnuts, sunflower seeds, cotton seeds, neem, their multimycotoxins (Table 1). castor, coconut, etc. Fungi growing on these (Binomials for oil cake source: Peanut Arachis substrates will produce secondary metabolites hypogea; Sesame Sesamum indicum; cotton called mycotoxins. It shows much diversified toxic Gossypium hirsutum; neem Azadirachta indica.; effects in humans and animals health and coconut Cocous nucifera; castor Ricinus productivity (Dmello & Macdonald, 1997; Ostry, communis). 1999). The economic impact of reduced Extraction of multimycotoxins (Sundaram et al., productivity, increased incidence of disease due to 2001) immunosuppression, damage to vital organs and Ten gram of the sample was added with 36 ml interferences with reproduction capacity are many of acetonitrile, 4 ml of 4% KCl and 0.8 ml of 5N HCl times greater than the impact caused by Table 1. Sample list and place of collection death due to mycotoxin poisoning. Oil cake Sample Name of Sample Place of collection and de-oiled cake have been used as no. excellent feeds for cattle and poultry. 1. Peanut oil cake (POC) Polur, Tamil Nadu However, they are found to be favourable 2. Sesame oil cake (SOC) Pernambut, Tamil Nadu substrate for the growth of fungus and toxin 3. Peanut oil cake (POC) Pernambut, Tamil Nadu production. It still retains a considerable 4. Peanut oil cake (POC) Vandavasi, Tamil Nadu proportion of the toxin, when used for 5. Sesame oil cake (SOC) Vandavasi, Tamil Nadu preparation of processed animal feed. 6. Cotton seed oil cake Lactating mammals such as cattle, sheep and Adyar, Tamil Nadu (CSOC) goats consuming processed oilcake and de7. Peanut oil cake (POC) Adyar, Tamil Nadu oiled cake contaminated with AFB1 and 8. Peanut oil cake (POC) Namakkal, Tamil Nadu AFB2, excrete them in milk and urine as 9. Peanut oil cake (POC) Namakkal, Tamil Nadu AFM1 and AFM2 (Allcroft & Carnaghan, 1962; Diaz et al., 1994). Consumption of such 10. Neem oil cake (NOC) Adyar, Tamil Nadu feed is thus highly dangerous primarily to 11. Sesame oil cake (SOC) Adyar, Tamil Nadu animals and secondarily to humans who 12. Copra oil cake (COC) Adyar, Tamil Nadu depend on the animals for milk (van Egmond, 13. Castor oil cake Adyar, Tamil Nadu 1989). These metabolites along with several (CASOC) other metabolites of AFB1, such as aflatoxicol 14. Peanut oil cake (POC) Adyar, Tamil Nadu have also been studied very extensively 15. Copra oil cake (COC) Alapuzha, Kerela (Busby & Wogan, 1981). Although both acute 16. Peanut oil cake (POC) Arakonam, Tamil Nadu (hepatotoxic) and chronic toxicity (liver 17. Sesame oil cake (SOC) Arakonam, Tamil Nadu cancer) of AFB1 is well established, its 18. Peanut oil cake (POC) Arakonam, Tamil Nadu
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Multimycotoxins in feed

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Indian Journal of Science and Technology and macerated using pestle and mortar for 3 minutes. The sample was filtered through Whatmans No.1 filter paper. Twenty ml of this filtrate was transferred into 250 ml of separating funnel. Twenty ml of distilled water and 20 ml of hexane were added to this and shaken well for few minutes. The upper hexane layer was discarded. To the lower layer 20 ml of hexane was added and shaken well for another 3 minutes. The resulting lower layer was again collected. The acetonitrile phase was extracted with 20 ml of chloroform. The chloroform layer was passed through anhydrous sodium sulphate. The chloroform was evaporated to dryness. The final residue was dissolved in 0.2 ml of chloroform. http://www.indjst.org Sterigmatocystin Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 T2 toxin Ochratoxin 0.74 0.73 0.71 0.68 0.66 0.66 0.00 0.57 0.22 0.20 0.15 0.12 0.33 0.46 Vol.1 No 4 (Sep. 2008) Brick red Blue Blue Green Green Dull bluish green spot Blue

Ten, 20, 50 and 100 l of sample extract were spotted onto pre-coated TLC sheets (0.20 mm thickness, Merck) together with standard spots. The plate was developed in chloroform:acetone (9:1) in one direction and toluene: ethylacetate: formic acid (5:4:1) in 2nd direction perpendicular to the first. After the development, the plates were viewed under long and short wavelengths. Sterigmatocystin (in small amounts) and T2-toxin exhibit no fluorescence. Sterigmatocystin is visible as an orange fluorescent spot in long UV light after spraying with 20% aqueous KOH. The same plate was allowed to dry at room temperature and 20% H2SO4 was sprayed. At Rf of T2-toxin, dry at 110 C in an hot air oven and viewed under long UV light. T2-toxin fluoresces as dull bluish green colour. Quantification of multimycoitoxins using long and short UV light was made by evaluating the plate itself by using the following calculations. It was expressed as parts per billion. Multimycotoxins were quantified by using the following calculations: SxCxD Multimycotoxins (ppb)= ------------- x 1000 TxE S = Standard volume which matches with test volume in fluorescence intensity C = Concentration of standard D = Dilution factor T = Test volume which matches with standard volume in fluorescence intensity E = Effective weight of the sample = (4.9)

Quantification of multimycotoxins by TwoDimensional Thin Layer Chromatography (2D-TLC)

Estimation of moisture content (Sundaram et al., 2001) To determine the moisture content of sample, 2 g of the sample was kept at 60C in an hot air oven for 24 h.
(Initial weight of paper + Sample) --------------------------------- - Final weight after drying Weight of sample Moisture content= ---------------------------------------------------------------------- x 100 Weight of the sample

Weight of dried sample Dry matter % = ----------------------------x 100 Weight of sample before Drying

Identification

The identification of the toxins is based on the Rf values and standard toxins. Rf values Mycotoxin Colour Sol I Sol II

Dry matter = 100 - % moisture. Results and discussion A total of eighteen different commercially available market feed samples were tested for their multitoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin, sterigmatocystin and T2toxins) (Table 1 & 2). Among the 18 different commercial feed samples analysed for multitoxins, 14 of them showed contamination with AFB1, AFB2 and OA. But all the 18 samples showed negative results to AFG1, AFG2, sterigmatocystin and T2-toxin. Aflatoxin B2 was absent in the samples of SOC 2, NOC 10, CASOC 13. The maximum concentration of AFB1 was observed for POC4 (120 ppb) and the minimum was observed for COC 12 and SOC 3 (5 ppb, each) (Table 2) Similarly, the maximum contamination of AFB2 was observed for the sample POC 14 (90 ppb) and the minimum for COC 12 (2 ppb). The contamination of OA was maximum in POC 4 and NOC 10 (40 ppb) and the minimum was in SOC 17 (10 ppb). Among the three toxins, the concentration of AFB1 was high compared with other two toxins. The peanut oil cake showed high incidence of toxin contamination than other samples. The COC 12 and SOC 17 showed low level of toxin contamination among the samples studied. The moisture content of feed samples ranged from 3% to 17.5%. The moisture content of POC 4 was 3.5% during analysis and from this sample 120 ppb of AFB1 was extracted. For SOC 2, the by Banu & Muthumary

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Indian Journal of Science and Technology moisture content was 17.5% but the AFB1 level was 12 ppb. Therefore it is concluded, that the moisture content had no effect on toxin production. Among the poultry and cattle feed, the toxin content of peanut cake was the highest followed by sunflower, sesame, cotton, neem, castor and coconut oil cake. The contamination by toxins and the growth of the fungi was influenced by many factors apart from moisture content. The maximum permissible limit of aflatoxin in food for animal and human is 20 ppb. It is apparent from this study that some of the samples crossed this action level set by the U.S. FDA and this will not be a suitable feed to feed the animals. In order to overcome this problem, it is essential to monitor these commodities periodically and evolve policies which discourage the marketing of toxin contaminated foods and feeds. Sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 Source Moisture content (%) 12.5 17.5 6.5 3.5 7.5 5.5 5.0 2.5 7.5 8 2.5 8 13 AFB1 (ppb) http://www.indjst.org Vol.1 No 4 (Sep. 2008)

after the contaminated feed is removed from the ration. In general, it is recommended that no dairy animal receive rations containing aflatoxin level greater than 20 ppb (Van Egmond, 1989). It is indicated that the environment of the refineries might act as a source of contamination as the seeds are brought to the site by various means of transport in gunny bags and stored before entering the extracting units. Also, the refineries should maintain a good sanitation ground to reduce the mycotoxin contamination. The permissible limit of mycotoxin in animal feed is <20 ppb especially AFB1, which varies with the country. However, the limit prescribed should be strictly enforced with regard to the aflatoxin produced in the animal feed. All these results show a possible danger for those animals consume the contaminated AFG1 (ppb) Sterigm atocystin (ppb) T2 (ppb) AFG2 (ppb) OA (ppb)

Table 2. Quantification of multitoxins from commercial feed samples by 2D-TLC

AFB2 (ppb)

POC 95 20 20 SOC 12 POC 60 20 30 POC 120 30 40 SOC 25 12 CSOC 25 12 30 POC 12 12 25 POC 40 50 30 POC 12 8 30 NOC 20 40 SOC 30 25 25 COC 5 2 CASO 10 C 14 POC 4.5 30 90 20 15 COC 12 20 25 25 16 POC 16.5 8 9 25 17 SOC 3 5 5 10 18 POC 7 25 20 30 The maximum level for aflatoxin fed to dairy feedstuffs. The best suggestion to livestock cattle is 20 ppb in the total ration dry matter. When producers is to be aware of the possible danger lactating cows consume aflatoxin , it not only can through the feed ingredient containing aflatoxin be toxic to the cow but also appears in the milk and other mycotoxins. within 24 hours. Generally, the levels of aflatoxin Acknowledgements We greatly acknowledge the Department of appearing in milk are 1 to 2 percent of the aflatoxin content of the feed. Lactating cows consuming 20 Science and Technology, Govt. of India for ppb or less of aflatoxin will have less than 0.1 ppb providing financial assistance to work in the of aflatoxin in the milk. Violation of the 0.5 ppb limit research project. will result in condemnation of the milk and References additional milk cannot be sold until it has been 1. Allcroft R and Carraghan RBA (1962) Groundnut toxicity Aspergillus flavus toxin tested and found free from aflatoxin. Research (aflatoxin) in animal products. Preliminary data indicate aflatoxin will clear the system of communications. Vet. Rec. 74, 863 864. lactating dairy cows within 48 hours to 96 hours iSee category: Research article Multimycotoxins in feed by Banu & Muthumary

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Indian Journal of Science and Technology 2. Busby WF and Wogan GN (1981) Aflatoxins. In: Shank R.C. (Ed.), Mycotoxins and NNitrosocompounds; Environmental risks, volume II CRC Press, Boca Raton, FL, pp. 3 - 45. 3. Dmello (1997) Handbook of plant and fungal toxicants. CRS Press, Boca Raton, New York. 4. Diaz S, Moreno MA, Prieta J and Dominguez L (1994) Aflatoxins in milk and milk products: review of analytical methods, with particular emphasis in dialysis methods. Alimentaria 31, 19-23. 5. Ostry V (1999) Microscopic fungi, Mycotoxins and Human health. Casopis Lekaru, Ceskych, 138, 515 521. 6. Sundaram TK, Natarajan A, Chandrasekaran D and Mani K (2001) Feed Analytical Techniques. Laboratory Manual pp:70 71. 7. Van Egmond HP (1989) Aflatoxin M1: Occurrence, toxicity, regulation. In: Van Egmond, H. (Ed.) Mycotoxins in Dairy products, Elsevier, London, pp. 11- 56. http://www.indjst.org Vol.1 No 4 (Sep. 2008)

Indian Society for Education and Environment

iSee category: Research article

Multimycotoxins in feed

by Banu & Muthumary

Ind.J.Sci.Technol.