Biochemical Tests

A wide variety of methods have been used to quantify the fungal activity in stored grain. Chitin, ergosterol, adenine triphosphate, immunofluorescence, immunoassays, and DNA probes have all been developed (Fleurat-Lessard 2002; Magan 1993). Since, ergosterol is the predominant sterol in most spoilage fungi (ascomycetes and deuteromycetes) and not found in insect pests it has been utilized extensively as an indicator of whether deterioration has occurred in grain. The method was first described by Seitz et al. (1977) and can now be performed relatively quickly and routinely using simple extraction and HPLC. It has thus been used extensively for the in vitro quantification of biomass of spoilage fungi which demonstrated that this does change with culture age (Marfleet

Changes in grain enzyme concentrations, e.g., amylases, due to fungal deterioration are important as they have an impact on processing and bread making quality of flour and dough. However, studies which examined a-amylase, b-amylase, and total amylases of wheat found no correlation with the time to microscopic/visible molding (Magan 1993). Fleurat-Lessard (2002) suggested that for both wheat and malting barley enzyme changes are too small and occur to late as functions of storage conditions and duration, especially with regard to incorporation in a model for decision support systems.

However, there are other studies which suggests the opposite. Fungi colonizing the rich grain substrate under conducive environmental conditions produce a battery of hydrolytic enzymes for degrading the grains and causing the dry matter losses discussed earlier in this Chapter. Flannigan and Bana (1980) and Magan (1993) showed that aw and temperature affect the production of enzymes by fungi during grain colonization, including cellulases, polygacturonase, pectin methyl esterase, 1-4-p-glucanase, b-glucosidase, b-xylosidase, and lipases. Jain et al. (1991) were the first to demonstrate that by using chromogenic 4-nitrophenol substrates in an ELISA well format, rapid quantification could be carried out for a range of hydrolytic enzymes, provided that substrates were available for them. They demonstrated that in both barley and wheat grain at different aw levels (0.85, 0.90, and 0.95) significant increases in N-acetyl-b-d-glucosaminidase were produced when compared to nonmolded dry harvested grain. Grain inoculated with the xerophile E. amstelodami also showed marked increases in a-d-galactosidase.

Magan (1993) extended this and examined stored dry grain with that at different aw levels and temperatures of incubation. This showed that a significant change in the production of some enzymes was evident at times of microscopic and visible molding. Of seven enzymes examined significant changes in b-d-glucoaminidase, b-d-glactosidase, and b-d-glucosidase were observed by the time microscopic growth had occurred. Similar results were obtained with fumonisin producing Fusaria (F. verticillioides, F. proliferatum) by Marin et al. (1998d). Changes could be monitored within 72 h of storage. They also suggested that these enzymes could be used as an early indicator of infection of maize grain by such species and that these enzymes were important in enabling rapid colonization over a wide range of environmental factors. Recent work by Keshri and Magan (1998) and Keshri et al. (2002) have also suggested similar hydrolytic enzymes are an early indicator of fungal activity in vitro on wheat flour-based media and in bread substrates. Thus, potential does now exist for the use of such relatively simple enzyme assays to be used as a possible tool for early detection of fungal activity in cereal grain.

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