Fig. 3.2 Utilisation of sugars during fermentation of an ale wort of original gravity 1.040 (Clutterbuck and Boulton, unpublished data).

tivation. The global effects on yeast metabolism of the presence of exogenous glucose and the mechanisms by which its effects are exerted are discussed in detail in Section 3.4. Some of these effects impinge on sugar uptake. Thus, there are specific and often multiple carriers for individual sugars. The activity of individual carriers is modulated by the spectrum and concentration of sugars present in wort. In particular, glucose appears to be the preferred substrate and when present in the medium its presence inactivates or represses carriers for the uptake of other sugars (Lagunas, 1993).

At least two glucose uptake systems have been recognised: low affinity and high affinity types, which apparently operate by facilitated diffusion (Bisson & Fraenkel, 1983a). The high affinity carrier requires the presence of a kinase although phosphorylation of glucose during uptake has been discounted in view of the evidence that the non-phosphorylable analogue, 6-deoxyglucose, had similar uptake kinetics to glucose (Bisson & Fraenkel, 1983b; Kruckenberg & Bisson, 1990). Both transporters are active with glucose and fructose. The low affinity system is constitutive, whereas the high affinity transporter is repressed in the presence of high glucose concentrations (Bisson & Fraenkel, 1984; Neigeborn et al., 1986). It follows that the role of the high affinity system is to provide an efficient scavenging mechanism in the event of competition for low concentrations of glucose. Repression of the high affinity system is associated with the general catabolite repression phenomenon and has been shown to occur only in fermentative yeast strains (Does & Bisson, 1989). It has been suggested that the low affinity system is merely passive diffusion. However, Gamo et al. (1995) refuted this on the basis that actual uptake rates using this transporter were 2-3 orders of magnitude higher than could be accounted for simply by passive diffusion.

The glucose carriers are influenced by components of the medium other than glucose itself. Thus, it is reported that exhaustion of nitrogen sources during batch growth brings about an irreversible inactivation of the glucose (and other) sugar transporters (Lagunas et al., 1982). This inactivation is apparently due to proteolysis of the carrier molecules (Busturia & Lagunas, 1986; Lucero et al., 1993). The consequences of these effects to brewery fermentation are unclear.

Maurico and Salmon (1992) concluded that differential patterns of loss of glucose transporters due to nitrogen starvation formed the basis of differences in ethanol productivity in two enological strains of S. cerevisiae. Thus, the strain that was capable of the greatest ethanol productivity had a putative second low affinity hexose transporter not subject to inactivation by nitrogen starvation. Presumably this phenomenon could be of significance to brewery fermentation depending on which component of the wort actually limits yeast growth. Should the sugar transporters be inactivated it follows that they would need to be switched on again during the initial lag phase in early fermentation. This would add further support to the contention that the carbon required for sterol synthesis during the aerobic phase of fermentation is supplied by dissimilation of endogenous glycogen reserves (Quain & Tubb, 1982). This could be due to the inability of yeast to assimilate sugars because of inactivation of the transporters, or equally to lack of membrane competence because of sterol depletion.

Sucrose is assimilated via the mediation of an invertase which is secreted into the cell periplasm. In S. cerevisiae the enzyme is encoded by the SUC2 gene and it hydrolyses both sucrose and raffinose (Carlson & Botstein, 1982). Once hydrolysed, the released fructose and glucose are taken up via glucose transporters. In the presence of high glucose concentrations the invertase is repressed via binding of a component Miglp to the SUC2 gene promoter (Neigeborn & Carlson, 1994). The same group have suggested that low levels of glucose (0.1%, w/v) are actually required for maximum transcription of the SUC2 gene (Ozcan et al., 1997).

Maltose utilisation is accomplished using the products of a multigene (MAL) family that occurs at several loci in the yeast genome and is not restricted to a single chromosome. Each locus consists of three genes: MALT which encodes for a maltose permease; MALS encoding for a maltase (a- glucosidase) and MALR which encodes for a post-transcriptional activator of the MALS and MALT genes (Needleman et al., 1984; Michels & Needleman, 1984; Cohen et al., 1985). Both the latter two genes are induced by maltose and repressed by glucose (Busturia & Lagunas, 1985; Cheng & Michels, 1991). The maltose uptake system is an active process requiring cellular energy. Uptake is via a proton symport system in which potassium (K + ) is exported to maintain electrochemical neutrality (Serrano, 1977). As with glucose uptake it has been reported that there are also low and high affinity uptake systems for maltose (Busturia & Lagunas, 1985; Cheng & Michels, 1991). More recently Benito and Lagunas (1992) concluded that the low affinity component was due to non-specific binding of maltose to the yeast cell wall.

Jiang et al. (1997) investigated the inactivation of maltose uptake by glucose. They concluded that in maltose grown yeast cells, two specific signalling pathways exist for sensing the presence of extracellular glucose and that these were responsible for inhibition of maltose uptake. The first pathway was independent of glucose transport and caused proteolysis of maltase permease. The second pathway required glucose transport into the cell and resulted in proteolysis of the maltose permease and rapid inhibition of maltose transport. Wanke et al. (1997) reported that glucose inactivation of the maltose uptake system was modulated via the RAS/adenyl cyclase, protein kinase A signalling system.

The maltose uptake system shows no activity with maltotriose, instead there is a specific constitutive facilitated diffusion carrier (Michaljanicova et al., 1982). Stewart et al. (1995) investigated the reasons for the observation that ale strains are frequently less effective than lager types at fully attenuating high gravity worts. They suggested that in some cases the strain-specific differences were due to the lack of a maltotriose permease. However, environmental effects were also important. Thus, elevated osmotic pressure inhibited uptake of maltose and maltotriose to a greater degree than glucose. High ethanol concentrations inhibited uptake of glucose, maltose and maltotriose. However, at modest concentration (5%, w/v) uptake of maltose and maltotriose were stimulated, an effect attributed to ethanol-induced changes in membrane configuration.

3.3.2 Uptake of wort nitrogenous components

Wort nitrogenous components are heterogeneous in nature. In a Canadian lager wort, Ingledew (1975) reported a rough distribution as: protein, 20%; polypeptides, 30-40%, amino acids, 30^10% and nucleotides, 10%. Of these the amino acid fraction is of most significance to fermentation performance and beer quality.

The uptake of wort amino acids uses a number of permeases, some specific for individual amino acids and a general amino acid permease (GAP) with a broad substrate specificity. Horak (1986) recognised 16 different amino acid transport systems in yeast. Of these 12 are constitutive and the remaining 4 are subject to regulation by the nitrogen sources present in the growth medium, a phenomenon termed nitrogen catabolite repression (Grenson, 1992). In other words, the presence of an exogenous supply of certain nitrogenous nutrients abolishes the utilisation of others by repressing the enzymes responsible for their assimilation. Uptake is an active process requiring energy (Hinnebusch, 1987). The patterns of uptake are complex, several regulatory mechanisms being evident. Thus, the spectrum of permeases present, their specificity, competition for binding to individual permeases and feedback inhibition of specific permeases by amino acids in the intracellular pool and other nitrogenous components are all influential.

The GAP permease is a high-affinity type, which is one of the group subject to nitrogen catabolite repression. Thus, maximum activity of this carrier is only expressed when nitrogen is limiting. Olivera et al. (1993) studied activity of amino acid permeases in chemostat cultures of S. cerevisiae, a technique which allows the effects of various nutrient limitations to be studied. These authors concluded that the specific permeases were likely to be involved in uptake of amino acids for anabolic pathways, notably protein synthesis, whereas the GAP permease and the others, which are subject to nitrogen catabolite repression, had catabolic roles. This provides an explanation as to why, for example, some amino acids are used in preference to ammonia when supplied as a mixture (see Table 3.1). Thus, although nitrogen is a preferred nitrogen source for catabolic reactions, certain amino acids may be used first for direct incorporation into proteins.

Table 3.1 Classes of wort amino acids in order of assimilation during fermentation (Pierce, 1987).

Class A

Class B

Class C

Class D








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