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Mould isolation and identification
A wide variety of moulds were isolated from the sorghum grain and malt (Table 2.2). The mould load of malted sorghum far exceeded that of the unmalted grains. Fusarium verticillioides, Mucor spp. Phoma sorghina and Rhizopus oryzae were the most abundant, being detected in all analysed sorghum malt samples (100% incidence level). A. alternata was also detected at high levels (39-74%), while the incidence of Fusarium chlamydosporum, Aspergillus flavus and Eurotium spp. was relatively low (12-35, 5-31 and 3-21%, respectively). Aspergillus niger, Penicillium spp. Culvularia spp. and Aspergillus fumigatus occurred at less than 2% (results not shown). Thus the fungal population consisted of field (Fusarium spp. P. sorghina and Alternaria alternata) and storage fungi (Mucor spp. Rhizopus spp. and Eurotium spp), suggesting that an increase in moisture content during malting (Williams and McDonald, 1983; Noots et al., 1999) is quite favourable for fungal growth of different genera. The contamination of sorghum malt by a wide range of fungal species agrees with findings by Rabie and Lübben (1984) who earlier demonstrated the contamination of South African sorghum malt samples 65 commercial (floor malting) and 22 industrial (pneumatic malting) by the above fungal species in almost the same order of incidence as was observed in the current study. The top layer of the unturned samples for both germination temperatures had, lower (p< 0.05) incidence of some mould spp. (A. flavus, Eurotium spp. F. chlamydosporum and A. alternata) compared to both the middle and bottom layers. Unlike unturned samples, the mould distribution of turned sorghum malt samples was not influenced (p> 0.05) by either grain bed depth (i.e. whether top, middle or bottom layers), or malting temperature. This is probably due to the fact that the mixing effects of turning reduced differences in temperature, moisture content and gas distribution between the three malt bed layers, ensuring that these conditions were nearly identical throughout the malt bed. Presumably, the blending effects of malt turning ensured even distribution of individual mould propagules across the malt bed. The high levels of mould contamination found in this work raise concerns because of the presence of the potentially mycotoxigenic moulds such as P. sorghina, F. verticillioides and A. flavus and suggests the possibility that the malts could be contaminated with unacceptable levels of mycotoxins. Mycotoxins produced by P. sorghina are involved in the aetiology of onyalai disease (Rabie, Van Rensburg, Van der Walt and Lübben, 1975), while toxins of F. verticillioides (fumonisins, fusarin C and moniliformin) are involved in several human and animal ailments (D’Mello and McDonald, 1997; Marasas et al., 1979). Beside potential mycotoxin contamination, high levels of moulds in malt may not be desirable for the simple reason that they could make products unpalatable, resulting in reduced consumer acceptance and therefore loss to the producer. From this perspective the high level of contamination of malts by even presumably innocuous moulds such as those of the genera Rhizopus and Mucor may not be acceptable (Rabbie and Lübben, 1984; Rabie et al., 1997).
Cytotoxicity and Mycotoxin analyses
The lowest concentration of the DON standard to cause cytotoxic effects in the MTTbioassay was found to be 1-2 g/g. ZEA and aflatoxin B1 did not show any toxicity against the SP 2/0 cells. Unmalted sorghum and the top layer of the unturned, 18-20°Cmalted grains showed no toxicity (100% cell growth) even at the highest concentration of 500 mg/kg examined. Generally, high sorghum malt IC50 values of 62.5–500 mg/kg were obtained which suggest that the malts are relatively non-toxic as those quantities are very large and not normally consumed by humans. However, it is important to note that this assumption pertains only to acute intoxication and does not preclude the possibility of intoxication due to continuous ingestion of sorghum malt over several years (chronic exposure).
Apparently very high levels of aflatoxins were detected in unmalted sorghum (42 g/kg) and malted samples (52–160 g/kg) using the Vicam AflatestTM (Table 2.3). These apparent levels are 8 to 32 times higher than the South African legal limit of 5 g/kg (Department of Health, 1972) and had never before been obtained in South African sorghum malts. It was therefore necessary to test for aflatoxins using a standard method. TLC was thus performed to confirm the Vicam results. One-dimensional TLC results contrasted with the Vicam AflatestTM results, indicating that sorghum samples contained about 0.5 g of aflatoxin B1/kg. However, in fact that was a false positive because the two dimensional TLC showed that the aflatoxin B1 suspected spots obtained on the single dimension TLC were not really aflatoxin B1 spots and that the unmalted sorghum and sorghum malt samples contained less than 0.3 g of aflatoxin B1/kg (minimum detection level). This was confirmed by the detection of a spot of 0.5 g/kg aflatoxin B1 spiked in an unmalted sorghum sample. These results agree with findings by Odhav and Naicker (2002), who reported the absence of aflatoxin in South African sorghum malt samples. In contrast, Trinder (1988) reported the presence of aflatoxins (2-18 g/kg) in South African sorghum malt that have been produced by indoor floor malting, so–called industrial malt. We can only hypothesize about the reason why the Vicam test gave false positive aflatoxin results. Perhaps the sorghum phenols bound to the Vicam Aflatest antibodies which resulted in a higher aflatoxin count. Unmalted and turned samples as well as the top layers of unturned 18-20°C-germinated malt contained less than 0.25 g fumonisin/g (the minimum detection limit). Conversely, middle and bottom layers of the unturned 14-17°C-germinated malt contained 2 g fumonisin/g (Table 2.3). Fumonisin levels seemed related to malt sample toxicity to SP2/0 cells. Both batches of malt contained DON values of 15-20 g/kg and ZEA values of 10-15 g/kg.
This study is the first report of the presence of fumonisin and DON in sorghum malt. Levels of ZEA obtained in this study (15-20 g/kg) are substantially lower than that (387 g/kg) reported by Rabie and Marais (2000), for South African sorghum malt. There are no regulatory levels of DON, ZEA and fumonisins in human foods in South Africa. The United States Food and Drug Aministration (FDA) has the regulatory levels of DON (500 μg/kg) and no specifications for ZEA (Food and Agriculture Organization, 1997). The regulatory levels of fumonisins are 2-3μg/g (FDA, 2001) and therefore fumonisin levels of 1- 2 g/g were within the limit. The levels of DON, ZEA and fumonisins detected in this work are considered to be too low to be of any concern.
1. INTRODUCTION
1.1. Statement of the problem
1.2. Literature review
1.3. Conclusions
1.4. Hypotheses
1.5. Objectives
2. RESEARCH
2.1. The microbial contamination, toxicity and quality of turned and unturned outdoor floor malted sorghum
2.2. Effect of dilute alkaline steeping on the microbial contamination and toxicity of sorghum malt
2.3. Antimicrobial activities of bacterial and yeast cultures in sorghum malting
3. DISCUSSION
3.1. Methodologies
3.2. Mechanisms of microbe inhibition
3.3. Relative merits of the technologies
4. CONCLUSIONS AND RECOMMENDATIONS
5. REFERENCES
6. APPENDIX: Published papers and oral presentations
