A Study on the incidence and nature of aflatoxin from animal feeds

Transcription

1 SIRJ-AZASN Volume 1 Issue 1 (2014) ISSN Scrutiny International Research Journal of Advanced Zoology, Animal Science and Nutrition (SIRJ-AZASN) A Study on the incidence and nature of aflatoxin from animal feeds Rajamalar. P and Ravikumar. K, Department of Biotechnology, S.N.R. Sons College, Coimbatore , Tamil Nadu, India. Article history: Submitted 29 April 2014; Accepted 11 May 2014 Abstract The safety characteristics of feed used in animals systems are an essential tool to assure the productivity of those animal exploitations. Safety of feed may be affected by different hazards, including biological and chemical groups. The aim of this preliminary study was to evaluate fungi contamination and the presence of aflatoxins in animal feeds. Total 50 samples were collected from various animal feed shops. These samples were classified as old samples (2 years old) and new samples (2months old). Around 25 old samples and 25 new samples were analyzed. Molds were found in 25,24 old sample showed as a positive results for the presence of fungal isolates (96%) samples and out of 25,11 new samples showed a positive result for the presence of fungal isolates (44%) of the old samples,100% incidence of fungal contamination was found in levels ranging from CFU/g.These fungal isolates were identified as Aspergillus flavus,aspergillus niger,penicillum sp and Rhizopus sp. All the fungal isolates feed samples screen for aflatoxin production using Thin Layer Chromatography. To analyze the different animal feeds based on the storage condition, statistical tools like student t-test, and Two-way ANOVA were used. Key words: aflatoxins, chromatography, feed, penicillum Corresponding author Introduction Rajamalar. P, Research Scholar, Department of Biotechnology, S.N.R. Sons College, Coimbatore , Tamil Nadu, India. E Mail ID: malarbj@gmail.com Different types of agricultural commodities like groundnut, maize, millet, wheat flour, sorghum, black pepper, coconut cake, soybean are contaminated with aflatoxin

2 producing fungi such as Aspergillu flavus, Aspergillus parasiticus etc.,these food grains are contaminated with aflatoxins in the field at pre-harvest and post-harvest stages during storage and in transport. Possible presence of aflatoxin in food and in feeds has a profound effect on their utilization and trade (Palmgren and Hayes, 1987; Patricia et al., 2006; Bauer and Gedek, 1983). Environmental factors affect mycotoxin presence in raw and stored commodities, with temperature, water activity for toxin production optimum ph and the quality of incoming ingredients for the production of foods and feeds (Hegazi and Hamouda, 1995). In the market place, mycotoxin can be a handle to international trade, leading to increased regulation of food that may contain them and removal of the commodity from the market (Line and Brackett, 1995). Aflatoxins are acutely toxic, immune suppressive, mutagenic and carcinogenic compounds. The main target is organ for toxicity and carcinogenicity in the liver. Result carried out in 1987 by the International Agency for Research Cancer (IARC) found that there is sufficient evidence in humans for the carcinogenicity of naturally occurring mixtures of aflatoxin. Several out breaks of aflatoxicosis have occurred in tropical countries, mostly among adults in rural populations with a poor level of nutrition (Peers and Linsell, 1973; Aziz and Refai, 1989; Aziz and Refai, 1990). At the farm level mould growth can result in reduced crop yields and livestock productivity stemming from illness to death due to consumption of contaminated feed (Refai and Sadek, 1968) when present in food in sufficiently high levels, these fungal metabolites can have toxic effect that range from acute to chronic damage even to death (Refai et al., 1990). In our present study, five different animal feeds were taken for the study. They are millet, coconut cake, sorghum, soybean and wheat drift.two types of each feed was used.two years old samples named as OLD and two months old sample named as NEW were used to study the impact of storage condition in the production of aflatoxin. Materials and Methods The total of 50 samples of different feedstuffs received by the market to detect aflatoxin contamination was used in this study. These were poultry ration, concentrates, processed animal feeds, Coconut cake, Wheat graft, Millet, Sorghum and Soybeans. The feeds were collected between August 2011 and September 2011.Two types of feeds was two year old sample and the other type was 2months old sample The media used were Potato Dextrose Agar medium (Shotwell et al., 1966), yeast extract sucrose broth (Bauer et al., 1983) Microflora analysis of different animal feeds The old and new types of different animal feeds were analyzed by spread plating the serially diluted samples (10-7,10-8,10-9 ) on PDA plates, incubating them at 28 paired diluted sample, (10-4,10-5,10-6 ) of each different animal feeds were used in the present study. One ml of each dilution was taken and poured, spreaded into PDA media under Laminar Airflow Chamber to isolate the micro organism such as Aspergillus and Penicillum species after those plates were kept at room temperature for three days. Depending upon the incubation period different types of colonies were observed. 8

3 Characterization and isolated from animal feeds Four different types of colonies are observed in some of the old and new samples of different animal feeds at various dilutions. The characteristics of the colonies were observed and colony was given below: A. colonies on potato dextrose agar at 25 o C were olive to lime green with a cream reverse rapid growth, texture was wooly to cottony to somewhat granular. (a) Colonies on potato dextrose agar at 25 o C were initially white, quickly becoming black with conidial production, reverse was pale yellow and growth produced radial fissures in the agar. (b) Colonies grew quite slowly (after7 days; 2080 mm diameter) sparsely produced pale grey green conidia, and exhibited yellow mycelium, soluble pigment and reverse colors: small clusters of phialides only, stipes and slender and not epically enlarged, and conidia are spherical, smooth walled and tiny ( um in diameter) conidia were observed. (c) Colonies grey very fast and matured within 4days.The pathogenic species grew well at 37 o C.It quickly filled a Petridis (agar surface) with a typically cotton candy like colony, initially white that turned grey to yellowish brown in time. The reverse was white to pale Screening of isolates for aflatoxin production The Aspergillus group, Penicillum group and other fungal isolates were grown on yeast extract sucrose broth for the production of aflatoxin. The broth was kept for incubation in shaker for ten days for the production of aflatoxin by different fungal isolates. After ten days using the mycelium was filtered by using what man no 1 paper. The thick mycelia mat was discarded and the filtrate was stored at 4 o C and used for further analysis (Bauer et al., 1983). Qualitative identification of aflatoxin by Thin Layer chromatography About 100ml of media filtrate was mixed with 5oml of chloroform and transferred to different centrifugation tubes (Singh et al., 1991). The tubes were vortexes at 20s and spun for 5min at maximum. The organic layer was pulled off into new 1.5ml tubes. In fume hood, the tubes were placed in a heating mantle at 65 o C for about 30min or until dry. 10ml of developing solvent was added to solvent tank, and the solvent tank bottom should be covered with 1cm or less depth of solvent and allowed for saturation. 10µ of chloroform was added to re- suspend dried sample and spotted onto TLC plate. The TLC plate were placed in tank, run until solvent front is 2-3 centimeters from top of plate (approx min) (Seitz and Mohr, 1977). Statistical Analysis of aflatoxin producing sample of different animal feeds were done by two methods. Analysis by student s t-test (paired samples). Analysis by Two-way ANOVA Results The animal feeds were classified into two types based on the storage condition two months period samples was considered as new sample and two years period sample was 9

4 considered as old sample.five different types of sample were taken for study-millet, coconut cake, sorghum, soybeans, and wheat grit. A total of 50 samples were screened (25 new samples and 25 old samples) for the production of aflatoxin. Incidence of fungal isolates in various animal feed conditions Table no. 1: Showing the incidence of fungal isolates of various animal feeds (2 years Old samples) Animal Storage Incidence feeds 2yrs (Old) 2mon (New) Old New Millet 5 2 5/5=100% 2/5=40% Coconut cake 5 1 5/5=100% 1/5=20% Sorghum 6 1 5/5=100% 1/5=20% Soybean 4 3 5/5=100% 3/4=75% Wheat grit 4 4 5/5=100% 4/6=66.6% Figure no. 1: Showing the incidence of fungal isolates of various animal feeds (2 months old samples new) OLD Millet NEW Millet Coconut cake Sorghum Coconut cake Sorghum Soybean Soybean Wheat grit Wheat grit The yeast extract sucrose broth flasks were inoculated with fungal isolates and incubated at 28 ± 2 0 C for ten days for the production of aflatoxin. 10

5 Figure no. 2: Screening of fungal isolates for aflatoxin production (a). Flask showing the growth of Aspergillus niger and Rhizopus in YES medium (b).flasks showing the growth of Aspergillus flavus and Penicillium and in YES medium (c) The plates showed fluorescence on exposure to UV light Qualitative identification of Aflatoxin production The Afalatoxin production was confirmed by spotting and running the sample (filtrate) on a silica gel plate. The plates were then observed for fluorescence upon exposure to long wave UV light (365 nm). 11

6 When the fungal isolates were screened all the isolates showed positive results for the production of aflatoxin. Among 25 old samples, 24 samples showed a positive result. Among 25 new samples, 11 samples showed a positive result for the aflatoxin production. Figure no. 3: Showing the aflatoxin production in old and new samples of animal feed MILLET COCONUT CAKE SORGHUM SOYBEAN WHEAT GRIFT NEW OLD Statistical analysis of aflatoxin production samples (Old and New) Analaysis by student s t-test for paired samples The old and new samples showing positive results for aflatoxin production was analyzed by student t-test for paired samples. t-test: Paired two samples for means. The probability value was found to be (one tail) which is less than 0.05.The data concludes that analysis is shows a significant result where the aflatoxin producing samples differ with storage conditions (Gupta et al., 2008; Sharma and Speck, 2000). Analysis of Two-way ANOVA The positive samples (old and new) of different animal feeds were analyzed by Twoway ANOVA using data Analysis Pak.Two factor were taken for study (Kothari et al., 2004; Saha and Sen, 1993). (a) Types of animal feeds : - millet, coconut cake, soybeans,sorghum, wheat grit (b) Storage conditions : - Old (2 years), New (2 months) Table no. 2: Anova: Two factors without replication Feeds Count Sum Average Varience A B C D E NEW OLD

7 Source of variation SS df MS F P- value F crit Feeds Storage conditions Error Total By analyzing the probability value for different animal feed it was found to be 1.7 which is equal / greater to 0.05 and is considered as insignificant where as the probability value for different storage condition was found to be 0.7 which is less than 0.05 and considered to be significant. This analysis concludes that the aflatoxin production does not vary in the different animal feeds but the aflatoxin production varies with the storage conditions. From the data we can conclude that the aflatoxin production is high in old samples, rather than in new samples. Hence the storage condition enhances the production of aflatoxin. Favorable stored condition to be responsible for higher fungal count and aflatoxin detection in these stored feeds and mushrooms (Mbata et al., 2008; Jonathan and Olowolafe, 2001; Dorner and Cole 2002). Discussion Five different animal feeds such as millet, coconut cake, sorghum, soybean, wheat grit were taken for our study. The samples were classified as old samples (2years old) and new samples (2months old).around 25 old samples and 25 new samples were analyzed. Five old samples and five new samples of millet,5 old samples and 5new sample of coconut cake,6 old samples and 3 new samples of Sorghum,5 old samples and 4 new samples of soybean,4 old samples and new samples of wheat grit were tested for the presence of aflatoxigenic fungi and the production of aflatoxin. The loads of various fungal isolates were in the range of CFU/g. The presences of incidence of fungal isolates in various new and old feeds were present in the feed condition table. From the table, we can infer that out of 25,24 old samples showed as a positive result For the presents of fungal isolates (96%) and out of 25, 11 new samples showed a positive result for the presents of fungal isolates (44%) of the old samples,100% incidence of fungal contamination was found in millet, coconut cake, sorghum, wheat grit. Only 80% was found in soybeans and 66.6% in wheat grit were observed. Highest incidence being in old samples irrespective of the feed used, a total of 4 different fungal isolates were isolates and characterized. These fungal isolates were identified as Asper gillus flavus, Aspergillus niger, Penicillum sp. All the fungal isolates from different animal feed samples were tested for aflatoxin production using Thin Layer Chromatography (TLC), which is considered to be a valuable technique and still widely applied in the developing countries. The advantaged being that large number of samples were analyzed and interfering substances contain in extracts obtain from naturally contaminated feeds do not interfere in the measurement of aflatoxin. All the fungal isolates identified showed result for aflatoxin production. Statistical tools like students t-test, Two way ANOVA were used to test the significance of storage condition on aflatoxin production.the probability value of student t- test and Two - way ANOVA confirmed the storage condition has an impact on the 13

8 increased production of aflatoxin. Thus the storage condition is one of the important environmental parameters that increased the aflatoxin production. Conclusion In conclusion storage animal feeds (over age periods) may increase microbial favorable conditions. Therefore necessary precautions in preventing contamination of dried and stored animal feeds with moulds must be ensured, in order to reduce the risk of aflatoxin and other mycotoxins that are deleterious to animal and human health. Reference Aziz, N. and Refai M., Effect of gamma irradiation on the growth of Aspergillus versicolor and activity of sterigmatocystin in dairy cattle feed. Vet. Med. J., 37(8): Aziz, N. and Refai, M., Occurrence of aflatoxins and aflatoxigenic moulds in coffee beans and decontamination by gamma irradiation. J. Egypt. Vet. Med. Ass., 50 (2): Bauer, J., Montgelas, A. V. and Gedek, B., Aflatoxin B1 production in presence of preservatives antimicrobial agents. Dorner, J. W. and Cole, R. J., Effect of application of nontoxigenic strain of Aspergillus flavus and A.parasiticus on subsequent aflatoxin contamination of groundnuts in storage. Journal of Stored Products Research, 38: Gupta, S. P., Statistical methods, Sultain chain and sons educational publishers, New Delhi 37: Hegazi, E. and Hamouda, A., Effect of antimicrobial agents on the growth of toxigenic Aspergillus ochraceus in poultry feedstuffs. J.Egypt.Vet.Med.Ass, 55(4): Jonathan, S. G. and Olowolafe, T. B., Studies on nutrient contents and microorganisms associated with dodo Ikire a plantain snack from Western Nigeria. NISEB Journal, 1(1): Kothari, C. R., Research methodology methods and techniques. 2: Line, J. E. and Brackett, R. E., Factors affecting aflatoxin B1 removal by flavobacterium aurantiacum. J.Food Protec, 58 (1): Mbata, T. I., Ogiehor, S. I. and Obeleagu, M. N., Isolation of filamentous fungi from Yardenit-Baptismal site on the Jordan River. Sudanese Journal of Public Health, 3(4): Palmgren, M. S. and Hayes, A. W., Aflatoxins in Foods: Mycotoxins in Food", Pallenkrogh Academic Press, London, pp Patricia, A., Suzanne, Handrich, P., Cindy Landgren and Bryane, M., Food mycotoxins. Journal of Food science, 6(71): Peers, F. G. and Linsell, C. A., Dietary aflatoxin and liver cancer - a population based study in Kenya. Br J. Cancer, 27: Refai, M. and Sadek, I., Studies on mould contamination of different foods in Egypt. Mykosen, 11(4):

9 Refai, M., Hammad, H., El-Far, F., Hegazi, E., Saleh, N., Abdel-Aziz, A. and Hassan, A., Fungal flora of grains and poultry feeds with reference to aflatoxin and ochratoxin contamination. J. Egypt. Vet. Med.Ass., 50(3): Refai, M., Niazi, Z. M., Aziz, N. H. and Khafaga, N. E. M., Incidence of aflatoxin B1 in the Egyptian cured meat basterma and control by γ-irradiation. Nahrung Food, 47(6): Saha, S. and Sen, J. N., Business statistics, published by New central agency, Calcutta 6: Seitz, L. M. and Mohr, H. W., New method for quantitation of aflatoxin in corn. Cerela Chem., 54: Sharma, K. V. S. and Speck, M. L., Compenmedium of methods for microbiological examination of foods. American Public association, 14 (7): Shotwell, O. L., Hesseltine, C. W., Stubblefield, R. D. and Sarsenoson, W. G., Production of aflatoxin on rice. Appl. Microbiol., 14: Singh, K., Frisvad, J. C., Thrane, U. and Mathur, S. B., An illustrated manual on identification of some seed borne Aspergilli, Fusaria, Penicillia and their mycotoxins. Hellerup, Denmark: Danish Government,Institute of seed pathology for developing countries. 15