Citrate Metabolism

Although rapid fermentation of lactose and production of lactic acid is a primary requirement for dairy lactic acid bacteria, the ability of selected strains to ferment citrate and form diacetyl is also an important property in many dairy products. Diacetyl contributes buttery aroma and flavor attributes in cultured butter, buttermilk, sour cream, and Gouda and Edam cheeses. Citrate fermentation also results in formation of CO2, which is responsible for eye development in Dutch-style cheeses. Despite the practical importance of this fermentation, however, only recently have the key biochemical and metabolic events been defined.

A. Diacetyl Synthesis

Under ordinary conditions, citrate fermentation and diacetyl formation occur only in those strains of lactic acid bacteria that contain genes coding for transport and metabolism of citrate. Among the dairy lactic acid bacteria, citrate utilization is most often associated with Leuconostoc spp. and selected strains of Lactococcus sp. Accordingly, plasmids containing genes coding for citrate transport have been found in those strains that ferment citrate (Lopez et al., 1998). In Lc. lactis subsp. lactis biovar diacetylactis, citrate fermentation is linked with an 8-kb plasmid, whereas in Leuconostoc, citrate genes are associated with plasmids as large as 22 kb. These plasmids contain a cluster of genes that encode citrate permease (CitP) in Lc. lactis subsp. lactis biovar diacetylactis and CitP and citrate lyase in Leuc. paramesenteroides (Martin et al., 1999).

How citrate-fermenting lactic acid bacteria actually form diacetyl has been the subject of considerable debate. Two pathways have been proposed. In both pathways, citrate is transported by the pH-dependent CitP that has optimum activity between pH 5 and 6. Transport is mediated by a PMF; however, as described below, the net bioenergetic effect of citrate metabolism may actually be an increase in the PMF. Intracellular citrate is then cleaved by citrate lyase to form acetate and oxaloacetate (Fig. 10). Although acetate is ordinarily released into the medium, oxaloacetate is decarboxylated to pyruvate by oxaloacetate decar-boxylase. Importantly, the evolved CO2 can cause eye formation in some cheeses. Although lactic acid bacteria could conceivably reduce all excess pyruvate to

Figure 10 Citrate fermentation pathway in lactic acid bacteria. The dashed line indicates the nonenzymatic, oxidative decarboxylation reaction.

lactate via lactate dehydrogenase, this does not normally occur. This is because pyruvate reduction requires NADH, which is made during glycolysis, but which is not formed in the citrate fermentation pathway. Using NADH to reduce citrate-generated pyruvate would quickly deprive cells of the NADH pool necessary to reduce pyruvate produced during glycolysis. Instead, excess pyruvate is decar-boxylated by pyruvate decarboxylase in a thiamine pyrophosphate (TPP)-depen-dent reaction, and acetaldehyde-TPP is formed. Some researchers have proposed that an enzyme (diacetyl synthase) is responsible for converting acetaldehyde-TPP (in the presence of acetyl-CoA) directly to diacetyl. However, no evidence for the presence of diacetyl synthase currently exists. Instead, the accepted alternative pathway for diacetyl synthesis involves first a condensation reaction of acetaldehyde-TPP and pyruvate catalyzed by a-acetolactate synthase. This enzyme apparently has a low affinity for pyruvate in Lc. lactis subsp. lactis biovar diacetylactis (Km = 50 mM); thus high concentrations of pyruvate are necessary to drive this reaction (Snoep et al., 1992). The product, a-acetolactate, is unstable in the presence of oxygen and is next nonenzymatically decarboxylated to form diacetyl. This oxidative decarboxylation pathway is now supported by substantial biochemical, genetic, and nuclear magnetic resource evidence.

Figure 11 Citrate transport in lactic acid bacteria. Citrate is transported via CitP and lysed to form oxaloacetate. Decarboxylation of the latter consumes a proton and forms pyruvate, which can be converted to diacetyl (dashed line). Lactate formed via sugar metabolism (or from citrate) is effluxed in exchange for citrate.

Figure 11 Citrate transport in lactic acid bacteria. Citrate is transported via CitP and lysed to form oxaloacetate. Decarboxylation of the latter consumes a proton and forms pyruvate, which can be converted to diacetyl (dashed line). Lactate formed via sugar metabolism (or from citrate) is effluxed in exchange for citrate.

Although utilization of citrate by lactic acid bacteria requires several enzymatic steps, it appears that citrate fermentation provides cells with no obvious benefits, as ATP-generating reactions are absent in this pathway and citrate consumption results only in excretion of organic endproducts and CO2. Why then do cells ferment citrate? As noted earlier, the driving force for transport of citrate is the PMF, with divalent citrate transported in symport with a single proton (Fig. 11). However, during the oxaloacetate decarboxylation reaction, a cytoplasmic proton is consumed, resulting in an increase in the cytoplasmic pH and an increase in the ApH component of the PMF. In addition, when citrate-utilizing bacteria are grown in the presence of a fermentable sugar and lactate is produced, efflux of monovalent (anionic) lactate can drive uptake of divalent (anionic) citrate. Thus, CitP acts as a electrogenic precursor-product exchanger, with a net increase in the Ay or electrical component of the PMF. Both of these mechanisms (electro-genic exchange and decarboxylation), therefore, result in an increase in the metabolic energy available to the cell (Bandell et al., 1998).

B. Enhancing Diacetyl Formation in Dairy Products

Even among citrate-fermenting lactic acid bacteria, the amount of diacetyl formed in dairy products is relatively low (<2 mg/L), and there is much interest in manipulating growth conditions and cultures in an effort to enhance diacetyl production in cheese and cultured milk products. Because citrate transport via CitP re quires low pH (see above), citrate-fermenting strains are usually combined with acid-producing strains during manufacture of cultured dairy products. Oxygen can also stimulate diacetyl formation by as much as 30-fold (Boumerdassi et al., 1996). Presumably, high atmospheric oxygen can reduce activity of lactate dehydrogenase and accelerate the oxidative decarboxylation reaction responsible for diacetyl synthesis. In addition, oxygen can oxidize NADH, thereby slowing the rate at which diacetyl is reduced to acetoin or 2,3-butanediol (see Fig. 10). Another strategy considered for enhancing diacetyl formation involves genetic modification of the cultures. Several metabolic steps have been identified at which mutations or blocks will lead to increased production of diacetyl. Inactivation of lactate dehydrogenase, for example, results in excess pyruvate, and such cells could theoretically produce more diacetyl than wild-type cells (even non-citrate-fermenting lactococci have been genetically manipulated to produce diacetyl). Enhanced expression of plasmid-borne copies of genes coding for a-acetolactate synthase or NADH oxidase in Lc. lactis also enhances diacetyl formation by increasing the concentration of a-acetolactate available for oxidative decarboxyl-ation (Benson et al. 1996; de Felipe et al., 1998). Similarly, inactivation of the gene coding for a-acetolactate decarboxylase, the enzyme that forms acetoin directly from a-acetolactate, also results in an increase in diacetyl production (Monnet et al., 1997; Swindell et al., 1996).

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