EA001075B1 - Medvedev V.N. (RU) - Google Patents

Abstract

1. A process for the production of a fermentable wort, comprising the steps of: (a) liquefaction of cereal material, with the aid of a-amylase and/or endoprotease activity, to obtain a liquefied mash, (b) saccharification of the said liquefied mash in the presence of amylase, and (c) filtration of the liquefied and saccharified mash to obtain a fermentable wort, wherein at least one of the steps (a) and (b) above is carried out in the presence of an enzyme having exo-peptidase activity. 2. A process according to claim 1, wherein the cereal material comprises at least 20% unmalted cereal. 3. A process according to claim 2, wherein the cereal material comprises at least 20% unmalted sorghum. 4. A process according to claim 3, wherein the cereal material comprises at least 50% unmalted sorghum. 5. A process according to any claims 1 to 4, wherein the endoprotease is obtainable from Bacillus amyloliquefaciens. 6. A process according to any of claims 1 to 5, wherein the exopeptidase is present during the liquefaction of the cereal material. 7. A process according to any of claims 1 to 5, wherein the exopeptidase is a fungal exopeptidase. 8. A process according to claim 7, wherein the fungus is an Aspergillus species. 9. A process according to claim 8, wherein the fungus is Aspergillus sojae. 10. A process according to any of claim 1 to 9, wherein the exo-peptidase is an amino-peptidase. 11. A process for producing alcohol, or an alcohol containing beverage, comprising the step of fermenting a wort obtainable in the presence of a yeast capable of producing alcohol, wherein the wort is used obtained according to any of claims 1 to 10. 12. A process according to claim 11, wherein an alcohol-containing beverage is beer.

Description

The invention relates to a method for producing a fermented wort, namely a wort obtained from cereal grains, especially from unmalted grains. Another object of the invention is a method for the production of alcohol and an alcohol-containing beverage using this method of producing wort.

Beer is produced by fermentation of either malted or unmalted grains. In the latter case, the grains are liquefied and saccharified using commercial enzyme preparations to produce a wort that contains fermentable sugars and amino acids or other forms of nitrogen (collectively referred to as forms of “freely available nitrogen” (SDA)) necessary for yeast fermentation.

So, for example, McFadden et al. (MasRabbep Ό.Ρ., s1 a1. Apb C1au1op. M. Vteitpd apb Veuetade 1pbi51pe5 1p1egpaPopa1 (1989), 1, 7781) suggest that when brewing beer, enzymes from unmalted sorghum, such as alpha-amylase, protease, beta-glucose, alpha-amylase of fungi, aminoglucosidase, etc. In addition, McFadden et al. when using unmalted sorghum, they also recommend adding baker's yeast for fermentation.

Bayomo et al. (Ba.) Oto, M.R. apb Woopd, T.A., 1. 1p§1. In gays. (1992) 98, 515-523; Va.) From, M.R. Apb Woipd, T.A., 1. (1994) 100, 79-84 3) data on brewing beer from 100% unmalted sorghum grains are given, despite the fact that the SDA level in wort from unmalted sorghum (51 mg / l) is lower than the value required for fermentation of wort obtained from malted barley.

International Patent Application AO 92/20777 describes a method for producing ethanol, comprising liquefying cereal material from unmalted grains of corn or sorghum using amylase, saccharification of mash using glucoamylase and mushroom acid protease. The method involves the addition of a protease at the fermentation stage and at the saccharification stage. The pH value in the fermented medium varies in the range of 4–5, which means that the fungal exopeptidases are inactive (LeBe, L.E., KeuutoPe, R., VusBut1e (1974) 56, 839-844; Beitap, K. ipb INID, N Norre 8eu1eg'5 2.Pu51oSiet. (1969) 350, 99-104). The disadvantage of this method is that under these conditions, the used proteases of the fungi carry out only endoproteolysis, leading to the release of mainly oligopeptides and a small amount of free amino acids.

The main source for obtaining a general idea in this area should be mentioned Palmer's review (O.N. Pa1teg, Segea1 8c1epse apb TesNpoodu. 1n: Segea1 8c1epse apb TesNpoodu, (1989) eb. Algebeep ishuegeu Rte55, Alboebep, 61 242).

SUMMARY OF THE INVENTION

The present invention relates to a method for making wort from unmalted cereal grains, which, as it turned out, has unexpectedly good properties, such as a high level of freely available nitrogen (hereinafter referred to as SDA), good filterability and good yield of wort. The specified method can also be used in the production of wort from malted grains of grain, but its advantages are especially noticeable when used in the production of wort from unmalted grains, such as unmalted sorghum or a mixture of unmalted sorghum and corn. The benefits are also noticeable when using wort obtained from the so-called “mixed mash”, which is a combination of malted cereals (such as malted barley) and unmalted cereals (such as corn, rice or sorghum).

Thus, the present invention relates to a method for producing fermentable wort, comprising the steps of:

(a) liquefying the cereal material with a substance having alpha-amylase and / or endoprotease activity to produce a liquefied mash, (b) saccharifying the liquefied mash in the presence of alpha-amylase, and (c) filtering the liquefied and saccharified mash to produce a fermented wort, moreover, at least one of the stages (a) or (b) is carried out in the presence of an enzyme having exopeptidase activity.

Suitable enzymes with exopeptidase activity of exopeptidases, preferably thermostable exopeptidases, such as fungal aminopeptidases, however, thermostable carboxypeptidases can also be used. Particularly preferred in accordance with the present invention are aminopeptidases endogenous for fungi from the genus Akretd Shik, and A. shdet, A. ogu / ae, or A.5o.) Ae are more preferred among them.

This method is especially useful when cereal grains are used, including at least 20% unmalted cereals, preferably more than 50% unmalted cereals, such as sorghum grains with additives from unmalted whole ears of corn, rice or other unmalted cereals. It was found that in accordance with the present invention the presence of exopeptidase is very beneficial in liquefying cereal grains.

Together with exopeptidase, an endoprotease is used, preferably obtained from Bacillus arteriosus.

Preferably, said exopeptidase, i.e. an enzyme having exopeptidase activity is an exopeptidase from fungi, preferably from fungi of the genus Accretde Shik, namely, Akretd Shik co.

Preferably, the exopeptidase is an aminopeptidase.

According to another aspect, the invention relates to a method for producing alcohol or an alcohol-containing cast, comprising the step of fermenting the wort in the presence of yeast capable of producing ethyl alcohol, using the wort obtained by the above method.

Preferably, the alcohol-containing beverage is beer, however, it is understood that other alcohol-containing beverage, for example, drinking alcohol, can be obtained from the wort.

The following is a description of the figures illustrating the present invention.

Description of figures

In FIG. 1 shows the pH profile for leucine mycopeptidases from Accret chic tdeg;

In FIG. Figure 2 shows the pH profile for phenylalanine aminopeptidase from Accretum Chic;

In FIG. 3 shows the temperature profile for both aminopeptidases.

The following is a more detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The method is designed for the manufacture of a fermented wort from cereals, the hallmark of which is that under the conditions used, at least one protein present has exopeptidase activity. Aminopeptidases endogenous for fungi are used, especially for AkregdShi8 fungi, since they are quite stable at pH values from 5 to 8, which coincides with the range of values to which the pH drops at the dilution stage. Under the conditions of such temperature and pH values, carboxypeptidases are less useful. At the saccharification stage, slightly more acidic conditions prevail than at the liquefaction stage, and therefore, carboxypeptidase can also be used successfully at this stage, provided that it has sufficient stability in the temperature range of 50-60 ° C, preferably 50-70 ° C.

The use of exopeptidases according to the method of the present invention not only leads to an increase in the level of freely available nitrogen (SDA) in the wort, but also affects the improvement of filterability and an increase in the yield of the wort in comparison with the process embodiment without adding exopeptidases.

The method of the present invention is particularly advantageous when preparing fermented wort from unmalted cereals, even more preferably from unmalted sorghum with optional additives of other cereals, such as corn, wheat, oats or rice. In the context of the present description, the term "cereal" includes sorghum, wheat, barley, oats, rice, corn, etc.

The use of exopeptidases of the present invention also provides benefits in the case of mixed mash, which uses, as a rule, malted cereal material, as well as unmalted material from cereal raw materials (containing, for example, up to 80% or even 90% malted cereal, whereas the remainder includes unmalted cereals), since it was shown that exopeptidases have a positive effect on organoleptic characteristics (taste and / or smell). It was shown in relevant studies that these advantages are manifested when using in brewing both unmalted and malted cereals, as well as their mixtures.

Those cereals, on which the advantages of using exopeptidases are especially obvious, such as the level of SDA, filterability, yield, and organoleptic characteristics, are cereals with a relatively high content of glutelin and prolamine protein fractions. In addition to sorghum, rice also belongs to this category (about 80% glutelin). When using sorghum, you need to pay attention to varieties in order to use those that contain relatively little polyphenol.

With the exception of the addition of exopeptidases, the process of making wort, such as beer wort, is carried out in the usual way. Basically, it involves liquefying the raw cereal material to produce a mash, then saccharifying said mash to produce a wort. It is important to filter before fermenting the wort.

The liquefaction step typically involves grinding the unprocessed cereal material to produce flour with particles of an acceptable size, hydrating them by adding from about 1 to about 4, preferably about 3 parts of water and optionally, depending on the type of endoprotease used, adding from about 50 to about 300 parts per million calcium, preferably 200 ppm Ca ²⁺ . At the same time, the enzymes from Bacteriacereptorium II8 are considered to be less dependent on calcium. And therefore, in this case, there is no need to add Ca ² +. The particle size of the crushed cereals should not exceed 3 mm; not more than 3.5% may exceed 1.3 mm; not more than 1.5% should be less than 0.25 mm. In addition to these, enzymes such as cellulases, β-glucanases and / or other enzymes that destroy plant cell walls can be used.

The liquefaction medium is usually adjusted to a pH of from about 5 to 8, preferably from about 6 to 7, using, for example, calcium hydroxide. It is important to add α-amylase, preferably thermostable α-amylase, as well as endoprotease, to a medium for liquefaction, in a dosage sufficient to carry out at least partial liquefaction of grain starch and cause at least partial protein degradation. Suitable dosages of α-amylase when using Β.Ά.Τ.δ. from about 0.5 to about 2.0, preferably in the range of about 1-1.5 kg per ton. Suitable dosages of proteases, when using the HCV / USG5 protease 2000, are greater than 0.5 kg / ton of grain (kg / ton), preferably greater than 1 kg / ton. When using Raiiktake 400, it should be added at a dose of more than 2 kg / t, preferably more than 5 and even more preferably more than 10 kg / t.

In the process of liquefaction, many stages are carried out at elevated temperature: after the addition of α-amylase and protease, the mixture is maintained at a temperature of from about 40 ° C to about 65 ° C, preferably from about 45 to 55 ° C, and most preferably at 50 ° C, until a sufficient degree of dilution is achieved. The time required for this depends on which cereal or which cereal mixture is used, and usually a period of from about 30 minutes to about 2 hours is satisfactory in this regard. After that, the temperature is gradually increased to about 90-95 ° C, while the rate of increase is not a significant factor, and maintained at this temperature from about 30 minutes to about 1 hour. Then the mixture is cooled to a temperature at which saccharification is carried out: usually it is from about 50 ° C to about 70 ° C, preferably from 55 ° C to 65 ° C, and most preferably about 60 ° C. It is possible to use slightly higher temperatures than 70 ° C, which depends on the thermal stability of the enzymes used in the saccharification stage. To achieve the preferred temperature, saccharifying enzymes are added, such as Wge \ whet Rettech (α-amylase) or Νοναιηνί (recombinant β-amylase), in amounts that usually vary from about 400 g / t to about 1 kg / t in the case of Wge \ hack Regtech. Glucoamylases are also often used. The saccharification process takes from about 30 minutes to about 2 hours, after which the temperature is raised to 75 ° C-85 ° C, in particular in order to inactivate enzymes and undesirable microorganisms, and the mixture is kept at the indicated preferred temperature for about 10 minutes; holding time is not a decisive factor.

The mash thus obtained is then filtered using equipment known in the art for this; satisfactory results are obtained by the funnel with filter paper BSYuyuyet & §syeie11. After filtering, the wort is fermented using suitable yeast, the conditions being selected depending on the yeast strain used and on the ultimate goal of the process; in addition to use in brewing, the present invention contemplates the use of the method in the production of alcohol as biofuel or as an alcoholic beverage. Suitable strains and suitable conditions are known to those skilled in the art.

It has been shown that the use of exopeptidase in the production of wort, for example, for the manufacture of beer, gives particularly good results at the stage of thinning the grain material. However, in comparison with the case of the absence of exopeptidase, its addition at the saccharification stage also leads to the appearance of the already mentioned positive results, such as an increase in the level of SDA forms, an improvement in filterability, and an increase in the yield of wort.

The method according to the invention allows you to choose a phase that has a pH and temperature conditions conducive to the manifestation of the activity of exopeptidases. Suitable are exopeptidases endogenous to fungi in general, especially representatives of the genus AkretdShik. It was shown that aminopeptidases from the genus AkretdShik, including A.pscheg, are especially useful. A.5o.) Ae and A.otu / ae.

As will be clear from the above description, the addition of enzymes exhibiting exopeptidase activity at the stage of liquefaction and / or saccharification in the manufacture of wort results in a wort with good filterability, high yield and high level of SDA. In turn, this allows us to use a significant percentage of unmalted cereal or even exclusively unmalted cereal in brewing, in the production of alcoholic beverages (liquids), and in the production of alcohol (as biofuel). The latter circumstance is especially beneficial for brewing in countries where imports of malt are limited or less economically viable. This is especially significant for Africa, where beer is produced from a (mixture) of cereal raw materials, which includes a large percentage of sorghum.

In addition, it was shown to improve the organoleptic characteristics (taste and smell) of beer made from must, in the process of which exopeptidases were used at the stage of liquefaction or saccharification (or both). Studies have shown that this advantage can be achieved in the production of beer from malted cereals, such as ordinary barley malt or mixed beer mash (for example, using a combination of malted and unmalted cereals).

Experimental data

A. Thermostable α-amylase (liquefaction step).

The aamylase used in the liquefaction step of the process of the present invention is generally an enzyme that breaks down a-1,4-glucose glucose bonds in starch. It is selected from the number of thermostable α-amylases. Very good results are obtained from α-amylase from Vaschiz Nsiet-Gogsh15, commercially available from O151-Vgosabe5 under the brand name Vgmeg'z Ashunts Tiegto 81aEe (B.A.T. 8.).

B. Endoprotease (liquefaction stage).

The endoprotease used in the liquefaction stage of the process according to the method of the present invention is generally an enzyme that breaks down peptide bonds in proteins under the conditions of pH and temperature at the beginning of the liquefaction stage (pH 5-6; 1 ° 4555 ° C). Very good results can be obtained using a neutral peptidase from Baehibium ashuodonium gas1e8, commercially available from O18! .

B. Exopeptidases (liquefaction and / or saccharification stage).

The exopeptidases used in the liquefaction step of the process of the present invention are generally enzymes that cleave the Ν-terminal bonds in peptides or proteins. Very good results can be obtained using preparations from the species Aureggidae. The following is a method for producing aminopeptidases from Argillidae.

B.1 Determination of enzymatic activity.

Exopeptidase activity is expressed in units of leucine aminopeptidase or units of phenylalanine aminopeptidase:

L-AR unit indicates the amount of enzyme required to obtain 1 μmol of p-nitroaniline per minute at pH 7.2 and at 20 ° C from L-leucine-p-nitroanilide.

* Phenylalanine aminopeptidase unit.

Phenylalanine aminopeptidase unit indicates the amount of enzyme required to obtain 1 μmol of p-nitroaniline per minute at pH 7.2 and at 20 ° C from L-phenylalanine p-nitroanilide.

B.1.1 Phenylanaminamine peptidase (RDe-AR).

Phenylalanine paranitroanilide is dissolved in 7.5 mm HCl to a concentration of 0.9 mm. 1 ml of such a solution of the substrate is mixed with 1.5 ml of 0.1 M phosphate buffer, pH 7.2. At time 1 = 0, add 0.5 ml of the enzyme and leave the mixture to undergo a reaction at 20 ° C.

After 15 min, add 1 ml of 1Ν HCl. A blind test is set with 1Ν HC1, introduced at time 1 = 0. The optical density of the blind sample (OD _sl . _Pr ) and the test sample (OD _test ) are measured at 400 nm. Activity is calculated by the following formula:

(OD _teS f) ~ (OD _sl . _P r) 4

A = - X - Pb-AP / ml

9.8 x 15 0.5

B. 1.2 Leucine-aminopeptidase (Le-AR). Leucine paranitroanilide is dissolved in water to a concentration of 9 mm. 1 ml of such a solution of the substrate is mixed with 1.5 ml of 0.1 M phosphate buffer, pH 7.2. At time 1 = 0, add 0.5 ml of the enzyme and leave the mixture to undergo a reaction at 20 ° C. After 1 to 5 minutes add 1 ml of 1 Ν HCl. A blind test is set with 1 Ν HC1, introduced at time 1 = 0. The optical density of the blind sample (OD _sl . _Pr ) and the test sample (OD _tesg ) are measured at 400 nm. Activity is calculated by the following formula:

(OD _test ) - (OD _sl . _Pr ) 4

A = - x - Ley-AR / ml

9.8 x 15 0.5

B. 1.3 Endoprotease (RI).

The indicated activity is measured by hydrolysis of casein at pH 6.0 and 40 ° C for 1 h. One unit of RI indicates the amount of enzyme required to release the equivalent of 1 μmol tyrosine per minute after precipitation of the remaining proteins with trichloroacetic acid.

B.2 Screening of strains 200 strains of Azregdis schizae, isolated from various sources or obtained from a culture collection, are grown in a medium containing 15 g / l of potato flour, 20 g / l of bactopeptone, 7 g / l of yeast extract, 4 g / l of potassium dihydrogen phosphate, 0.5 g / l of magnesium sulfate, 0.5 g / l of calcium chloride and 0.5 g / l of zinc chloride. The pH of the medium is 4.8. Supernatants obtained by centrifugation at 30 ° C of a 24-hour preculture at 240 rpm and a 96-hour culture at 275 rpm (same temperature) were collected and analyzed for the presence of leucine, phenylalanine and valinaminopeptidase activities, as was described earlier. As follows from the following table 1, a number of strains of Azregdis schizae have a high potential for producing at least one of these enzymatic activities (each of the values is an average of four individual results):

Table 1

room strain Aminopeptidase activity in supernatants Endopeptidase RI / ml Ley-ar / l RDE-AR / L Уа1-АР / l 1053 25 170 32 <0.1 1085 23 135 48 0.1 1103 37 285 40 0.1 1108 60 435 29th 0.1 1444 40 192 fifty 0.1 1497 25 105 75 0.1 1502 sixteen 44 63 0.1

Of these strains, strains 1108 and 1502 were obtained from the culture collection and were deposited under the numbers, respectively, \ HH1. 3112 and CB8 115.39. Strain \ HH1. 3112 was used to obtain amyloglucosidase, α-amylase and glucoamylase. Strain CB8 115.39 was used to produce amylase.

B.3 Obtaining exopeptidases on a laboratory scale.

Several strains from the screening kit in Example 1 were used for fermentation in laboratory fermenters (10 L). The example below presents the results obtained with this.

Spores L8regdy1i8 where strain No. 1502 are collected on plates with PDA (paraphenylamine diamide) after incubation for 7-10 days at 30 ° C. Inoculation is carried out in a rocking flask, which contains a medium comprising glucose (20 g / l) and corn extract (20 g / l) at a pH of 4.8 for 24 hours.

The main fermentation is carried out batchwise. For this, the following nutritional ingredients are used: 1 00 g / l maltodextrin, 40 g / l soy flour, 40 g / l hydrolyzed casein, 5 g / l corn extract, 2 g / l gelatin, 2 g / l potassium dihydrogen phosphate, 1, 3 g / l sodium nitrate, 1 g / l ammonium chloride, 0.01 g / l iron sulfate and 0.5 g / l antifoam. Initially, all the nutritional ingredients, with the exception of maltodextrin, are mixed together. The pH is adjusted to 4.8 ± 0.1. Then the fermenter is sterilized at a temperature of 125 ° C for 40 minutes. The maltodextrin solution is sterilized separately and added to a sterile and chilled environment.

The main fermentation is carried out in a laboratory fermenter filled with 6 liters of the medium described above and the culture from the flask is inoculated into it. Mixing and aeration are carried out in order to maintain the highest possible concentration of dissolved oxygen. The temperature is maintained at 30 ° C. As soon as all the nutrient components are consumed, after about 130 hours, the fermentation process is stopped.

After fermentation, the broth is filtered to remove microorganisms. The aminopeptidase and endoprotease activities are measured in the filtrate:

0.15 Ley-AR / ml

1.0 Rye-AR / ml <0.05 Va1-AR / ml <0.1 RI / ml

To obtain a preparation of liquid aminopeptidase with glycerol (50%), concentrations after ultrafiltration are used as a stabilizing agent. The resulting solution of the so-called "Peptidase L2" has the following activities:

0.5 Ley-AR / ml

3.2 Rye-AR / ml <0.05 Va1-AR / ml <0.1 RI / ml

The results show that the selected strain AkregdShik shdeg when grown under these conditions produces aminopeptidases that do not contain significant amounts of endoprotease.

B.4 pH profiles of enzymatic activities.

The activity of Lye-AP and Rye-AP is determined based on the peptidase L2 (see example 2), but using different buffers for screening activity in the pH range from 2.5 to 9.0.

The pH profile of leucine aminopeptidase from L8regdy1 and 8 is shown in FIG. one.

The pH profile of phenylalanine aminopeptidase from L8regdy1 and 8 is shown in FIG. 2.

The above figures demonstrate that Ley-AP is active in the pH range from 5 to 8.5, while Pye-AP is active in the pH range from 5.5 to 9, which corresponds to the data obtained for aminopeptidases from other Acregschik species.

B.5 Temperature profiles of enzymatic activities.

The activity of Lye-AP and Rye-AP is determined based on the peptidase L2 (see example 2), but using different incubation temperatures to screen activity in the temperature range from 5 to 70 ° C.

Temperature profiles are shown in FIG. 3. The results show that each enzyme has an optimum temperature for activity, i.e. 50 ° C for Lye-AR and 60 ° C for Rye-AR.

The following discloses a method for producing aminopeptidases from the culture of AkregdSik π deg. These aminopeptidases have optimal activity in the pH range of 6-8 and in the temperature range of 50-60 ° C; Moreover, under the considered culturing conditions, aminopeptidases that do not contain significant or significant amounts of endoprotease can be obtained. This method allows to achieve an excess of the activity of aminopeptidase over the activity of endoprotease, preferably the first of them is 10 times higher, more preferably 30 times higher.

G. Maltogenic amylase (saccharification stage).

The amylase used in the saccharification step of the method of the present invention is an enzyme that cleaves a-1,4-glucose-glucose bonds in dextrins or starch to produce maltose and / or glucose as the main products. Very good results can be achieved by using α-amylase from AkregdSikogu / ae, commercially available from Ci1-Vgosaye5 under the brand name Vrg eg'8 Regteh, or by using recombinant β-amylase from Bashik atu1o1k | IsGas1SP5. commercially available from Νονο under the brand name Νοναιηνί.

Example 1

Sorghum (RARA RAKA grade) and corn cobs are ground according to the standard beer production method. One part of the grains (60% sorghum + 40% corncobs) is hydrated with 3 parts of water. Calcium chloride is added so as to ensure that 200 ppm Ca ² + is present in the liquid medium. Using calcium hydroxide, the pH was adjusted to 6.5. Then add B.A.T. 8. at a dose of 1.5 kg per ton of grains. Other enzymes are also added in the amounts indicated below in table 2:

table 2

Test No. Enzymes Dose per ton of grains one Missing 0 2 Vgehuegk Rgo1eake 2000 1.2 kg 3 Vgehuegk Prgo1eake 200 Exopeptidase from A.co | ae 1.2 kg 73000 Lei-AR 4 Raitake 400 10 kg 5 Raitake 400 Exopeptidase from A.co | ae 10 kg 73000 Lei-AR

The mixture is kept at a temperature of 50 ° C for 1 h; then the temperature is raised to 95 ° C (the rate of temperature increase is 1 ° C / min) and maintained at 95 ° C for 45 minutes. After that, the mixture is cooled to 60 ° C for 5 minutes. Then add Vgs \ usg5 Regteh (600 g / t). For 45 minutes spend mash saccharification at 60 ° C. The temperature was raised to 76 ° C and held at this temperature for 10 minutes. Congestion is poured into a funnel containing a paper filter ZsYeyuyet apb 8s1she11. Then measure the volume and specific gravity of the filtered wort. Based on these data, extract characteristics and yield are calculated. Using a ninhydrin reagent using glycine as a standard, the level of amino acids is measured. The obtained amino acid concentration values are adjusted accordingly to compare wort characteristics at the same sugar content (12 ° P1a1o).

The results are presented below in table 3.

Table 3

Test No. Filtered Volume (ml) Exit (%) Amino acids at 12 ° (P1aTo) (mg / L) one 310 85,4 26.9 2 370 86.9 54.7 3 382 90.2 119.5 4 365 86.9 76,2 5 372 91.0 102.3

The results show that the use of endoprotease in combination with exopeptidase in the method of the present invention improves not only the number of amino acids, but also the yield and filterability.

Example 2

In this series of tests, the same fermentation is carried out as in Example 1, but using a combination of a neutral protease from Bacillus acinella | the research scheme shown in table 4.

Table 4

Test No. Exopeptidases Used Biological origin Ley-AR / T Rye-AR / T one Not biological. origin. 0 0 2 AkregdShik ko | ae 73000 49000 3 AkregdShik ogu / ae 73000 53000 4 AkregdShik sh§eg 25,000 100,000

The results are presented in table 5.

Table 5

Test No. Filtered Volume (ml) Exit (%) Amino acids at 12 ° (P1a! O) (mg / l) one 340 86.1 61,4 2 400 90.0 116.0 3 385 87.9 95.7 4 382 89.8 99.8

All combinations of endoprotease + exopeptidase work better than a single endoprotease, regardless of the biological origin of the used exopeptidase. And in this case, also in addition to increasing the amino acid content, it is possible to achieve an increase in yield and improve filterability.

Example 3

In order to verify the applicability of the obtained wort in the production of beer, sorghum and corn ears are fermented according to the method of example 1; at the same time, another fermentation is carried out in which they do not use the enzyme and completely replace sorghum with malt. Table 6 illustrates the scheme of the study:

Table 6

Test No. Enzymes Dose one Malt Replaced sorghum 2 Vgehuegk Prgoeke 2000 Exopeptidase. \. To | ae 1.8 kg / t 42000 Rays -. \ R T 3 Vgehuegk Prgoeake 2000 Exopeptidase. Woo / ae 1.8 kg / t 40000 LIE-AR / T 4 Vgehuueg Prgoeche 2000 Exopeptidase A. Schdeg 1.8 kg / t 25000 Lei-AR / T

The results are presented in table 7.

Table 7

Test No. Filtered Volume (ml) Exit (%) Amino acids at 12 ° (P1a! O) (mg / l) one 400 82.3 222.0 2 395 89.5 127.0 3 390 87.9 109.0 4 375 91.1 113.1

The exact amino acid composition of each mash was determined by HPLC. Amino acids are classified into groups depending on the rate of their assimilation by representatives of the genus 8assyatus:

Group A: rapid assimilation;

Group B: medium assimilation;

Group B: slow assimilation. The results are presented in table 8: (Note: the values in mg / l do not coincide with the total number of amino acids shown in table 6, due to the fact that the data shown in table 7 were obtained using the glycine standard, whereas calibration for HPLC carried out in a different way.)

Table 8

Amino acids (mg / l) Test No. one 2 3 4 Group A Osh 36 38 thirty 39 Acre 53 29th 26 29th 8eg 143 64 51 56 Bj 54 28 21 22 1, UK 95 65 45 47 Agd 133 114 67 74 Total 513 340 260 268 Group B Ya1 102 36 32 28 Me1 26 37 thirty 28 Bie 158 216 205 166 11th 75 41 42 28 Ηίδ 58 26 sixteen fifteen Total 418 356 325 265 Group C O1u 29th nineteen thirteen 17 RNe 134 89 85 95 Tug 107 67 58 57 Tgr 55 eleven eleven 12 A1a 97 80 66 73 Total 421 265 233 253

Each wort is boiled for 45 minutes; aseptically, boiled distilled water is added to each wort to obtain a sugar content of 12 ° P1a1o. 350 ml of each standard wort is aseptically poured into sterile flasks and then inoculum yeast (5 g / l) is inoculated into each flask. Fermentation lasts for 8 days at 11 ° C. After 8 days, a clear fermentation is observed, determined by density.

The results are presented in table 9.

Table 9

Test No. Marked fermentation at 8 days (%) one 82.0 2 81.0 3 80,4 4 Not determined

The combination of endoprotease + exopeptidase of the present invention, used in the case of sorghum, allows to obtain wort with better characteristics for fermentation in beer production, compared with wort obtained from malt.

It was found that the combination of an endoprotease from Bashik atu1okcisGas1sp5 with an exopeptidase from AkregdShikko) e allows to achieve a significant increase in the amino acid content of Group A + Group B.

Example 4

This example illustrates the advantage of administering exopeptidases according to the method of the present invention in the liquefaction step rather than in the saccharification step. Fermented according to the method of example 1 sorghum + corn cobs. In all brews at the liquefaction stage, the enzyme Bg \\ cr5 Prgo1eake 2000 (1.8 kg / t) is added. Exopeptidase from Akregschikcoe (40000 Le-AP / T) is added either at the dilution stage (tests n ° 1-2) or at the saccharification stage (tests n ° 34). The results are presented in table 1 0.

Table10

Test No. Filtered Volume (ml) Exit (%) Amino acids at 12 ° (P1a! O) (mg / l) one 380 89.3 115.1 2 385 89.5 119.8 3 370 89.4 109.1 4 370 89.8 112.0

The standard deviation values for the filtered volume, yield and amino acid content are determined by repeating identical tests. They were installed as follows: 10 ml, 0.5% 0.9 mg / L, respectively.

Thus, the above results show that the introduction of exopeptidases at the dilution stage makes a significant positive contribution, primarily in terms of the production of amino acids and the ability to filter.

Example 5

This example illustrates the effect of increasing doses of exopeptidases from A.ogu / ae on mash characteristics.

Sorghum + corn cobs are fermented in accordance with the method described in the example

1. At the liquefaction stage, Add Br ekgo Protein 2000 and exopeptidase are added (Table 11).

Table 11

Test No. Enzymes Dose one No enzyme 0 2 Vgeuegk Rgo1eake 2000 1.0 kg / t 3 Vgeuegk Rgo1eake 2000 Exopeptidase A.ogu / ae 1.0 kg / t 62000 LIE-AR / T 4 Vgeuegk Rgo1eake 2000 Exopeptidase A.ogu / ae 1.0 kg / t 109000 LIE-AR / T 5 Vgeuegk Rgo1eake 2000 Exopeptidase A.ogu / ae 1.0 kg / t 155000 LIE-AR / T 6 Vgeuegk Rgo1eake 2000 Exopeptidase A.ogu / ae 1.0 kg / t 234000 LIE-AR / T 7 Vgeuegk Rgo1eake 2000 Exopeptidase A.ogu / ae 1.0 kg / t 313000 LIE-AR / T

The results are presented in table 1 2.

Table 1 2

Test No. Filtered Volume (ml) Exit (%) Amino acids at 12 ° (P1a! O) (mg / l) one 188 88.4 39.1 2 275 88.9 59.8 3 280 89.1 81.5 4 280 89.6 90.5 5 280 89.7 95.3 6 280 90,4 101.0 7 280 89.5 114.0

The more exopeptidases are introduced, the higher the yield and higher amounts of free amino acids can be obtained. While the effect of filterability reaches its optimal value even at a low level of exopeptidase.

Example 6

This example illustrates the effect of increasing doses of exopeptidases from A guide g on fermentation.

In accordance with the methodology of example 1, sorghum + corn cobs are fermented. The enzyme Vr \ veg8 Prgo1ea8e 2000 is added at the liquefaction stage.

Table 13

Test No. Enzymes Dose one without enzyme 0 2 All-in-one 1.0 kg / t 3 Brycephalonium Protein 2000 Exopeptidase A.ogu / ae 1.0 kg / t 51000 Rye-AR / T 4 Vgrrrrr8 Protein 2000 Exopeptidase A.ogu / ae 1.0 kg / t 90000 Rye-AR / T 5 Vg \ ue8 Prgo1ea8e 2000 Exopeptidase A.ogu / ae 1.0 kg / t 127000 Rye-AR / T

The results obtained are presented in table 14.

Table 14

Test No. Filtered Volume (ml) Exit (%) Amino acids at 12 ° (P1a! O) (mg / l) one 188 88.4 39.1 2 240 88.6 60.1 3 246 89.7 63.7 4 258 89.5 64,2 5 232 89.7 65.7

And here also, the more exopeptidases are introduced, the higher the yield and higher amounts of free amino acids can be obtained. These results show that exopeptidase from A.ogu / ae works slightly better than exopeptidase from A. shdet.

Example 7

Barley (grade ΡΤΑΙδΑΝΤ) is crushed into a fine flour, suitable for entering the filter press in the cooking shop. Suspended at 50 ° C 57 mg of flour in 30 ml of water containing:

mg Β.Α.Τ.8.

mg of Rhtake L3000 (+) β-glucanase from BaeBi8 atuAcerea extinguish. commercially available from C181-Wgosabe8);

mg Br \\ ex8 Prgo1ea8e 2000; and the corresponding amount of exopeptidase from A8retd8b8 8o) ae. Then the temperature is raised to 63 ° C (the rate of temperature increase is 1 ° C / min) and maintained at 63 ° C for 30 minutes. After that, the temperature is increased to 90 ° C (the rate of temperature increase is 1 ° C / min) and maintained at 90 ° C for 20 minutes. Then the fermentation mixture is cooled to 25 ° C and centrifuged at 7000§ for 1 5 min. The supernatant is analyzed for soluble proteins and free amino acids.

The results obtained are presented in table 15.

Table 1 5

Test No. Dose of exopeptidase (Ley-AR / T) Amino acid level at 1 2 ° Р1а! О (mg / l) The level of soluble proteins (% of dry matter one 0 77.9 4.21 2 25,000 95.3 4.75 3 50,000 118.4 4.99 4 100,000 133.2 5.42 5 200,000 203.6 6.63 6 400,000 295.0 7.55

The above results show that the use of exo-peptidase from AkretdShik 8o.) Ae, when applied at the liquefaction stage, allows to achieve the same high level of free acids in barley mash, as when working with malt mash.

Example 8

Barley is fermented, as described in Example 7, but with the replacement of the exopeptidase from AccretdShik 8o) expects the exopeptidase from AccretdShik.

The results are presented in table 1 6.

Table 1 6

Test No. Dose of exopeptidase (Ley-AR / T) Amino acid level at 1 2 ° Р1а! О (mg / l) The level of soluble proteins (% of dry matter one 0 68,2 4.37 2 10,000 69.8 4.20 3 50,000 75,2 4.25 4 200,000 78.0 4.16 5 500,000 89.1 4,50 6 1,000,000 101.0 4.18

Even though the exopeptidase from Akretdshik looks, it seems to work somewhat less efficiently than the enzyme from Akretdshik ogu / ae, it allows to obtain up to 1 00 mg / l amino acids in barley wort at 1 2 ° Р1а! О, which quite enough for fermentation.

Deposition of microorganisms

The strains used as producers of exopeptidases were deposited in the Collection of cultures in the Netherlands (Sep1gaa1 Vigeai uoot §Stte1si11ite8, OochegChgaa! 1, Vaagp, THe No. 1Neg1apY8) under deposit numbers CB δ 115.39 (state collection) ae (Ό δ 8351); date of deposit February 12, 1996) and СΒδ 210.96 (A.otu / ae (ϋδ 23617); date of deposit February 12, 1996).

Claims (12)

one . A method of producing a fermented wort, comprising the steps of: (a) liquefying the cereal material with a substance having alpha-amylase and / or endoprotease activity to produce a liquefied mash, (b) saccharifying the liquefied mash in the presence of alpha-amylase, and (c) filtering the liquefied and saccharified mash to produce a fermented wort, characterized in that at least one of the stages (a) or (b) is carried out in the presence of an enzyme having exopeptidase activity. 2. The method according to claim 1, characterized in that the cereal material contains at least 20% unmalted cereals. 3. The method according to claim 2, characterized in that the cereal material contains at least 20% unmalted sorghum. 4. The method according to claim 3, characterized in that the cereal material contains at least 50% unmalted sorghum. 5. The method according to any one of claims 1 to 4, characterized in that the use of an endoprotease derived from Bacillus arteriocephalus. 6. The method according to any one of claims 1 to 5, characterized in that the enzyme having exopeptidase activity is used at the stage of thinning cereal material. 7. The method according to any one of claims 1 to 6, characterized in that the enzyme having exopeptidase activity is an exopeptidase from fungi. 8. The method according to claim 7, characterized in that the mushrooms are fungi of the genus Azregilles. 9. The method according to claim 8, characterized in that the fungi of the genus AzregdSchiz are Azregschiz zo] ae. 10. The method according to any one of claims 1 to 9, characterized in that the exopeptidase is an aminopeptidase. eleven . A method for producing alcohol or an alcohol-containing beverage, comprising the step of fermenting the wort in the presence of yeast capable of producing ethyl alcohol, characterized in that the wort obtained by the method according to any one of claims 1 to 10 is used. 12. The method according to claim 11, characterized in that the alcohol-containing beverage is beer.