SI8111916A8 - Process for producing fermented alcohol products - Google Patents

Description

Process for the production of fermented alcoholic products

Technical field of the invention

C 12C 11/00

The present invention relates to the field of biotechnology, specifically to a new process for the production of low-diacetyl fermented alcoholic products by fermentation of a carbohydrate-containing substrate by a microorganism.

A technical problem

There was a need to find a new, technologically advanced process for the production of low-diacetyl fermented alcoholic beverages, which has a strong and unaccented odor even at very low concentrations, which makes the products obtained an unacceptable aroma and taste, and diacetyl is difficult to separate from of ethanol by distillation.

The state of the art

When substrates containing carbohydrate, such as malt or grape juice, are fermented with yeast or other microorganisms, various pleg fermentation processes are performed

- 2 alcohols that can cause unwanted by-products. An example is the formation of diacetyl, which has a strong and unpleasant odor even at very low concentrations.

Alcoholic beverages ^, \ as beer or wine, can thus have an unacceptable aroma and taste if the diacetyl content exceeds significantly certain limits, which, for beer, are about 0.1 ppm.

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Diacetyl formation is also unsuitable in the industrial production of ethanol because it is difficult to separate diacetyl from ethanol by distillation. A particular problem arises in the preparation of absolute ethanol, where ethanol is dehydrated by azeotropic distillation with benzene. Diacetyl will accumulate in the benzene phase during azeotropic distillation, which may cause the formation of a mixture of diacetyl and benzene, from which it is difficult to obtain the benzene used for azeotropic distillation.

Typically brewing beer involves fermenting malt with a suitable yeast species, such as Saccharomyces cerevisiae or Saccharomyces carlsbergensis.

Fermentation is usually carried out in two stages, namely in the main fermentation, which lasts normally 7 to 10 days, and in the secondary fermentation - the so-called · maturation process, which can take 3 to 12 weeks. During main fermentation, most carbohydrates in malt are converted to ethanol and carbon dioxide. They reach maturation at low temperature in the presence of a small residual amount of yeast. The purpose of maturation is, inter alia, to precipitate undesirable compounds of high molecular weight and

- 5 diacetyl conversion, 2,3-pentanedione _; Ja-acetolactate and aaceto-α-hydroxybutyrate into compounds, as diols, which do not affect taste and aroma. So e.g. butanediol, which is the end product of the conversion of α-acetolactate and diacetyl into beer, does not affect the taste and aroma at concentrations below 5θ0 horn per liter

The enzymatic and chemical reactions relevant to the diacetyl content of the beer are shown in the following scheme

(diacetyl) (acetoin) (2,3-butanedi Ol) '(ot, β-dihydroxy (val in);

isovalerate)

The precursor of diacetyl, namely α-acetolactate, is produced in fermented yeast by enzymatically catalyzed condensation of pyruvate and thiamine pyrophosphate acetaldehyde, and is an intermediate in the biosynthesis of valine amino acid. α-acetolactate can also be degraded spontaneously by oxidative de-4-carboxylation to give diacetyl [reaction (4)], which is then reduced by reductases in yeast cells present during the brewing process. Decarboxylation of α-acetolactate is a temperature-dependent reaction that takes place relatively slowly at low temperatures, while the subsequent conversion of diacetyl to acetoin and 2,3-butanediol is relatively fast. from beer, which determines the rate of reaction. Similarly, the rate-determining step in the removal of 2,3-pentanedione and α-aceto-α-hydroxy-butyrate spontaneously decarboxylates the precursor of 2,3-pentanedione.

In order to achieve maximum precipitation of high molecular weight substances and beer of satisfactory quality, ripening must take place at a temperature as low as about 0 ° C. At this temperature, however, it may take several months before the acetolactate is completely removed and the resulting diacetyl is reduced by yeast. However, the ripening time can be reduced by allowing the process to proceed at higher temperatures, e.g. 4 or 2 weeks at 40 ° C, 4 or 2 weeks at 5 ° C and 4 or 2 weeks at -4 ° G. Such a process will accelerate the conversion of acetolactate to diacetyl.

With regard to accelerated brewing processes, however, they have been proposed to accelerate the degradation of acetolactate and α-aceto-α-hydroxy-butyrate by briefly heating the beer to 60 or 80 ° C, at J. Inst. Brew., Vol. 79, 4973? p. 43-44.

- 5 Reaction (1) can essentially be completed within 4 to 15 minutes at these temperatures. However, the taste and aroma will be affected to some extent by such rough processing. Heat treatment is also not suitable for conventional brewing operations, and it is inappropriate and economically disadvantageous to heat and re-refrigerate large quantities of beer during the main fermentation and ripening process, as all yeast must be removed before heat treatment and fresh yeast added after heat treatment and cooling. to convert diacetyl to butanediol.

The present invention is based on the knowledge that the slow decomposition of acetolactate to diacetyl shown in reaction (1) of the scheme can be avoided by using the enzymes-ζθ decomposition of acetoi lactate. In principle, any enzyme that causes the conversion of acetolactate can be used for this purpose. An example is the decarboxylation of acetolactate to acetoin; see reaction (4) in the scheme mentioned above.

Other examples are the conversion of acetolactate to α-ketoβ-hydroxy isovalerate by isomerase reaction (6) or the conversion of acetolactate to α, β-dihydroxy isoValerate by a reductive-isomerase reaction (5). The reaction products in both reactions are precursors to the amino acid valine.

Description of solution to a technical problem with implementation examples

The process of the invention is therefore carried out by treating the substrate with an acetolactate-converting enzyme during or after fermentation. An example of a suitable enzyme for the purposes of the present process is acetolactate decarboxylase, which can be obtained from the microorganism Aerobacter aerogenes.

- 6 This enzyme has been described by E. Juni in J. Biol. Chem. 195 (1952), p. 715-734. However, it was not foreseeable that the enzyme could be conveniently used in fermentation processes such as malt fermentation for brewing low diacetyl beer.

In carrying out the process in the sense of the invention, acetolactate is enzymatically decarboxylated to acetoin, thereby avoiding the formation of an undesirable diacetyl having a strong acetolactate odor. Similarly, in other embodiments, the degradation of α-acetolactate to diacetyl is avoided by conversion of the diacetyl precursor by acetolactate reducto-isomerases or isomerases (reactions 5 and 6).

The process can be carried out in connection with conventional brewing of beer. So e.g. acetolactate decarboxylase can be added during the main fermentation or during the ripening process.

The use of this enzyme makes it possible to significantly shorten the ripening process, as acetolactate rapidly decarboxylates to fermentable acetoin without the formation of diacetyl. According to a specific embodiment of the invention, an enzyme is added during the maturation process, whereby the formation of acetolactate is substantially complete after the main fermentation. However, if desired, the enzyme may be added before or during the main fermentation when the pH is higher than during the ripening process.

Instead of using the enzyme in the free state, it can be used in the immobilized state, adding the immobilized enzyme to the malt during or after fermentation. It may also be maintained in the column through which fermented malt or beer passes. The enzyme can be immobilized separately or co-immobilized yeast cells and acetolactate decarboxylase can be used.

The use of an immobilized enzyme enables accelerated continuous brewing of beer, since the fermentation of beer can be achieved by passing the malt dcozi columns containing immobilized yeast and immobilized enzymes, optionally in a co-immobilized state. In this case, the main fermentation and the maturation process are combined into a continuous conversion of the malt to the finished beer, the capacity depending on the volume and diameter of the columns. Such a process saves effort and reduces investment in a manufacturing plant.

The process of the invention may not be used only for brewing beer, but is also suitable for the production of wine, where similar advantages are achieved, in particular reducing the ripening period and simplifying the process. In this context, the use of enzymes for the conversion of acetolactate in combination with so-called low-lactic fermentation is particularly interesting.

This process, which is achieved by Leuconostoc Lactobacillus or Pediococcus micro-organisms, is carried out after the main fermentation of the wine to increase the pH of the product as well as its biological stability and develop the taste of the wine. However, it is strongly desirable to carry out the fermentation as it allows for rapid bottling and

-δε. this greatly improves the revenue of the winemakers. Unfortunately, the process may provoke diacetyl experiments, the formation of which can be reduced by aceto lactate conversion enzymes. Furthermore, the process can be used appropriately for the industrial production of ethanol, as fermentation products are obtained with or without diacetyl-free content, which simplifies the distillation process, especially for azeotropic distillation in the preparation of absolute ethanol, i.e. of pure anhydrous ethanol.

As mentioned) acetolactate decarboxylase, which is e.g. isolated from Aerobacter aerogenes. However, enzymes that convert acetolactate from other sources can also be used, e.g. Bacillus, Enterobacter, Klebsiella, Leuconostoc, Serratia and Streptococcus species, and some Actinomycetes and fungi species.

Suitable acetolactate reducto-isomerase and isomerase can be isolated from bacteria such as E. coli or Aerobacter aerogenes, or from Neurospora Crassa, yeast, Salmonella or plants. Since the enzymes discussed are such that they have an effect on amino acid metabolism, they can be expected to occur in almost all living cells.

Since alcohol fermentation processes are often carried out at a pH of 4 to 5 and many readily available acetolactate decarboxylases derived from microorganisms have optimal stability and activity at pH values above 5,

- 9 it is advisable to use & cetoXactate decarboxylase in the chemically modified state of the invention in order to achieve great stability and / or optimum activity in the pH range between 4 and 5. kdt is indicated in Biochem. 11, no. 22, 1972.

The process of the invention is illustrated in the following by some examples.

EXAMPLE 1?

Batch fermentation and rapid maturation of beer using acetolactate decarboxylase liter stenylbego Fruit The inoculum with malt strength 10.7 P is inoculated with brewer's yeast (Saccharomyces carlsbergensis) in an amount of 20 χ 10θ cells per ml. After 6 days at 10 ° C, the main fermentation is complete and the strength (apparent beer extract is 2.0 ° P. The measured content of free and bound diacetyl (ie α-acetolactate derived diacetyl) in the beer is 0.12 ppm or 0, respectively. 71 ppm (cf. Haukeli, AD and Lie

S.: Journal of the Brewing Institute 1971, 22 »558).

The beer is then decanted from the precipitated yeast and mixed with 100 ml of Kreuzen, i.e. strongly fermented beer made from malt inoculated with yeast 48 hours ago. To this was added 25 mg of acetolactate decarboxylase isolated from Aerobacter aerogenes as described in European Journal of Biochemistry 1970, 14, 133- After 24 hours at 10 ° C, the content of free and bound diacetyl was determined as 0, for secondary fermentation and maturation of beer. 05 ppm oz. 0.10 ppm.

The fivo is then cooled to -1 ° C and allowed to stand for another 2 days at this temperature, then the beer is filtered. '

EXAMPLE 2

Continuous fermentation and rapid maturation of beer

350 g of brewer's yeast (Saccharomyces carlsbergensis) isolated by centrifugation are suspended in 350 ml of 3% sterile sodium alginate solution. Mixture

- 11 • i) added to 10 liters of sterile CO ^ ljmCaC ^ to gelatinize <calcium; ' alginate and form globular particles about 3 mm in diameter containing yeast.

These are allowed to stand in calcium chloride solution for 4 hours at a temperature of 4 ° C and then filled into a 19 cm diameter column.

_......................... j -.......

and 5 ^{cm high} · Sterile malt is pumped through this reactor at 10 ° C at a rate of 0.15 liters per hour, with a malt strength of 10.7 ° P. The dilution in the eluate from the reactor was 2.1 ° P, and gas chromatography showed a free and bound diacetyl content of 0.15 ppm. oz. 2.21 ppm. To one liter of beer from the reactor was added 25 mg of acetolactate decarboxylase produced as described in Example 1. The beer was allowed to stand for 24 hours at 10 ° C and then pumped through another reactor by immobilizing it. yeast, so the holding time is 5 hours. The content of free and bound diacetyl in the eluate was found to be 0.05 ppm, resp. 0.10 ppm. The temperature was then brought to -1 ° C and, after a two-day condition, the beer was decanted from the precipitated material at this temperature and finally filtered.

EXAMPLE 3

Batch fermentation and rapid maturation of beer using acetolactate reducto-isomerase liter of sterile malt were fermented with Saccharomyces carlsbergensis as described in Example 1. After 6 days of main fermentation, the free and bound diacetyl content of beer was measured as 0.10 ppm, respectively. 0.61 ppm. The beer was then decanted from precipitated yeast, and 100 ml of Kreuzen was added as described in Example 1, together with 100 mg of α-acetolactate reductase isomerase produced by E. coli as described by H.E. Umbarger, B. Brown, and E.J. Eyring in the Journal of Biological Chemistry, vol. 235, p. 1425-1432. The beer was then allowed to stand for a further 24 hours at 10 ° C, and the free and bound diacetyl content was determined to be 0.03 ppm. oz. 0.09 ppm. The beer was then cooled to-1 C for a further 2 days at Jtj temperature, after which the beer was filtered.

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An indication of the best known applicant for the economic 'exploitation of the invention'

Batch fermentation and rapid maturation of beer using acetolactate decarboxylase liter of sterile malt with a strength of 10.7 ° P malt were inoculated with brewer's yeast (Saccharomyces carlsbergensis) in an amount of 20 χ 10θ cells per ml. After 6 days at 10 ° C, the main fermentation is complete and the strength (apparent beer extract is 2.0 ° P. The measured content of free and bound diacetyl (ie α-acetolactate derived diacetyl) in the beer is 0.12 ppm or 0, respectively. 71 ppm (cf. Haukeli, AL and Lie

S .: Journal of the Brewing Institute 1971, 77¾ 53θ) ·

The beer is then decanted from the precipitated yeast and mixed with 100 ml of Kreuzen, i.e. strongly fermented beer made from malt inoculated with yeast 48 hours ago. To this was added 25 mg of acetolactate decarboxylase isolated from Aerobacter aerogenes as described in European Journal of Biochemistry 1970, 14; 133. After '24 hour condition at 10 ° C for secondary fermentation and maturation of beer, determine the free and bound diacetyl content as 0.05 ppm or. 0.10 ppm.

The beer was then cooled to -1 ° C and allowed to stand for another 2 days at this temperature, after which the beer was filtered. ·

Claims (5)

A process for the production of low-diacetyl fermented alcoholic products by fermenting a carbohydrate-containing substrate with a microorganism, characterized in that the substrate is treated as malt, during or after fermentation with an acetolactate-converting enzyme, such as acetolactd; decarboxylase or reducto-isomerase, whereby the substrate can be subjected to major fermentation and maturation. Process according to claim 1, characterized in that the acetolactate decarboxylase enzyme which converts acetolactate is obtained from the aerobacter aerogenes microorganism. Process according to claim 1, characterized in that the maturation process comprises a small-lactic fermentation during which treatment is carried out with an acetolactate-converting enzyme. Process according to any one of claims 1 to 3, characterized in that the acetolactate-converting enzyme is used in the immobilized state. Process according to claim 4, characterized in that the enzyme in the form of co-immobilizate with yeast is used.