Pyruvate, diacetyl production from

Figure 13.4 Diacetyl production from citrate. CL, citrate lyase OD, oxaloacetate decarboxylase LDH, lactate dehydrogenase AS, a-acetolactate synthase PDHC, pyruvate dehydrogenase complex.

Figure 13.5 Diacetyl production from pyruvate. The chemical oxidative decarboxylation of a-acetolactate into diacetyl is shown by a dotted arrow. ALS, a-acetolactate synthase PDHC, pymvate dehydrogenase complex DS, diacetyl synthase.

Hydroxy-2-butanone (acetoin) is a characteristic constituent of butter flavour used for flavouring margarine and can be obtained as a by-product of molasses-based and lactic acid fermentations [49, 71]. The closely related 2,3-butanedione (diacetyl) has a much lower organoleptic threshold than acetoin and is an important strongly butter-like flavour compound in butter and other dairy products [72] in buttermilk, for instance, the diacetyl concentration is only about 2-4 mg [73]. a-Acetolactate (a-AL) is an intermediate of lactic acid bacteria mainly produced from pyruvate by a-acetolactate synthase. In most lactic acid bacteria, a-AL is decarboxylated to the metabolic end product acetoin by a-AL decarboxylase (ALDB) [71] (Scheme 23.5). 

Also acetic acid may arise from a reaction of this type. Most important compounds of this pathway are pyruvic aldehyde, diacetyl, hydro-oxyacetone and hydroxydiacetyl which can easily react with amino acids. The Strecker degradation is a reaction where the amino acid is de-carboxylated and loses its amino group. Reaction products are the Strecker aldehyde and - as an intermediate - an aminoketone which forms a pyrazine by dimerization. This pathway is considered to be most important for the origin of pyrazines in thermal aromas. However, only limited knowledge is available about the fate of the Strecker aldehydes. As we will demonstrate they are very reactive. 

Diacetyl, and its reduction products, acetoin and 2,3-butanediol, are also derived from acetaldehyde (Fig 8D.7), providing additional NADH oxidation steps. Diacetyl, which is formed by the decarboxylation of a-acetolactate, is regulated by valine and threonine availability (Dufour 1989). When assimilable nitrogen is low, valine synthesis is activated. This leads to the formation of a-acetolactate, which can be then transformed into diacetyl via spontaneous oxidative decarboxylation. Because valine uptake is suppressed by threonine, sufficient nitrogen availability represses the formation of diacetyl. Moreover, the final concentration of diacetyl is determined by its possible stepwise reduction to acetoin and 2,3-butanediol, both steps being dependent on NADH availability. Branched-chain aldehydes are formed via the Ehrlich pathway (Fig 8D.7) from precursors formed by combination of acetaldehyde with pyruvic acid and a-ketobutyrate (Fig 8D.7). 

Some species of the LAB group such as Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. dextranicum, and Lactococcus lactis subsp. lactis biovar diacetylactis, are known for their capability to produce diacetyl (2,3-butanedione) from citrate, and this metabolism appears especially relevant in the field of dairy products (Figure 13.4). Actually, selected strains belonging to the above species are currently added as starter cultures to those products, e.g., butter, in which diacetyl imparts the distinctive and peculiar aroma. Nevertheless, in particular conditions where there is a pyruvate surplus in the medium (e.g., in the presence of an alternative source of pyruvate than the fermented carbohydrate, such as citrate in milk or in the presence of an alternative electron acceptor available for NAD+ regeneration) (Axelsson, 2(X)9, pp. 1-72), even other LAB such as lactobacilli and pediococci can produce diacetyl by the scanted pyruvate (Figure 13.5). Thus, in addition to butter and dairy products, diacetyl can be present in other fermented foods and feeds, such as wine and ensilage (Jay, 1982). 

Diacetyl, butane-2,3-dione CH3-CO-CO-CH3, a diketone produced as a byproduct of carbohydrate degradation, m.p. -3°C, b.p. 88.8 °C.D. is a component of butter aroma, and has been found in many biological materials. It is produced by dehydrogenation from acetoin, the decarboxylation product of pyruvate. In microorganisms D. is also produced by reaction of active acetaldehyde with acetyl-CoA. Little is known about the further metabolism of D.It is used as an aroma carrier in the food industry. 

Acetolactic acid, obtained from the condensation of two pyruvate molecules, is the intermediary product in the biosynthetic pathways of valine and leucine (Fig. 5.28). However, 2-acetolactic acid can be decarboxylated in a side reaction into acetoin, the precursor of diacetyl. At a-keto-3-methylbutyric acid, the metabolic pathway branches to form methylpropanal and... 

Other secondary products of fermentation are also derived from pyruvic acid acetic acid, lactic acid, butanediol, diacetyl and acetoin. Their formation processes are described in the following paragraphs. 

Acetoin is the compound at the intermediary oxidation level in the group of three acetoinic molecules present in wine diacetyl, acetoin and butanediol. It is formed from pyruvate, itself a metabolic intermediary product having different origins in microorganisms. 

The main pathway used for the production of BDO from two pyruvate moelcules is combination of these molecules to form carbon dioxide and a-acetolactate, followed by decarboxylation of a-acetolactate to form carbon dioxide and acetoin, and then reduction of acetoin by NADH to form BDO and NAD (Juni and Heym, 1956). The reduction of acetoin is reversible (Syu, 2001). If acetoin is oxidized to form diacetyl instead of being reduced, BDO can be formed by combining two diacetyl molecules to form acetate and acetylacetoin, which is then reduced by NADH or NADPH to acetylbutanediol followed by another reduction to form acetate and BDO (Juni and Heym, 1956). The sum of these reactions forms a cycle known as the BDO cycle (Juni and Heym, 1956). 

Citrate is present in milk, fruit, and vegetables. It can be co-metabolized with sugars by citrateutilizing LAB. Citrate utilization results in an excess of pyruvate, which is thus converted to diacetyl (2,3-butanedione), acetoin (2-hydroxy-3-butanone), and 2,3-butanediol to equilibrate the redox balance of cellular metabolism (Collins 1972 Bartowsky and Henschke 2004). Some LAB can also synthesize 2,3-pentanedione from pyruvate and threonine (Ott et al. 2000). Diacetyl and 2,3-pentanedione are associated with a buttery aroma, which positively contributes to the flavor of a range of fermented dairy products such as butter (MalUa et al. 2008), yogurt (Routray and Mishra 2011), and cheese (Curioni and Bosset 2002). Diacetyl also contributes to wine style, while it is responsible for flavor defects in beer. Diacetyl is widely produced by LAB, including species of the Lactococcus, Streptococcus, Leuconostoc, Lactobacillus, Pediococcus, and Oenococcus genera. 

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