Diacetyl from pyruvate

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). 

Scheme 23.5 Metabolic pathways of lactic acid bacteria leading from pyruvate to a-acetolactate and acetoin and chemical diacetyl formation. ALS a-acetolactate synthase, ALDB a-acetolactate decarboxylase, DDH diacetyl dehydrogenase. (Adapted from [72])...

Ketols can also be formed enzymatically by cleavage of an aldehyde (step a, Fig. 14-3) followed by condensation with a second aldehyde (step c, in reverse). An enzyme utilizing these steps is transketolase (Eq. 17-15),132b which is essential in the pentose phosphate pathways of metabolism and in photosynthesis. a-Diketones can be cleaved (step d) to a carboxylic acid plus active aldehyde, which can react either via a or c in reverse. These and other combinations of steps are often observed as side reactions of such enzymes as pyruvate decarboxylase. A related thiamin-dependent reaction is that of pyruvate and acetyl-CoA to give the a-diketone, diacetyl, CH3COCOCH3.133 The reaction can be viewed as a displacement of the CoA anion from acetyl-CoA by attack of thiamin-bound active acetaldehyde derived from pyruvate (reverse of step d, Fig. 14-3 with release of CoA). 

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, pyruvate dehydrogenase complex DS, diacetyl synthase.

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. 

Because of the slow rate of flavin synthesis by the crude enzyme system, it has been difficult to determine the origin of the four-cari)on unit which condenses with 6,7-dimethyl-8-ribityllumazine to yield the o-xylene ring of the flavin. Two possibilities are apparent, either of which is conristent with the data obtained with radioactive glucose (86) (see Table III and Fig. 4). (a) Acetoin and diacetyl arise from pyruvate in metabolic qrstems and either one will condense chemically with 6,7-dimethyl-8-ribitylluma-zine to yield riboflavin. However, addition of diacetyl to the enzyme system from A. gosaypii did not enhance the incorporation of label from 6,7-... 

Three enzymes are involved in the synthesis of 2,3-BD a-acetolactate synthase (EC 4.1.3.18), a-acetolactate decarboxylase (EC 4.1.1.5), and butanediol dehydrogenase (also known as diacetyl [acetoin] reductase Larsen and Stormer 1973 Johansen et al. 1975 Stormer 1975). Two different enzymes form acetolactate from pyruvate. The first, termed catabolic a-acetolactate synthase, has a pH optimum of 5.8 in acetate and is part of the butanediol pathway. The other enzyme, termed anabolic a-acetolactate synthase or acetohydroxyacid synthetase, has been well studied and characterized and will not be discussed here. This enzyme is part of the biosynthetic pathway for isoleucine, leucine, and valine and is coded for by the ilvBN, ilvGM, and ilvH genes in E. colt and Salmonella typhimurium (Bryn and Stormer 1976). 

A novel mechanism for stereoisomer formation was described for Bacillus polymyxa. The RR-acetoin formed from pyruvate is converted into RR-butanediol by diacetyl (acetoin) reductase. The same enzyme reduces diacetyl to RR-acetoin. An S-acetoin-forming diacetyl reductase converts diacetyl to SS-acetoin. The racemic acetoin molecules are acted upon by a butanediol dehydrogenase, which generates either RR-butanediol or meso-butanediol (Ui et al. 1986). 

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. 

Amino-5-hydrazinopyrazole dihydrochloride 300 is a good source for the synthesis of this type of heterocyclic compound [78JCS(P1)885] and it was prepared by reaction of malononitrile with two equivalents of hydrazine. Reaction of 300 with ethyl pyruvate afforded 301. Unstable hydra-zone 302 formed when 300 was boiled with diacetyl rapidly cyclized to 303. Reaction of 300 with benzil gave 304 directly, which gave an acetyl derivative and resisted reductive deamination. On the other hand, a polymer was isolated from the reaction of 300 with glyoxal (Scheme 65). 

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). 

Precursors. Precursors for this reaction are compounds exhibiting keto-enol tau-tomerism. These compounds are usually secondary metabolites derived from the glycolysis cycle of yeast metabolism during fermentation. Pyruvic acid is one of the main precursor compounds involved in this type of reaction. During yeast fermentation it is decarboxylated to acetaldehyde and then reduced to ethanol. Acetone, ace-toin (3-hydroxybutan-2-one), oxalacetic acid, acetoacetic acid and diacetyl, among others, are also secondary metabolites likely to participate in this kind of condensation reaction with anthocyanins. 

Unfortunately diacetyl formation is still not well understood. Acetoin formation occurs either by nonspecific interaction of acetaldehyde with the a-hydroxyethyl thiamine pyrophosphate intermediate in pyruvate decarboxylation (209) or by decarboxylation of a-acetolactate (210), which in turn arises either from interaction of pyruvate with a-hydroxyethyl thiamine pyrophosphate (211) or as a specific intermediate in valine biosynthesis (212, 213). Diacetyl does not appear to be formed directly from acetoin (208, 214). It is formed from a-acetolactate, in absence of cells, by O2 oxidation (215), and even under N2 (216), although an oxidation must occur. It is also formed from acetyl CoA (217, 218), probably by interaction with a-hydroxyethyl thiamine pyrophosphate [cf. stimulation by acetyl CoA addition to a solution of pyruvate and pyruvate decarboxylase (2i5)]. It is not known whether this involves a specific enzyme or is a mere side reaction. 

Bertrand, 1994 Allen, 1995) decanal and ( )-2-nonenal, on the other hand, are associated with sawdust or plank odour (Chatonnet and Dubourdieu, 1996 1998). The principal carbonyl compound formed in MLF is 2,3-butanedione (diacetyl), whose level can improve, or affect, the wine with its butter-like or fat note (Davis et al., 1985). Diacetyl and 3-hydroxy-2-butanone (acetoin, the reduced form of diacetyl) are produced by pyruvate metabolism of yeasts and lactic bacteria, and their levels may increase two or three fold with MLF depending on the lactic bacteria strain involved (Davis et al., 1985 Martineau and Henick-Kling, 1995 Radler, 1962 Fornachon and Lloyd, 1965 Rankine et al., 1969 Mascarenhas, 1984). For diacetyl in wine sensory thresholds ranging from 0.2mg/L (in Cbardonnay) to 0.9mg/L (Pinot noir), and 2.8 mg/L (Cabernet Sauvignon wine), are reported (Martineau et al., 1995). 

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. 

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