Diacetyl fermentation

Ishii, N., Matsumura, M., Tanaka, H. and Araki, K. 1995. Diacetyl fermentation coupled with pervaporation using oleyl alcohol supported liquid membrane. Bioprocess Biosyst. Eng. 13 119-123. 

Diacetyl, acetoin, and diketones form during fermentation. Diacetyl has a pronounced effect on flavor, with a threshold of perception of 0.1—0.2 ppm at 0.45 ppm it produces a cheesy flavor. U.S. lager beer has a very mild flavor and generally has lower concentrations of diacetyl than ale. Diacetyl probably forms from the decarboxylation of a-ethyl acetolactate to acetoin and consequent oxidation of acetoin to diacetyl. The yeast enzyme diacetyl reductase can kreversibly reduce diacetyl to acetoin. Aldehyde concentrations are usually 10—20 ppm. Thek effects on flavor must be minor, since the perception threshold is about 25 ppm. 

Genetic manipulation or cloning offers many possibiUties and perhaps there will be yeast strains especially designed for special beers, ie, types, which are usehil because of low diacetyl formation, high—low ester formation, and insensitivity to pressure or high fermentation temperatures or extracellular enzymatic abiUties (P-glucanases). 

The presence of diacetyl at any stage of the process does not necessarily iadicate an infection by pediococci, because diacetyl is normally formed duting fermentation by oxidation of the precurser 2-acetolactate, which reaches a peak (1—1.2 ppm) at 24—36 h fermentation. The concentration of 2-acetolacetate is usually reduced to values of 0.01 ppm or less, and the diacetyl is reabsorbed by the yeast cells and en2ymatically transformed through acetoia to butanediol. It is extremely important that 2-acetolactate as diacetyl is reduced below the threshold of 0.05—0.10 ppm (ia terms of diacetyl). 

Coffee bioconversions through enzymatic hydrolysis have been used to modify green coffee and improve the finished product (60). Similarly, enzymes have been reported which increase yield and improve flavor of instant coffee (61). Fermentation of green coffee extracts to produce diacetyl [431 -03-8] a coffee flavor compound, has also been demonstrated (62). 

After phytochemical reduction was noted in the case of aldehydes and ketones, interest arose in the behavior of fermenting cells toward compounds containing several carbonyl groups per molecule, such as diketones and quinones. This class deserves special consideration because the simplest representative, diacetyl, as well as its products of reduction, acetylmethylcarbinol (3-hydroxybutanone) and 2,3-butylene glycol, are connected with the metabolism of numerous cells quinones also are biologically important. 

The first experiments made by Neuberg and Nord with the simplest diketone, diacetyl, showed at once that this substance can be hydrogenated phytochemically with comparitive ease. Acetylmethylcarbinol appears as an intermediate (see below), and the end product of reduction is asymmetric and levorotatory. Reduction was effected by the action of fermenting yeast on diacetyl. The 2,3-butanediol that is formed can be isolated by alcohol-ether extraction of the fermentation mixture after concentration in the Faust-Heim apparatus or by steam distillation in an atmosphere of carbon dioxide under ordinary pressure it is then carefully concentrated with the birectifier and obtained in the pure state by final fractionation. 

The phytochemical reducibility of quinones was first demonstrated in the case of p-xyloquinone. This compound is worthy of interest since it is very easily formed from diacetyl by purely chemical means through a type of aldol condensation followed by ring closure. It is reduced to p-xylohydroquinone by fermenting yeast. Benzoquinone, thymoquinone and a-naphthoquinone similarly yield the corresponding hydroquinones. Tetrabromo-o-quinone and anthraquinone proved resistant to attack, while phenanthraquinone could be reduced phyto-chemically to phenanthrahydroquinone in poor yield (9%). Phytochemical reduction can also be accomplished in the dicyclic terpene series. According to unpublished experiments by Neuberg and Peiser, 2,3-dihy-... 

Quantitative determination of diacetyl and 2,3-pentanedione during beer fermentation was achieved by GC/MS method as 2,3-dimethylquinoxaline and 2-ethyl-3-methylquinoxaline, respectively, after reacting with 1,2-diamino-benzene <2006MI52>. 

The enzyme, df-acetolactate decarboxylase (ALDC), has been developed and coimnercialized in the beginning of the nineties. ALDC catalyzes the decarboxylation of hf-acetolactate to acetoin during the primary fermentation, thereby redncing the prodnction of diacetyl and, consequently, eliminating or greatly redncing the need for a matnration period. 

Some of the volatile substances which are produced during fermentation, like acrolein, diacetyl, 2-butanol, allyl alcohol, or acetic acid, are a result of enhanced microbiological activities and may cause an unpleasant flavour (off-flavour) at certain levels thus, elevated concentrations of such compounds are markers for spoilage of the raw material, negative microbiological influences during or after the fermentation process, or a poor distillation technique. 

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). 

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). 

The highly flavorable compound diacetyl is an important by-product of lactic acid bacterial fermentation. The mechanism of its formation has recently been unraveled (35). Diacetyl (measured as diacetyl rather than as diacetyl plus acetoin) is present in higher concentrations in wines with malo-lactic fermentation (cf. Ref. 36). At approximately threshold levels, this compound might contribute favorably to the flavor of wine (7) since increased complexity has been shown to enhance the quality of wine (37). 

Cultured buttermilk is manufactured by fermenting whole milk, reconstituted nonfat dry milk, partly skimmed milk, or skim milk with lactic acid bacteria. Most commercial cultured buttermilk is made from skim milk. Mixed strains of lactic streptococci are used to produce lactic acid and leuconostocs for development of the characteristic diacetyl flavor and aroma. Buttermilk is similar to skim milk in composition, except that it contains about 0.9% total acid expressed as lactic acid. The percentage of lactose normally found in skim milk is reduced in proportion to the percentage of lactic acid in the buttermilk. According to White (1978), the fat content of buttermilk usually varies from 1 to 1.8%, sometimes in the form of small flakes or granules to simulate churned buttermilk, the by-product of butter churning. Usually 0.1% salt is added. 

Michaelian, M. B., Hoecker, W. H. and Hammer, B. W. 1938. Effect of pH on the production of acetylmethylcarbinol plus diacetyl in milk by the citric acid fermenting streptococci. J. Dairy Sci. 21, 213-218. 

The newest enzyme for use in beer is acetolactate decarboxylase, used to decrease the fermentation time, by avoiding the formation of diacetyl. Externally or internally produced a-acetolactate decarboxylase transforms the a-acetolactate to acetoin (acetylmethylcarbinol) without the enzyme, acetolactate goes to diacetyl, and then a secondary fermentation slowly reduces it to acetoin. Avoiding or reducing the secondary fermentation results in significant reduction in storage capacity and money tied up in inventory Q). Normally acetolactate forms by the thiaminepyrophosphate-catalyzed acyloin condensation of acetaldehyde and pyruvic acid (2) or by the condensation of two pyruvic acid molecules to yield acetolactate and CC. Acetolactate is important in the synthesis of isoleucine and valine by the yeast. The acetolactate left at the end of the primary fermentation is oxidized spontaneously in a nonenzymatic reaction to diacetvl and C0.> (Eqn. 1)... 

Typically the level of diacetyl might be 0.10-0.15 mg/L, well above the taste threshhold level of 0.05 mg/L. The purpose of the secondary fermentation is to allow the excess acetolactate that diffused out of the yeast cells to diffuse back in and be reduced to acetoin, before it is spontaneously converted to diacetvl that secondary fermentation can take several weeks (J. Power, J.E. Siebel Sons, Personal Communication). If acetolactate decarboxylase is used it then decarboxylat.es the acetolactate to produce C07 and acetoin in... 

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