Diacetyl from citrate

Members of three genera are used as cheese starters. For cheeses that are cooked to a temperature below about 39°C, species of Lactococcus, usually Lc. lactis ssp. cremoris, are used, i.e. for Cheddar, Dutch, Blue, surface mould and surface-smear families. For high-cooked varieties, a thermophilic Lactobacillus culture is used, either alone (e.g. Parmesan) or with Streptococcus salivarius ssp. thermophilus (e.g. most Swiss varieties and Mozzarella). Leuconostoc spp. are included in the starter for some cheese varieties, e.g. Dutch types the function is to produce diacetyl and C02 from citrate rather than acid production. 

The production of fermented milks no longer depends on acid production by the indigenous microflora. Instead, the milk is inoculated with a carefully selected culture of LAB and for some products with LAB plus lactose-fermenting yeasts (Table 10.12). The principal function of LAB is to produce acid at an appropriate rate via the pathways summarized in Figure 10.12. The yoghurt fermentation is essentially homofermentative but the characteristic flavour of cultured buttermilk is due mainly to diacetyl which is produced from citrate by Lactococccus lactis ssp. lactis biovar diacetylactis, which is included in the culture for this product (Figure 10.31). 

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). 

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

After adding diacetyl dioxime (dimethylglyoxime) solution, nickel ions are extracted from citrate-buffered solution with chloroform, re-extracted with 1 m hydrochloric acid from the organic phase and determined by means of atomic-absorption analysis. 

Fig. 10.33. Formation of diacetyl and butanediol from citrate by Streptococci. 1 citratase, 2 oxaloac-etate decarboxylase, 3 pyruvate decarboxylase, 4 a-acetolactate synthase, 5 diacetyl reductase, 6 a-acetolactate decarboxylase, 7 2,3-butanediol dehydrogenase...

Cultured butter is made from milk fat to which a mesophilic starter culture has been added to enhance its flavor, principally that of diacetyl. Diacetyl, made from citrate by LAB, enhances buttermilk s storage properties. Lactobacillus lactis or mixed cultures that contain Lb. lactis, Leuconostoc citrovorum, and Leu. dextranicum are used (Early 1998). Fat (cream) is separated from skim milk by centrifugation of milL The cream is pasteurized and inoculated with selected starter cultures. The ripened cream is then churned. The cream separates again into cream butter and its byproduct sour buttermilk, which has limited use because of its high acidity. 

In addition to sugars, several LAB species can metabolize citrate. Citrate fermentation in LAB leads to the production of volatile compounds. In fermented dairy products, these confounds are compounds, such as diacetyl, acetoin, and butanediol, which are responsible for the typical aroma of many fermented dairy products. Therefore, citrate metabolizing LAB, such as L. lactis subsp. lactis biovar. diacetylactis (L. diacetylactis) and some Leuconostoc and Weissella species, are currently used as starter and adjunct cultures for the production of these compounds. However, in other fermented products, such as wine, beer, and sausages, the volatile compounds produced from the fermentation of citrate by LAB are considered off-flavors, and their presence should be avoided. Citrate utilization by LAB has been previously described in detail (Quintans et al. 2008) and summarized in the first edition of this book (Mayo et al. 2010). Therefore, in this chapter, we are presenting only citrate metabolism in LAB in the context of recent achievements. 

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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