Screening of aflatoxigenic property of some Aspergillus flavus isolated from sunflower seeds and its products at sunflower oil refineries

Narasimhan Banu¹*, and John Paul Muthumary²

¹Department of Biotechnology, Vels University, Pallavaram, Chennai – 600 117, Tamil Nadu, India

²Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai – 600 025, Tamil Nadu, India

ABSTRACT:

Aspergillus flavus has become the most widely reported food-borne fungus, reflecting its economic importance and relative ease of recognition as much as its ubiquity. It is especially abundant in the tropics, and it has a particular affinity for nuts and oilseeds as substrates, although the reason is not understood. It is the main source of aflatoxins, the most important mycotoxins in the world’s food supplies. It is not necessary that all A. flavus isolates are toxigenic. Some are non-aflatoxigenic. Their toxin producing capability and the amount of aflatoxin production will vary among the A. flavus isolates from various substrates of sunflower oil refinery. Fifty-seven different isolates of Aspergillus flavus were isolated from different samples viz., sunflower seeds, kernels, oil cake, de-oiled cake, raw oil and filtered oil of sunflower oil refineries namely, Nayan Proteins, Lakshmi industries, Raviprakash refinery and Tamil Nadu Agro Industries Corporation Limited. The isolates were grown in YES medium containing 20 % sucrose and 2 % yeast extract. Among these, 52 isolates were aflatoxigenic and produce only aflatoxin B1. The other 5 isolates were non-aflatoxigenic. But all the 57 isolates were not known to produce aflatoxin B2, aflatoxin G1 and aflatoxin G2. Presence of aflatoxins in food and feed and strong evidence of their association with human carcinogenesis, the problem of mycotoxins especially aflatoxigenic property of Aspergillus flavus from sunflower seeds and its products were regarded as worthy of investigation.

KEYWORDS: Aflatoxigenic, aflatoxins, Aspergillus flavus, non-aflatoxigenic, sunflower seed

INTRODUCTION:

Aflatoxins are produced in nature only by A. flavus, A. parasiticus and the recently described A. nominus. Aflatoxins are most toxic, teratogenic, carcinogenic and immunosuppressive agents. The four major naturally produced aflatoxins are known as aflatoxins B1, B2, G1 and G2. 'B' and 'G' refer to the blue and green fluorescent colours produced under UV light on Thin Layer Chromatography plates, while the numbers 1 and 2 indicate major and minor compounds respectively. Aflatoxins are both acutely and chronically toxic to animals, including man. They produce four distinct effects: acute liver damage, liver cirrhosis, induction of tumors, and teratogenic effects (Stoloff 1977). When aflatoxin B1 and B2 are ingested by lactating cows, a proportion of Ca 1.5 % is hydroxylated and excreted in the milk as aflatoxins M1 and M2 (Frobish et al. 1986), compounds of lower toxicity than the parent molecules, but significant because of the widespread consumption of cow's milk by infants. Because of their high toxicity, low limits for aflatoxins on food and feeds have been set by most countries (van Egmond 1989)

In 1974 an outbreak of hepatitis that affected 400 Indians, of whom 100 died, almost certainly resulted from aflatoxins (Krishnamachari et al. 1975). The outbreak was traced to maize heavily contaminated with A. flavus, and containing up to 15 mg/Kg of aflatoxins. Consumption of toxins by some of the affected adults was calculated to be 2-6 mg in a single day. It can be concluded that the acute lethal dose for adult humans is of the order of 10 mg.

Aflatoxin contamination in oilseeds has been reported by Goyal (1992). Aflatoxin contamination of groundnut has been widely reported (Flach et al. 1992; Isa and Abidin 1996). Aflatoxin contamination is also known to be common in oilseeds other than groundnut such as cottonseed, mustard seed, coconut and palm kernel, soybean, coconut oil (Philip and Menon 1980), olive (Letutour et al. 1983) and other vegetable oils. Aflatoxin contamination of sunflower seeds and corn has been reported from different regions of the world (Christensen and Dorworth 1966; Subramanian and Rao 1974; Rao et al. 1979; Reddy and Reddy 1983; Singh 1983; Basha and Pancholy 1986; Sterzelecki and Cadestrazelecka 1988).

Sunflower seeds were shown to support aflatoxin production. This was first reported by Nagarajan et al. (1974). They studied the toxin production in five varieties of sunflower seeds. Under laboratory conditions, considerable variation in toxin production was seen among the five varieties. The possible causes for the low toxin production on whole seeds have been examined. The hard seed coat of the whole seeds impedes the penetrability of the fungus thus resulting in low toxin production. High levels of aflatoxins were found in sunflower seeds in Tunisia (Anonymous 1978), and oilseeds in Natal, Union of South Africa (Dutton and Westlake 1985). Vijayalakshmi and Rao (1985) and Banu and Muthumary (2005) isolated some mycotoxin producing fungi from sunflower seeds. Chulze et al. (1985) studied the aflatoxin production in sunflower varieties and hybrids. They selected 4 varieties and 11 hybrids to determine aflatoxin susceptibility. The samples were analysed after 7 days of inoculation with Aspergillus parasiticus NRRL 2999. They concluded that the varieties were more susceptible than the hybrids.

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and A. parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium with and without zinc enrichment by Chulze et al. (1987). On a synthetic culture medium the strains behaved in different ways according to the zinc concentrations. In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates. Dalcero et al. (1989) studied the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus in sunflower seeds. A mixture of spores of both strains was inoculated in sunflower seeds at 0.90 aw and incubated for 42 days at 28±1 ºC. The cultures were observed and analysed every 7 days to determine the infection level of the seeds and the production of aflatoxins. In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.

Aspergillus flavus isolated from sunflower seeds were tested for their aflatoxin content by Suryanarayanan and Suryanarayanan (1990). The presence of mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds used for human consumption were carried out by Jimenez et al. (1991).

Sunflower seed-borne fungi play an important role in the physico-chemical properties of the oil (Vijayalakshmi and Rao 1993). The oil samples extracted from fungal infected seeds contain a high amount of free fatty acid. The increase in free fatty acid contents which is an indication of seed deterioration may be attributed to the activity of lipases produced by the seed-borne fungi. Abdel-Mallek et al. (1993) studied sixty-three isolates of Aspergillus, Penicillium, and Fusarium isolated from corn grains and sunflower seeds for the production of mycotoxins. Eighteen isolates of Aspergillus, 18 isolates of Penicillium and 6 isolates of Fusarium proved to be toxic and produced mycotoxins. Eleven different known mycotoxins were detected in the chloroform extracts of the different isolates tested and these are aflatoxin B1, B2, G1 and G2, sterigmatocystin, ochratoxin A, citrinin, penicillic acid, rubratoxin B, diacetoxyscirpenol and zearalenone.

Isolates of Aspergillus flavus were screened for aflatoxin production on peanuts and in a nutrient solution (Diener and Davis, 1966). About 80 % of the A. flavus isolates produced aflatoxin. Ninety per cent of the isolates produced primarily AFB1, whereas about 10 % produced both AFB1 and AFG1. Davis et al. (1966) studied the production of AFB1 and AFG1 by isolates of A. flavus in 20 % sucrose and 2 % yeast extract medium incubated as stationary cultures for 6 days at 25 ºC.

Aflatoxin production of nine strains of Aspergillus flavus and one each of A. versicolor, A. penicilliformis and A. niger isolated from Delhi soils on different media was studied by Maggon et al. (1969). Seven strains of A. flavus produced AFB1 and AFB2 but no AFG. The other five isolates produced no detectable aflatoxins. Hesseltine et al. (1970) examined 12 selected strains for their ability to produce aflatoxin on rice and wheat with constant agitation. No aflatoxin was formed by two A. orzyae and two A. flavus var. columnaris strains. One strain of A. parasiticus, which had been in continuous pure culture for 46 years, produced as much as 378 µg/g of total aflatoxin on wheat.

The distribution of aflatoxin producing isolates of Aspergillus flavus group in feeds was studied by Moreno et al. (1988). Twenty-seven of 32 samples contained A. flavus and 21 of them had at least one aflatoxicogenic isolate of A. flavus. Of the 115 isolates analysed, 65 produced aflatoxins, mainly B aflatoxins.

Production of aflatoxins B1, B2, G1 and G2 in pure and mixed cultures of Aspergillus parasiticus and Aspergillus flavus were compared by Wilson and King (1995). Differing percentages of A. parasiticus (NRRL 2999) and A. flavus (NRRL 5520) conidia were used as inoculum and allowed to grow in static liquid culture for 10 days. Production of aflatoxin B1 and B2 increased slightly as the percentage of A. flavus in the A. parasiticus / A. flavus mixed inocula increased. Aspergillus flavus is apparently capable of suppressing accumulation of aflatoxin G1 and G2 by A. parasiticus when these fungi are grown in mixed culture. Milanez et al. (2002) screened 13 strains of Aspergillus spp. isolated from the terrestrial environment in the Brazilian Atlantic Rain forest (São Paulo State/Brazil) for aflatoxin B1, B2, G1 and G2, ochratoxin A and sterigmatocystin on coconut agar medium and moistened corn but none of the tested strains presented any of the mentioned mycotoxins.

Various factors influence the growth, development and toxin production by the fungi in various substrates. These include moisture content, temperature, relative humidity, hydrogen ion concentration, light, infestation by insects and mites, grain condition, drying and aeration of grains during storage (Christensen and Kaufmann 1965). Nutritional and other aspects of the substrate also influence growth and production of toxins by the fungi (Scott 1957).

MATERIALS AND METHODS:

Isolation of culture

Fifty-seven isolates of A. flavus isolated from different samples like seeds, kernels, oil cake, de-oiled cake, raw oil and filtered oil of sunflower oil refineries namely, Nayan proteins (factory 1), Lakshmi industries (factory 2), Raviprakash refinery (factory 3) and Tamil Nadu Agro Industries Corporation Limited.

Inoculation of Aspergillus flavus culture

Two-fifty ml Erlenmeyer flasks each containing 100 ml of YES medium was inoculated with a disc of 10mm diameter of 3 weeks old A. flavus and incubated for eight days at 25 ºC as a stationary culture.

Extraction and estimation of aflatoxin

After incubation, the cultures were killed and the aflatoxins were extracted from culture filtrates and estimated by using the procedure of Davis et al., 1966. Five, ten, twenty and forty microliter of sample extracts were loaded on the TLC plate depending on the concentration of the sample. The plates were developed in a tank containing chloroform:acetone in the ratio of 88:12 for 30 minutes at room temperature (30±1 ºC). After the development the plates were viewed under UV at 365 nm. The blue fluorescence corresponding to the authentic aflatoxin B1 and B2 indicated the presence of aflatoxin B1 and B2 in the sample. The green fluorescence corresponding to the authentic G1 and G2 with lower Rf than aflatoxin B1 and B2 indicated the presence of aflatoxin G1 and G2 in the sample and the aflatoxin content was quantified by evaluation on the plate itself.

RESULTS AND DISCUSSION:

Altogether 57 isolates (isolate nos. 1-50 isolated from the sunflower samples of Nayan proteins (factory 1), Lakshmi industries (factory 2) and Raviprakash refinery (factory 3) and isolate nos. 51-57 were isolated from the Tamil Nadu Agro Industries Corporation Ltd.) were tested for the aflatoxin producing ability. Among these, 5 isolates were non-aflatoxigenic and the remaining 52 isolates were aflatoxigenic (Table 1).

The aflatoxins are undoubtedly the most documented of all mycotoxins and have a wide product presence. Production of aflatoxin is confined to certain strains of Aspergillus flavus and A. parasiticus as well as the newly identified species A. nominus. Aflatoxins are most toxic, teratogenic, carcinogenic and immunosuppressive agents. There are four major aflatoxins namely, B1, B2, G1, G2 and two additional metabolic products namely, M1 and M2, that are of significance as direct contaminants of foods and feeds. In this group of toxins, AFB1 is most potent and is produced most abundantly under certain natural conditions by the fungi. Consequently, extensive studies on the toxicity, biological and biochemical effects of aflatoxins were primarily made with AFB1 in the last several decades (Goldblatt 1969; Busby and Wogan 1981; Anonymous 1989). Consumption of AFB1 contaminated feed by dairy cows results in the excretion of a hydroxylated metabolite, AFM1, in milk. Thus, contamination of AFM1, in milk is a great concern for human health (Cullen et al. 1987; Van Egmond 1989). Although both acute (hepatotoxic) and chronic toxicity (liver cancer) of AFB1 is well established, its carcinogenic effect is of the most concern. Because of their presence in foods and feed and strong evidence of their association with human carcinogeneis, aflatoxins are still a serious threat to human health (Anonymous 1989).

The study on the aflatoxin production from A. flavus showed that they are beyond the tolerance level (20 µg/kg) fixed by the World Health Organization. It is well established that the fungal contaminated food and feed are responsible for animal mycotoxicoses. The fungi reported in the present study including A. flavus shows their ecological importance. However studies on the synergistic or antagonistic effects of all other fungi occurring along with the mycotoxin producing fungi like A. flavus were not carried out in the present investigation. Investigation by other workers (Roy and Chourasia 1990) showed that the growth of A. flavus and the aflatoxin production by the fungus is affected by the biological properties of co-invading fungal partners.

Table 1. Quantification of AFB1 of Aspergillus flavus isolates by TLC

Source	Isolate No.	Aflatoxin B1 (ppm)	Dry weight of mycelium (g)
SSS 2	1	0.53	3.53
OC 1	2	Negative	4.77
SSS1	3	30	4.23
FSK 2	4	1	4.0
OC 3	5	8	3.55
FO1	6	0.66	3.79
SSK 1	7	4	4.56
FSK 2	8	0.33	4.83
OC 1	9	Negative	3.93
OC 1	10	12	0.28
OC3	11	0.53	4.96
RO 2	12	11.2	5.66
FO 1	13	0.33	4.3
SSS 1	14	0.83	3.0
SSD 2	15	0.53	2.67
SSS 2	16	0.33	3.45
RO 3	17	4.16	2.49
RO 1	18	16	2.96
FO 1	19	12	2.79
FO 1	20	0.53	4.08
RO 3	21	7.46	3.58
FO 2	22	6.66	4.31
OC 1	23	2.66	1.93
SSD 1	24	24	1.29
RO 2	25	0.53	3.67
OCD 3	26	4.16	3.28
OC 1	27	1	3.15
RO 3	28	9.33	4.47
DSS 1	29	Negative	3.24
RO 1	30	0.53	4.06
RO 2	31	1	4.1
FO 2	32	0.93	3.1
OC 3	33	Negative	1.34
OC 1	34	Negative	4.6
FKD 1	35	6	2.64
SKD 1	36	1.4	4.3
SKD 1	37	12	3.46
FO 1	38	0.53	4.45
DSS 1	39	17.5	4.03
DFS 2	40	15	5.0
DO 2	41	6	3.57
FO 1	42	30	3.18
DSS 2	43	4	4.85
RO 3	44	11.5	4.45
DFS 2	45	0.83	3.30
FO 1	46	7.5	3.69
FO 2	47	12	3.50
SSS 1	48	5	2.5
SSS 2	49	0.33	2.5
FSS 2	50	8	2.8
SEED	51	1	3.1
SEED	52	8	2.7
KERNEL	53	0.33	2.4
SEED	54	0.4	2.6
DOC	55	15	3.8
SEED	56	0.53	3.0
SEED	57	0.2	2.9

DFS- Direct fresh seed (Factory 1); DSS 1- Direct sundried seed (Factory 1); FO 1- Filtered oil (Factory 1);

FO 2- Filtered oil (Factory 2); FSK 2- Fresh surface sterilized kernel (Factory 2); OC 1- Dilution plate oil cake (Factory 1);

RO 2- Raw oil (Factory 2); RO 3- Raw oil (Factory 3); SSS 1 - Sundried surface sterilized seed 1

SSS2- Sundried surface sterilized seed (Factory 2)

Fifty-seven different isolates of A. flavus were isolated from different samples viz., seeds, kernels, oil cake, de-oiled cake, raw oil and filtered oil of sunflower oil refineries namely, Nayan proteins, Lakshmi industries, Raviprakash refinery and Tamil Nadu Agro Industrial Corporation Limited. They were grown in a nutrient solution consisting of 20 % sucrose and 2 % yeast extract (YES) medium which appeared to be suitable for both production of aflatoxin and for screening fungi for their ability to produce aflatoxins (Davis et al. 1966). Among these, 52 isolates were aflatoxigenic and produce only AFB1. The other 5 isolates were non-aflatoxigenic. But all the 57 isolates were not known to produce AFB2, AFG1 and AFG2.

Isolates of A. flavus vary widely in the amount of aflatoxin produced on natural substrates. Several investigators have made collections of isolates of the A. flavus group from several natural substrates and qualitatively determined aflatoxin production on natural and nutrient media (Wallbride 1963; Rao et al. 1965).

Isolates of A. flavus and A. parasiticus vary in their capacity to produce the different components of aflatoxins. Hiscocks (1965) states that “some isolates of A. flavus produced only the B compound, some only the G, but the majority produced both B and G components”. Schroeder and Ashworth (1966) reported that A. flavus strains produced B1 and B2 but no G1 and G2 on peanuts and rough rice, whereas NRRL 2999 produced all 4 aflatoxins on both substrates. Taber and Schroeder (1967) tested more than 100 A. flavus isolates from Spanish peanuts and did not find an isolate that produced G1 and G2.

Diener and Davis (1966) screened isolates of A. flavus for aflatoxin production on peanuts in a nutrient solution. Ninety percent of the isolates produced primarily AFB1, whereas about 10 % produced both AFB1 and AFG1.

Nagarajan et al. (1974) observed for the first time that the sunflower seeds showed the aflatoxin production by using five varieties of sunflower seeds. A considerable variation in toxin production was seen among the five varieties. From his observation, the hard seed coat impedes the penetrability of the fungus thus resulting in low toxin production. Chulze et al. (1985) studied the aflatoxin production in sunflower varieties and hybrids. The samples were analysed after 7 days of inoculation with A. parasiticus NRRL 2999. They concluded that the varieties were more susceptible than the hybrids. Aspergillus flavus isolated from sunflower seeds were tested for their aflatoxin content by Suryanarayanan and Suryanarayanan (1990).

By using TLC quantification, it was observed that the range of AFB1 production by toxigenic isolates varied from 0.2 to 30 ppm (Table 1). Aspergillus flavus isolate no. 3 isolated from surface sterilized seeds from Nayan proteins and isolate no. 42 isolated from filtered oil collected from Nayan proteins showed maximum level of AFB1 production and isolate no. 57 isolated from sunflower seed of Tamil Nadu Agro Industries Corporation Limited showed minimum level of AFB1 production.

In the present study, 5 isolates of A. flavus was reported as non-producer of aflatoxins (Table 1). It is not necessary to produce aflatoxins by all the isolates of A. flavus. Some of them were found to be non-aflatoxigenic. Among these 5, four of them were isolated from oil cakes and one from sunflower seed.

The production of aflatoxin is usually proportional to the weight of mycelium formed in culture, being a maximum when the biomass reaches its optimal value and rapidly declining from the moment that the mycelium starts to autolyse (Schroeder 1966). At the onset of lysis, degradation of aflatoxin occurs, both being favoured by high aeration and strong agitation of the culture (Ciegler et al. 1966).

In the present study, the weight of the mycelia ranged from 0.28 g to 5.66 g. The dry weight of the mycelium had no effect on the aflatoxin production by A. flavus isolates (Table 1).

CONCLUSION:

In the present study, it is not necessary that all the isolates of A. flavus produce aflatoxins, but the majority produced B1 not B2, G1 and G2. And also the level of AFB1 production of all these 57 isolates of A. flavus were comparatively low than the action level (20 ppb) set by the FDA. This low level may be due to the fact that fungi may find some difficulty to metabolize the oil content as a substrate which provides nutrients for its basic requirement for the growth on sunflower seeds and its products. The oil content of seeds, kernels and oil cake were high compared to de-oiled cake. The oil content of different sunflower samples used for the isolation of A. flavus is supposed to be related to the aflatoxigenic property and the level of aflatoxin production.

ACKNOWLEDGEMENTS:

We greatly acknowledge the Department of Science and Technology, Govt. of India for providing the financial support to carry out the research work.

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Received on 30.08.2010

Accepted on 20.09.2010

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Research J. Science and Tech. 2(5): Sept –Oct. 2010: 102-107