Aspects of the Microbial Ecology of Aflatoxin Production in Nigerian Staple Foods..

Abstract

The examination of the influence of seasonality on the ocurrence patteh of hnyi, bacteria and aflatoxins in the various varfeties of eighteen (18) Nigerian staple Foods and their products; the clarification of the effects of microbial interaction and the establishment of the influence of the bivalent metals, zinc (znZ'), iron (~;~.~'),man~anese ( ~ n ~ 'an)d copper (cu2') on atlatoxin production and nicrobial distribution both qualitatively and quantitatively, was carried out within a two year period - 1996 to 1998 in Enugu and its environs. The results show a significant difference in seasonality of fungal genera (P<0.05) but none for bacteria {P>0.05). Fungal isolations were made in 39.0%, 26.5% and 24.0% of food san~plesd uring the rainy, hat-dry and cold harmattan periods, respectively, while that of bacteria was 9.3%, 5.5% and 4.6% for the hot dry, rainy and cold harmattan seasons, respectively. Of the 15 mould grcups encountered in the study, A.sperg~fhj7 urw.r group [A.j?~r~uosc)c ur red [nost frequently, account in2 for 34.8% of all fullgal isolates in the study, being followed by Perrrcrliirm species (16.8%) and A-rliger (12.0%) among the co~nmonest ones with; Bacil/us strh/ilis accounting for 14.7%, Sfreplo~wccrrsfu eccif'is (14.3%) and Iis~~herichiaci di (13.9%) amongst [he commonest bacteria. The A. j?uvus group also showed the highest incidence during the rainy seasons being isolated in 14.4% of the samples in that season. The incidence declined considerably in samples taken in the hot dry seasons (4.43%) and in the cold harmattan seasons (7.25%). Most other hngi also showed a rainy season prepondence, including A.triger, C/adosyorirrm, 'liicl~odernru(,' urvrrlirriu, Geotrichntu. Succharont~~v.~ and Cwdida species. A.fr~rnigufr~.sa nd Mucor. r~rucedoh owever showed a hot season preponderance while Pcnici/lrtmr and IZhodo/orrr/t~s pecies occurred mostly in the cold harmattan periods of the study. For the bacteria however, 48.1% of all isolates were recorded in the hot dry season, with 28.l%and 23.8% it1 the rainy seasons and culd harmattan seasons, respectively. Maize (252 samples) and peanuts (234 samples) were the foodstutt's most frequently contaminated by fungi while acha (lh,qi/clr.ict csilis Stapf) grains (47 samples) and oil bean seeds (sliced and fermented 42 samples) were those most frequently contaminated by bacteria. DiRerences were observed in terms of microbia! and aflatoxin contamination according to food varieties (seed colour, class and cultivation practice). Thus eg., white maize (49.6%) was more contaminated by moulds than the ye1 low variety (43.5%). White testae peanuts (68.8%) were significantly more contaminated by tnoulds than those with red testae (23, I%)., (P<0.05). The Abakaliki (swamp) rice variety (57.8%) was significantly more contaminated by moulds than the Ugbawk~ (upland rice) (42.2%) (PC0 05) Also, white peanuts had a tnean aflatoxin 0, (AFBI) level of 876 ppb while the red variety had a mean of 510 ppb; Abakaliki rice had 193 ppb against 38ppb for Ugbawka rice. Of all the classes of bods, the highest level of natural contamination with total aflataxins was recorded in oil seeds, eg. coconut seeds (2.732. I ppb); followed by the cereal grains, ey white sorghum (4 l6.4ppb); Protein foods. eg, white cowpeas (70.5ppb) as against a load of 1 1 Zppb for the carbohydrate foods, eg. unripe plantain. Of the 369 A.J/~ivt~sis olates encountered in the study, 126were aflatoxigenic with lnore numbers of the afIatonigenic strains being encountered in hot dry seasons (42. I%), followed by the cold harmattan seasons (29.4%) and the rainy seasons (28.6%) of the study period even though 46.3% of the total A,./lavn~.s was actually sviii encountered in the rainy season as against 30.3% and 29.4% in the hot dry and cold harmattan seasons, respectively. Also, though more food numbers were contaminated by aflatoxins in the hot dry seasons, generally higher concentrations of aflatoxins were recorded in these foodstuffs during the rainy season. Again, varietal differences in seed crops were also recorded in the distributio~ip atterns of both total and aflatoxigenic A. flavrrs eg, white peanuts significantly contained more (6 1) total A&vus, than the red counterpart (231, but only 29.0% of these were aflatoxigenic. The red peanuts, 43.5% were aflatoxigenic. Similarly, the white maize again contained more total A,fivr/.s organisms (42), from which 19.0% were aflatoxigenic while the yellow maize contained 33, from which 48.5% were aflatoxigenic. Some foodstuffs,eg. pigeon peas, oil bean seeds, and cassava tubers contained aflatoxigenic A,J7ctvrrs but contained no aflatoxins, while some, eg, moin-moin and okpa cake (wet-milled steamed cowpeas and bavnbara peanuts) contained no toxiyenic A.JIcrvrr.s but contained some detectable levels of aflatoxins. It was also observed that the majority of food crops associated with the soil environment, eg, cocoyam, yam, peanuts produced largely both the B and G toxins while others that are not associated with this environment, eg. maize, sorghum, millet, produced largely only the B toxins. In coniparison with AJknw.s in monoculture, co-culture studies showed marked reduction of aflatoxin levels by some Fungi eg. Rl?izopus and total inhibition by others, eg. Trichodmncr, still other groups eg. C.Ic~do.spr,riurng, ave a mixed trend of either stimulation or no effect depending on the food substrate. Some bacterial species, eg. P.serrtlomonc~.sa nd J3cherichic1 coli reduced while others like Acetulmcker stimulated. Proter/.s spp generally reduced aflatoxin levels in the solid media (foodcrops) and in the synthetic liquid medium (low salt sucrose . . . ,,. . medium) but stimulated in the semi-synthetic medium (yeast extract sucrose mediurn- YES). Also ~~~~cpfococcus hctis, reduced aflatoxin levels in the synthetic medium but showed no effects in the semi-synthetic medium,while significantly reducing'aflatoxin levels (P<0.05), in all foodcrops (solid media). Some moulds also gave differing responses according to the substrate eg. f~it.art?rrtr~s pp which significantly reduced aflatoxin levels in the YES medium and the general foodstuffs (Pe0.05) completely inhibited the production of aflatoxins in the synthetic medium. Generally, 2n2' and ~ n ~ ' at concentration 5 550ug/g OF food stimulated Af production, both the level of stimulation varied from one food to the other except that with zn2' in oil seeds, a progressive inhibition of aflatoxin production was rather recorded while for the protein foods and oil seeds with ~ nlev~els,' th e stimulation started at metal levels of lOOug and 25uglg respectively and continued till the highest metal concentration (1000ug ~ n ~ ' / ~ roaf n ~ food substrate). A rather constant level of aflatoxins was maintained in all cereals and carbohydrate foods at all concentrations of ~ e " , while in the protein foods, a slight increase in aflatoxin levels occurred till ~ e ' ' concentration of 25ugIg of food substrate, and then a decrease thereafter tilt the highest metal level, but in oil seeds, after the initial increase in aflatoxin levels, at ~ e "co ncentration of IOug/g of food, a decrease followed thereafter till 250-400ug iron concentration. This started increasing again till the highest metal level. With copper in the cereals and carbohydrate foods, after the initid increase in toxin levels up to 250ug metal concentration, a progressive decrease till the highest metal level was recorded, while the progressive decrease with increasing metal concentration occurred from metal concentration of 25-50ug/g protein foods. For the oil seed, after the initial decrease in aflatoxin levels~recorded at cu2' level of 25- 1 OOudg of food, another drop was recorded again at 550ug copper levels which increased again till the highest metal level for coconut seeds, continue to decrease as in peanuts or levels up at a constant lower values as in melon seeds. With metal studies in the liquid media (semi synthetic and the synthetic media), at 2n2' levels 1-25ug/ml, aflatoxin production increased with a slight depression at 25ug/nil and mat weight of the fi111.y~a lso increased. A similar event occurred with hIn2' though the inycelial lnat weight of the A.flavus was essentially constant. With iron in the range 1-25ug/ml, aflatoxin production decreased progressively with mat weight almost constant and with copper levels from 1- 25ug/ml,a depression of Af production progressively occurred even though the mat weight was unaffected. Because of the tcsxicological and economic implications of aflatoxin contamination in these fbod crops, control proyramrne focusing on the knowledge of seasonality, microbial interactions and probably bivalent metal applications to reduce the incidence in these crops is hereby advocated.