Fungal enzymatic activity

Fungi are an important source of enzymes for the brewing industry, both as producers of the generic enzymes (0stergaard and Olsen, 2011) and as a host for the transgenic production of non-fungal industrial enzymes (Olempska-Beer et al., 2006). Fungal enzymes are useful as additives to enhance hydrolysis during mashes involving partial or sole addition of raw cereals (Bajomo and Young, 1993) or cereal adjuncts (Linko et al., 1998) and to enhance filterability of wort for mashes of less than fully modified malt. By contrast, fungal enzymatic activity can have quite negative effects if developed as natural contaminants through the endogenous fungal community of barley and malt. Fungi were estimated to make up approximately 0.1% of the microbial biomass present on or in naturally contaminated cereal grains (Van Nierop et al., 2006). Fungal enzymatic activity has repeatedly been linked to poor quality of cereals and malt e.g. insufficient malt modification, malt yield reduction, or reduction of diastatic power of malts (Van Nierop et al., 2006). Such mycobiota-associated modification of the malt parameters may, among other things, result in abnormal fermentation, intensification of wort and beer colour, gushing of beer, off taste and off flavour (Spicher, 1989). Information available on fungal enzymes produced and excreted by fungi in or on cereal grains is limited to the few species showing the most deleterious effects on the commodity, i.e. Fusarium graminearum and F. culmorum, Alternaria spp., Drechslera spp. It can, however, be assumed that excretion of any amylolytic, proteolytic or lipolytic fungal enzyme has a potential to modify the composition of cereal grains and, in turn, may potentially influence the quality of the cereal raw material used in malt production and beer fermentation. Changes that occur range from complete decomposition of components to various degrees of chemical modification. Also, the colour and odour of mould-contaminated barley and malt may be negatively influenced by enzymatic activity, especially when the grain is stored under suboptimum conditions (Christensen and Kaufmann, 1965). Fungi have been demonstrated to produce cellulases (Hoy et al., 1981), xylanases, в-glucanases, and proteases during infection of barley and other cereals (Schwarz et al., 2002). These authors detected an increase in ^-glucanase, xylanase, and protease activities in barley grain and in barley malt upon inoculation with Fusarium graminearum and F. poae, respectively. It was concluded that the enzymes were of fungal origin and that their activity levels appear to be such that they may affect the quality of the malt and the wort produced from it. The quality of beer produced from such worts is also affected. Fungal proteases play an important role here since they have considerable influence on the protein concentration and its composition in the grain and in products made therefrom (Nightingale et al., 1999). Fungal proteases produced by Fusarium spp., which belong to the so-called Fusarium head blight complex of species, have been particularly well characterized. Pekkarinen et al. (2003) detected several alkaline proteases in barley samples after field infection with Fusarium species. The presence of these proteases correlated well with the degradation of barley grain storage proteins (C- and D-hordeins) in the infested grains. Moreover, Hecht and Hippeli (2007) postulated the presence of a heat-stable protease and its degradation of the beer foam protein nsLtp1 as a major cause for beer gushing since they observed considerably lower concentrations of the foam protein in gushing beers as compared to nongushing beers.

Activity of fungal xylanases and glucanases has been suspected as the major reason for the presence of factors leading to premature yeast flocculation (PYF) during beer fermentation. Van Nierop et al. (2004) postulated that degradation of the malt husk arabinoxylan by fungal enzymes produces polysaccharides of sufficient size to cross-link yeast cell lectins resulting in flocculation and precipitation of fermenting yeast at still high sugar concentrations. Resultant beers are of low quality since they have high sugar contents and low end-of-fermentation cell counts (Verstrepen et al., 2003).

Fungal lipases play a major role as pathogenicity factors in many plant pathogenic fungi but have also great importance in biotechnology (Subramoni et al., 2010). Secretion of lipases by F. graminearum has been demonstrated as a virulence factor during the infection of cereals (Voigt et al., 2005).

In response to the secretion of fungal lytic enzymes, the plant reacts by changing its metabolic and gene expression profile (Geddes et al., 2008) and by producing antimicrobial substances to defend itself against attack (van Nierop, 2006). Besides a battery of enzymes that may directly attack microbial cells (^-1,3-glucanases, chitinases, proteases), the products of some enzymes (peroxidases, oxalate oxidase, ammonia lyase) may have adverse effects on microorganisms as well (van Loon et al., 2006). Moreover, pathogenesis-related (PR) proteins (Sels et al., 2008), which are antimicrobial peptides including the thionins, plant defensins, hevein- and knottin-like proteins and non-specific lipid transfer proteins (nsLTP), have been identified as having antifungal properties (Selitrennikoff, 2001). Interestingly, the heat-denatured and glycosylated form of the latter protein is supposed to be one of the major foam-stabilizing proteins in beer (Douliez et al., 2000) and its degradation by fungal proteases has been discussed as a causative agent of beer gushing (Hippeli and Elstner, 2002).

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