SK108698A3 - Process for producing fermentable wort - Google Patents

Abstract

The invention provides a process for the production of a fermentable wort from cereal grains, comprising the steps of: (a) liquefaction of cereal material, with the aid of alpha -amylase and/or endoprotease activity, to obtain a liquefied mash; (b) saccharification of the said liquefied mash in the presence of alpha -amylase; (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. Useful as enzymes having exo-peptidase activity are exopeptidases, preferably thermostable exopeptidases, such as the fungal amino-peptidases, but also thermostable carboxy-peptidases are useful. Particularly preferred according to the invention are amino-peptidases endogenous to Aspergillus fungi, more in particular A. niger, A. oryzae or A. sojae.

Description

Technical field

The present invention relates to the treatment of fermentable malt from cereal grains, in particular from non-malted cereal grains. The invention also relates to malt produced by this process. The invention further relates to the fermentation of the malts obtained according to the invention in the alcohol industry, in the distillery industry or in the brewing industry.

BACKGROUND OF THE INVENTION

Beer is produced by fermentation of either malt or non-malt grains. In the latter case, the grains are liquefied and saccharified by conventional additional enzymes to yield malt containing fermentable sugars and amino acids or other forms of nitrogen (totally calculated as FAN - Freely Available Nitrogen), which is essential for yeast fermentation.

For example, MacFadden et al. (MacFadden, D. P. and Clayton, M. Brewing and Bewerage Industries International (1989), 1, 77-81) propose to add non-malt sorghum enzymes, such as e.g. alpha-amylase, protease, beta-glucanase, cellulose, fungal alpha-amylase, amyloglucosidase and others. MacFadden et al. Also recommend the addition of nutrients for yeast when non-malt sorghum is used.

Bajomo et al. (M. F. and Young, T. W., J. Inst Brew. (1992) 98, 515-523; Bajomo M. F. and Young, T. W., J. Inst.

Brew. (1994) 100, 79-84, 3) speaks of brewing beer from 100% non-malt sorghum grains despite the fact that the FAN level present in the malt sorghum malt (51 mg / l) is much lower than the malt fermentation base prepared from malt malt barley.

International patent application WO 92/20777 describes a process for the production of ethanol which involves liquefying whole non-malt maize or sorghum by amylase, followed by the saccharification of the mash by glucoamylase and acid fungal protease. The process comprises the step of adding a protease during fermentation as well as during saccharification. The pH of the fermentation environment is in the range of 4-5, which means that fungal exo-peptidases are not active (Labbe, JP, Rebeyrotte, P. Biochemistry (1974) 56, 839-844, Lehman, K. and Uhlig, H. Hoppe Seyler's Z. Physiol Chem (1969) 350, 99104). A disadvantage of the above process is that under these conditions the fungal proteases present exhibit only endoproteolysis, thereby producing mainly oligopeptides and low free amino acids.

For a general overview of the art, reference should be made to the Palmer report (G. H. Cereal Science and Technology. In: Cereal Science and Technology. (1989) ed. Aberdeen University Press, Aberdeen, Scotland, 61-242).

SUMMARY OF THE INVENTION

The present invention includes a process for obtaining malt from unmalted cereal grains with unexpectedly good malt properties, e.g. high amount of available nitrogen (here calculated as FAN), good filterability and yield of malt obtained. The process may also be used for the production of malt from malt cereals, but is particularly advantageous for the production of malt from non-malted grains, such as e.g. malt sorghum, or a mixture of malt sorghum and corn. The stated advantages are also in the existence of malts that are made from malted cereals (such as malt barley) in combination with non-malted cereals (such as corn, rice or sorghum), which are called as a blend batch.

Thus, the invention provides a process for producing fermentable malt from cereal grains and comprises the steps of:

(a) liquefying the cereal material with the addition of a co-amylase and / or endoprotease to obtain liquefied mash, (b) saccharifying said liquefied mash in the presence of α-amylase; of steps (a) and (b) above is carried out in the presence of an enzyme having exo-peptide activity. Suitable enzymes with exo-peptide activity are exopeptidases, e.g. fungal aminopeptidases, but also temperature stable carboxy peptidases. Particularly preferred according to the invention are aminopeptidases endogenous to Aspergillus fungi, more particularly to A. niger, A. oryzae, A. sojae.

Particularly suitable for the process are cereal grains which consist of at least 20% unmalted cereal, preferably more than 50% unmalted cereal, such as sorghum.

- 4 cereal grains supplemented with non-malting ears of maize, rice or other non-malting cereals. It is found that according to the invention it is very advantageous if the cereal grains are liquefied in the presence of exopeptidases.

According to another aspect of the invention there is provided a fermentable malt obtained by the process of the invention. Yet another aspect of the invention is a brewing process characterized in that the fermentable malt obtained according to the invention is used.

Further provided is a process for producing an alcoholic beverage, e.g. beer, which comprises the steps of producing malt using the process of the invention and subsequently, or simultaneously, fermenting said malt to obtain e.g. beer.

In another embodiment, there is provided a process for obtaining enzymatically releasable, freely available nitrogen from cereals containing high levels of glutellin and / or prolamine, comprising the step of adding endoprotease and exo-peptidase to the grains or liquefied mash obtained from said grains.

In another embodiment, there is provided an alcohol production process comprising the step of fermenting the FAN obtained by the process of the invention in the presence of yeast capable of forming alcohol.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by the following figures.

Fig. 1 shows the pH diagram of Aspergillus niger leucine aminopeptidase.

Fig. 2 is a diagram pH phenylalanine- aminopeptidase Aspergillus niger. Fig. 3 show e temperature diagram both

aminopeptidase.

The invention will be explained in more detail below.

The process provides for the preparation of fermentable malt from cereals, provided that at least one protein with an exo-peptide effect is present. Suitable amiriopeptidases endogenous to fungi, in particular to Aspergilli, are sufficiently stable at pH 5-8 with sufficient activity, which is about the area where the pH drops during liquefaction. Carboxypeptidases are less suitable at this temperature and pH. In the saccharification step, a slightly acidic environment predominates over the liquefaction step, and therefore a carboxy peptidase is preferably used here, which ensures that the environment is sufficiently stable over a temperature range of 50-60 ° C, preferably at 50-70 ° C.

The presence of the exopeptidases of the present invention not only leads to an increase in malt of free nitrogen (FAN), but also to an improvement in filterability and malt yield as compared to a process where no exopeptidases are used.

and

A particular advantage of the process according to the invention is the preparation of fermentable malt from unmalted cereals, more particularly unmalted sorghum, which is optionally supplemented with another cereal material, e.g. maize, wheat, oats or rice. Cereals in the context of the present invention include sorghum, wheat, barley, oats, rice and corn and the like.

The presence of exopeptidases according to the invention is advantageous in the case of mixed batches where malt cereal raw materials as well as non-malt cereal raw materials are regularly used (e.g. having more than 80% or even more than 90% malt cereals, the remainder being non-malting cereals), as exopeptidases have been found to have a beneficial effect on organoleptic properties (ie, taste and / or odor). It is believed that these advantages are common in brewing beer from both malt and malt cereals, as well as in mixtures thereof.

Cereals in which the use of exopeptidases are particularly advantageous in terms of FAN, filterability, yield and organoleptic properties have relatively high proportions of prolamine and glutelin proteins. In addition to sorghum, this category also includes rice (with about 80% glutelin). If sorghum is used, consideration should be given to selected varieties having a relatively low polyphenol content.

In addition to exopeptidase addition, malt preparation e.g. for brewing beer, can be carried out in conventional ways. Generally, the preparation involves liquefying the cereal raw material to obtain the mash, followed by the saccharification of the mash and the recovery of the malt. Filtration is important, it takes precedence over fermentation.

Typically, the liquefaction step comprises crushing the cereal feedstock to obtain a flour having a suitable particle size which hydrates from about 1 to about 4, preferably about 3 parts of water and optionally, depending on the endoprotease used, with about 50 to about 300 ppm calcium, preferably 200 ppm Ca ²⁺ . Bacillus stearothermophilus enzymes have been shown to reduce calcium dependence. In this case, no additional Ca ²⁺ ion additions are required. The particle size of the crushed cereals should not exceed about 3 mm; the number of parts larger than 1.3 mm should not exceed 3.5%; smaller particles than 0.25 mm should not be more than 1.5%. In addition, enzymes such as cellulases, β-glucanases, or other enzymes that degrade the plant cell wall may be present.

The pH of the liquefying medium is usually adjusted to a value between about 5 and 8, preferably between about 6 and 7, which is ensured e.g. with calcium hydroxide. It is important to add α-amylase, preferably the thermostable α-amylase as well as the endoprotease are added to the liquefying medium at a dose necessary to at least partially liquefy the cereal starch and to at least partially degrade the protein. A suitable dose of α-amylase is from about 0.5 to 2.0, preferably about 1-1.5 kg / tonne when B.A.T.S. In the case of Brewers Protease 2000, a suitable protease dose is greater than 0.5 kg / ton of grains (kg / T), preferably greater than 1 kg / T. In the case of Panstimase 400, a dose of greater than 2 kg / T, preferably greater than 5 kg / T, most preferably greater than 10 kg / T should be used.

During liquefaction, usually a number of steps take place at elevated temperature: after addition of α-amylase and protease, the mixture is maintained at a temperature between about 40 ° C and

65 ° C, preferably between about 45 and 55 ° C, and most preferably at 50 ° C, until the necessary liquefaction is achieved. The time to react depends on the cereal or cereal mixture used, but usually takes from about 30 minutes to about 2 hours. Subsequently, the temperature is gradually increased, the rate of temperature increase is not critical up to about 90-95 ° C, and at this temperature the mixture is allowed to react from about 30 minutes to about 1 hour. Then the mixture is cooled to the saccharification temperature: usually at a temperature of about 50 ° C to about 70 ° C, preferably between about 55 ° C and 65 ° C, most preferably at about 60 ° C. In the saccharification step, a slightly higher temperature can be used depending on the temperature stability of the enzymes. Once the necessary temperature has been reached, saccharifying enzymes, e.g. Brewers Fermex (α-amylase) or Novamyl (recombinant β-amylase) in an amount that is typically in the range of about 400 g / T to about 1 kg / T for Brewers Fermex. Glucoamylases are also used frequently. The saccharification phase lasts from about 30 minutes to about 2 hours, and then the temperature rises to from about 75 ° C to about 85 ° C inter alia to inactivate the enzymes and unwanted organisms, and is preferably maintained at an elevated temperature of 10 minutes; the length of time is not too critical.

The mash obtained in this way is subsequently filtered on a device well known in the art; with a Schleicher & Schuell filter paper which is sufficient for this purpose. After filtration, the malt is fermented with the appropriate yeast, depending on the strain used and the final intention; furthermore, brewing beer, making alcohol using it as a biofuel or as an alcoholic beverage is understood as a separate invention.

Suitable yeast strains and suitable fermentation conditions are well known to those skilled in the art.

It has been found that the application of exo-peptidase during the liquefaction step of the malt preparation cereal raw material, e.g. when brewing beer is particularly preferred. However, in addition to the absence of exo-peptidase, its addition during the scavenging step also leads to the aforementioned advantages such as higher FAN levels, improved filterability and higher malt yield.

The process of the invention takes into account the phase where pH and temperature of the environment allow the fungal exo-peptidases to be active. As suitable exo-peptidases, these endogenous fungi are generally, more specifically, Aspergillus species. Aspergillus species aminopeptidases, including A. niger, A. sojae and A. oryzae, are known to be particularly useful herein.

As will be clear from the above description, the addition of enzymes that demonstrate the effect of exo-peptidase during the liquefaction and / or saccharification step of the sequence preparation produces malts with good filterability, high yield and high FAN. This predestines it to be used for brewing beer, for the production of alcoholic beverages (spirits) and for the production of alcohol (as biofuels) from cereals with a significant percentage of non-malting cereals, or even exclusively from non-malting cereals. Furthermore, the solution is very advantageous for beverages brewed in countries where malt import is limited or economically disadvantageous. This is particularly the case in Africa, where beer is produced from (from mixtures) cereal raw material, which comprises a large percentage of sorghum.

In addition, it is known that the organoleptic properties (taste and smell) of beers made from malt have been improved, where δ-peptidases were used in the liquefaction step or in the saccharification step (or both). This advantage is believed to be achieved when exo-peptidases are used in the production of malt cereal beer, e.g. from traditional barley malt, or in the production of beer from mixed brews (i.e., a combination of malt and non-malt cereals).

DETAILED DESCRIPTION OF THE INVENTION

A. The thermostable α-amylase (liquefaction) α-amylase used in the liquefaction step of the process of the invention is generally an enzyme that cleaves α-1,4-glucose-glucose starch bonds. It is ^! selected from thermostable α-amylases. Very good results can be obtained with Bacillus licheniformis α-amylase, which is commercially available from Gist-Brocades under the trademark Brewers Amyliq Thermo Stable (BATS).

B. Endoprotease (liquefaction)

The endoprotease used in the liquefaction step of the process of the invention is generally an enzyme that cleaves peptide bonds of proteins at a certain pH and temperature at the start of the liquefaction step (pH 5-6; t ° 45-55 ° C). Very good results can be obtained with the neutral protease Bacillus amyloliquefaciens, which is commercially available from Gist-Brocades under the trademark Brewer's Protease 2000. Streptomyces fradiae enzymes, which are commercially available from Panstimase SARL under the trademark Panstimase 400, can also be used.

C. Exo-peptidases (liquefaction and / or saccharification)

The exo-peptidases used in the liquefaction step of the process of the invention are generally enzymes that cleave N-terminal bonds of peptides or proteins. Very good results can be obtained with Aspergillus species. Further described is a process for obtaining aminopeptidases from Aspergillus niger.

C.I. Determination of enzymatic activities

Exo-peptidase activity is explained by the Leucine aminopeptidase unit or the Phenylalanine aminopeptidase unit:

Leu-AP unit is the amount of enzyme required to generate 1 gmole of p-nitroaniline per minute at pH 7.2 and at 20 ° C from L-leucine-p-nitroanilide * Phenylalanine aminopeptidase unit Phe-AP unit is the amount of enzyme required to produce 1 µmol of p-nitroaniline per minute at pH 7.2 and 20 ° C from L-phenylalanine-p-nitroanilide.

C.I.1 - Phenylalanine aminopeptidase (Phe-AP)

Phenylalanine paranitroanilide was dissolved in 7.5 mM HCl to a final concentration of 0.9 mM. 1 ml of this substrate solution was mixed with 1.5 ml 0.1 M phosphate buffer solution at pH 7.2. At t = 0, 0.5 ml of enzyme was added to the solution and allowed to react at 20 ° C. After 15 minutes, 1 mL of 1N HCl was added. The HCI was made blank, when the acid is added at time t = 0. For the blank test _(p and _{the bolts)} and the sample (ODvzorka) was determined by optical density / absorbance at 400 nm. The activity was calculated as follows:

(OD Pattern _with Sip)

9.8 x 15

Phe-AP / ml 0.5

C.1.2 - Leucine aminopeptidase (Leu-AP)

Leucine paranitroanilide was dissolved in water to a final concentration of 9 mM. 1 ml of this substrate solution was mixed with 1.5 ml phosphate buffer pH 7.2. At t = 0, 0.5 ml of enzyme was added to the solution and allowed to react at 20 ° C. After 15 min. 1 ml of 1N HCl was added. The 1 N HCl was made blank, when the acid is added at time t = 0. For the blank test _(p iepý) and the sample (ODvzorka) was determined by optical density / absorbance at 400 nm. The activity was calculated as follows:

(ODsample _with x _e p)

9.8 x 15

C. 1.3 - Endoprotease (PU)

Leu-ÄP / ml 0.5

This activity is measured by casein hydrolysis at pH 6.0, 40 ° C for 1 hour. 1 PU is the amount of enzyme required to release the equivalent of 1 gmole of tyrosine per minute after precipitation of the remaining proteins with trichloroacetic acid.

C.2 - Sorting of Aspergillus niger 'strains

200 strains of Aspergillus niger isolated from various sources or from a set of cultures were grown in a culture medium containing 15 g / l of flour, 20 g / l of bacteropeptone, 7 g / l of yeast extract of yeast, 4 g / l of potassium dihydrogen phosphate 0.5 g / l magnesium sulfate, 0.5 g / l calcium chloride, 0.5 g / l zinc chloride and at pH 4.8. After 24 hours cultivation at 240 rpm at 30 ° C and after 96 h. Growth at 275 rpm and 30 ° C, the supernatant was collected and sampled for leucine, phenylalanine, and valine aminopeptidase activity as described above. Several Aspergillus niger strains showed high potential generation for at least one of the following enzyme activities, as shown in Table 1 (each value is a set of values of four separate results):

Table 1

number aminopeptidase activity in} supernatant cvapaline nad endopeptidase strain Leu-AP / 1 Phe-AP / 1 Val-AP / 1 PU / ml 1053 25 170 32 0.1 1085 23 135 '48 0.1 1103 37 285 40 0.1 1108 60 435 29 0.1 1444 40 192 50 0.1 1497 25 105 75 0.1 1502 16 44 63 0.1

From the above strains, strains 1108 and 1502 were obtained from the culture collection and deposited under the accession numbers NRRL 3112 and CBS 115.39, respectively. The NRRL 3112 strain was used to produce amylglucosidase, α-amylase and glucoamylase. The CBS 115.39 strain was used to make amylase.

C.3 - Production of exopeptidase on a laboratory scale

Some screened strains as described in Example 1 were subjected to fermentation in laboratory fermenters (10 liters). The results obtained with species 1502 are shown in the Example.

Aspergillus niger No 1502 spores were captured on PDAdos after a 7-10 day incubation at 30 ° C. The inoculation step was carried out in a shake flask at pH 4.8 and for 24 hours. with medium consisting of glucose (20 g / l) and macerated corn (20 g / l). The main fermentation process was carried out by a batch process. The nutritional preparations were as follows: 100 g / l maltodextrins, 40 g / l soybean flour, 40 g / l hydrolysed casein, 5 g / l macerated corn, 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 sulphate and 0.5 g / l antipoaming agent.

All nutrients were first mixed together except maltodextrin. The pH was adjusted to 4.8 + 0.1. The fermenter was then sterilized at 125 ° C for 40 min. The maltodextrin solution was sterilized separately and added to a sterile but chilled fermentation broth.

The main fermentation was carried out in a laboratory fermenter which was filled with 6 L of the medium described above and inoculated with a seed vessel. In order to keep the dissolved oxygen concentration as high as possible, the air supply and mixing were adjusted. The temperature was maintained at 30 ° C. when all nutrients were consumed, fermentation was stopped, i. after about 130 hours

The fermented broth was filtered to remove all microorganisms. Aminopeptidase and endoprotease activity was measured in the filtrate:

0.15 Leu-AP / ml

1.0 Phe-AP / ml

0.05 Val-AP / ml

0.1 PU / ml

Ultrafiltration (UF) was then performed to form the liquid aminopeptidase, the stabilizing agent was glycerol (50%). This gave a solution called Peptidase L2 and had the following activity:

0.5 Leu-AP / ml

3.2 Phe-AP / ml

0.05 Val-AP / ml

0,1 PU / ml,

These results indicate that the selected strain of Aspergillus niger grew under the conditions selected herein and produced aminopeptidases without the necessary amount of endoprotease.

- 16 C.4 - pH diagrams of enzymatic activities

Leu-AP and Phe-AP activities were determined using peptidase L2 (see Example 2) using various buffers to maintain pH ranges from 2.5 'to 9.0.

The pH diagram of Aspergillus niger leucine aminopeptidase is shown in FIG. First

The pH diagram of the phenylalanine aminopeptidase from Aspergillus niger is shown in FIG. Second

It is evident from the figures that Leu-AP is active in the pH range of 5 to 8.5, while Phe-AP is active in the pH range of

5.5 to 9 and is similar to aminopeptidases from other Aspergillus species.

C.5 - Temperature diagrams of enzymatic activities

Activities Leu-AP and Phe-AP were fixed by peptidase L2, but at use different vaccines temperatures to maintain temperature interval from 5 to 70 ° C. temperature diagrams FIG. Third The results show that every enzyme has its own different

optimal temperature, which is 50 ° C for Leu-AP and 60 ° C for Phe-AP. Further described is a process for producing aminopeptidases from an Aspergillus culture. These aminopeptidases have optimal activity in the pH range of 6-8 and in the temperature range of 50-60 ° C; moreover, due to growing conditions, aminopeptidases can be formed without observable or substantial amounts of endoprotease. Preferably, the aminopeptidase activity is higher than that of the endoprotease, more preferably 10 times higher, even more preferably 30 times higher.

D. Maltogenic amylase (saccharification)

The amylase which is used in the saccharification step of the process of the invention is an enzyme that cleaves in dextrins or starch α-1,4 glucose-glucose bonds with terminal predominant products maltose and / or glucose. Very good results are obtained with α-amylase from Aspergillus oryzae commercially available from Gist-Brocades under the trademark Brewer's fermex or with recombinant β-amylase from Bacillus amyloliquefaciens commercially available from Novo under the trademark Novamyl.

Example 1

Sorghum (var. FAFA FARA) and corn cobs were ground according to standard technical conditions related to beer production. One part of the grains (60% sorghum + 40% corn cobs) is hydrated with 3 parts water. Calcium chloride was added to guarantee a total of 200 ppm Ca ²⁺ ions in a liquefied environment. The pH was adjusted to 6.5 with calcium hydroxide. BATS at a rate of 1.5 kg per ton of grains was added. Additional proteolytic enzymes were added in amounts as shown in Table 2:

Table 2

Test no. enzymes Dose / T grains 1 no 0 2 Brewers Protease 2000 1,2 kg 3 Brewers Protease 2000 1,2 kg Exo-peptidase from A. sojae 73000 Leu AP 4 Pastimase 400 10 kg 5 Pastimase 400 10 kg Exo-peptidase from A. sojae 73000 Leu AP

The mixture is maintained at 50 ° C for 1 hour; the temperature is then raised to 95 ° C (1 ° C / min) and held at this temperature for 95 minutes. It is then cooled to 60 ° C over 5 minutes. This was followed by the addition of Brewers Fermex (600 g / T). The mash is saccharified for 45 minutes at 60 ° C. The temperature was then raised to 76 ° C and maintained at this temperature for 10 minutes. The mash was then poured into a funnel containing Schleicher and Schuell filter paper. The volume of filtered malt was then measured and its specific gravity was determined. These values allow the extract and malt yield to be calculated. Using the ninhydrin reagent with glycine as standard, amino acids were measured. The amino acid concentrations obtained were corrected for the same sugar content (12 ° Plato) for comparison of the malts.

The data are shown in Table no. 3:

Table 3

Test no. filtered volume (ml) yield (%) Amino acids at 13 ° Plato (mg / 1) 1 310 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 obtained show that the use of an endoprotease in combination with an exo-peptidase in the process of the invention allows not only the amount of amino acids in the malt to be increased, but also its yield and filterability.

Example 2

In this test set, the same brewed drinks were tested as in Example 1, but using a neutral protease from Bacillus amyloliquefaciens (1.8 kg Brewers Protease 2000 per T grain) with various exo-peptidases from Aspergillus species as shown in Table 4 :.

Table 4

Test no. Exo-peptidases used Biological, origin Leu-AP / T Phe-AP / T 1 no 0 0 2 Aspergillus sojae 73000 49000 3 Aspergillus oryzae 73000 53000 4 Aspergillus niger 25000 100000

The data found are shown in Table 5:

Table 5

Test no. filtered volume (ml) yield (%) Amino acids at 13 ° Plato (mg / 1) 1 340 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 + exo-peptidase show better than endoprotease alone, regardless of the biological origin of the exo-peptidase. In addition to the amino acid content, filterability and yield are improved in this assay.

Example 3

To check the usability of the obtained malts for beer production, sorghum and corn cobs were boiled as described in Example 1; at the same time the sample is brewed in which the enzyme is not contained but the malt is replaced by sorghum, as shown in Table 6:

Table 6:

Test no. enzymes dose 1 malt replaces sorghum 2 Brewers Protease 2000 Exo-peptidase from A. sojae 1.8 kg / t 42000 Leu AP / T 3 Brewers Protease 2000 Exo-peptidase from A. oryzae 1.8 kg / t 40000 Leu AP / T 4 Brewers Protease 2000 Exo-peptidase from A. niger 1.8 kg / t 25000 Leu AP / T

The data are shown in Table 7:

Table 7

Test no. Filtered volume (Ml) yield (%) Amino acids at 13 ° Plato (mg / 1) 1 400 82.3 222, 0 2 395 89.5 127, 0 3 390 87, 9 109.0 4 375 91.1 113.1

The individual amino acid compositions of each brewed beverage were determined by HPLC. Amino acids are classified according to assimilation rate on Saccharomyces sp .:

Group A: rapid assimilation

Group B: moderate assimilation

Group C: slow assimilation

The results obtained are shown in Table 8:

(The N.B. values in mg / l do not match the summary of amino acid values shown in Table 6, since glycine was used as the standard in Table 7, while HPLC calibration is quite different.)

Table 8:

Amine acid (mg / l) An exam No. 1 2 3 4 Group A ¹ Glu 36 38 30 39 Asp 53 29 26 29 Ser 143 65 51 56 Thr 54 28 21 22 Lys 95 65 45 47 Arg 133 114 87 74 Pretty 513 340 260 268 Group B wall 102 36 32 28 Met 26 37 30 28 Leu 158 216 205 166 Ile 75 41 42 28 His 58 26 16 15 Pretty 418 356 325 265 Group Gly 29 19 13 17 Phe 134 89 85 95 Tyr 107 67 58 57 Trp 55 11 11 12 Ala 97 80 66 73 Total ¹ 421 265 233 253

Each malt was boiled for 45 minutes; boiled distilled water was added sterile to each of the malts to adjust the sugar content to 12 ° Plato. 350 ml of each standardized malt was poured aseptically into sterile containers; each vial was inoculated with brewer's yeast (5 g / l). The fermentation was carried out for 8 days at 11 ° C. The apparent dilution of each fermented malt was determined from the density after 8 days of fermentation.

The results obtained are shown in Table 9.

Table 9

Test no. Apparent dilution after 8 days (%) 1 82, 0 2 81.0 3 80,4 4 not determined

In the process according to the invention, the combination of endoprotease + exopeptidase was applied to sorghum, the invention makes it possible to produce malts that have a satisfactory ability to ferment beer compared to malt obtained by malt.

The surprising combination of Bacillus amyloliquifaciens endoprotease + Aspergillus sojae exo-peptidase provides a significant improvement in the A + group of amino acids.

Example 4

This example illustrates the advantages of artificially present exopeptidases in the liquefaction step rather than the saccharification step in the process of the invention. The sorghum and ears of corn are brewed as described in Example 1. Brewers Protease 2000 was added to all brewed beverages at a liquefaction step (1.8 kg / T). Aspergillus sojae exo-peptidase (40000 Leu-AP / T) was added either at the liquefaction step (assay n ° 1-2) or at the saccharification step (assay n ° 3-4).

The results obtained are shown in Table 10:

Table 10

Test no. Filtered volume (Mg / one) yield (%) Amino acids at 13 ° Plato (mg / 1) 1 380 89, 3 115.1 2 385 89, 5 119, 8 3 370 89, 4 109, 1 4 370 89, 8 112, 0

Repeated by the same. Standard deviations of filtered volume, yield and amino acid content were determined by assay. The values obtained are 10 ml, 0.5% and 0.9 mg / l, respectively.

The above results show that the introduction of exopeptidases in the liquefaction step presents a significant advantage in amino acid production and in particular contributes to filterability.

- 25 Example 5

This example illustrates the effect of increasing doses of A. oryzae exopeptidases in brewed beverages.

Sorghum + corn cobs are boiled as described in Example 1. In the liquefaction step, Brewers Protease 2000 and exopeptidase were added (Table 11).

Table 11

Test no. enzymes dose 1 no 0 2 Brewers Protease 2000 1.0 kg / T 3 Brewers Protease 2000 Exopeptidase from A. oryzae 1.0 kg / T _; 62000 Leu AP / T 4 Brewers Protease 2000 Exopeptidase from A. oryzae 1.0 kg / T 109000 Leu AP / T 5 Brewers Protease 2000 Exopeptidase from A. oryzae 1.0 kg / T 155000 Leu AP / T 6 Brewers Protease 2000 Exopeptidase from A. oryzae 1.0 kg / T 234000 Leu AP / T 7 Brewers Protease 2000 Exopeptidase from A. oryzae 1.0 kg / T 313000 Leu AP / T

The results are shown in Table 12:

Table 12

Test no. Filtered volume (Ml) yield (%) Amino acids at 13 ° Plato (mg / 1) 1 188 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

It follows from the above ^; that the greater the amount of exopeptidase, the higher the yields and the greater the amount of free amino acids. The optimum conversion-filtering effect is achieved even at a lower level of exopeptidase.

Example 6

The example illustrates the effect of increasing doses of A. niger exopeptidases in brewed beverages.

The sorghum and corn cobs were cooked as described in Example 1. Brewers Protease 2000 and exopeptidase were added in a surprising step.

Table 13

Test no. enzymes dose 1 no 0 2 Brewers Protease 2000 1.0 kg / T 3 Brewers Protease 2000 Exopeptidase from A. niger 1.0 kg / T 51000 Phe-AP / T 4 Brewers Protease 2000 Exopeptidase from A. niger 1.0 kg / T 90000 Phe-AP / T 5 Brewers Protease 2000 Exopeptidase from A. niger 1.0 kg / T 127000 Phe-AP / T

The results are shown in Table 14:

Table 14

Test no. Filtered volume (Ml) yield (%) Amino acids at 13 ° Plato (mg / 1) 1 188 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

It can also be seen that the greater the amount of exopeptidase, the higher the yields and the greater the amount of free amino acids. These results illustrate that A. oryzae exopeptidase appears to be slightly better than A. niger exopeptidase.

Example 7

Barley (var. PLAISANT) was ground to fine flour, which was treated in a brewery for filter press filtration. 57 g of flour was suspended at 50 ° C for 1 hour in 3000 ml of water containing:

mg B.A.T.S.

mg filtrase L3000 (+) (β-glucanase from Bacillus amyloliguiefaciens commercially available from Gist-brocades) 4 mg Brewers Protease 2000 and the relevant amount of exo-peptidase from Aspergillus sojae. The temperature was then raised to 63 ° C (1 ° C / min) and held at that temperature for 30 minutes. The temperature was then raised to 90 ° C (rate 1 ° C / min) and maintained at that temperature for 20 minutes. The brew is then cooled to 25 ° C and centrifuged at 7000g for 15 minutes. The supernatant is finally analyzed for soluble proteins and free amino acids.

The results are shown in Table 15:

Table 15

An exam no. Exo-peptidase dose (Leu-AP / T) Amino acids at 13 ° Plato (mg / 1) Soluble proteins (% dry substances) 1 0 77, 9 4,21 2 25000 95, 3 4, 75 3 50000 118, 4 4, 99 4 100000 133.2 5, 42 5 200000 203, 6 6, 63 6 400000 295, 0 7, 55

The results show that the exopeptidase from Aspergillus sojae allows to obtain the same level of free amino acids in the brewed barley beverage as in the brewed malt beverage where the enzyme is applied during the liquefaction steps.

Example 8

The barley was cooked according to Example 7, but the exo-peptidase from Aspergillus sojae was replaced by the exo-peptidase from Aspergillus niger.

The results are shown in Table 16:

Table 16

An exam no. Exo-peptidase dose (Leu-AP / T) Amino acids at 13 ° Plato (mg / 1) Soluble Teine (% dry substances) 1 0 68.2 4.37 2 10000 69.8 4.20 3 50000 75.2 4.25 4 200000 78, 0 4.16 5 500 000 89.1 4, 50 6 1000000 101.0 4.18

Although the Aspergillus niger exo-peptidase appears to be less potent than Aspergillus sojae, it also allows to achieve 100 mg / l amino acids in 12 ° Plato barley malt, a satisfactory value for beer fermentation.

Storage of microorganisms

The strains used for the production of exopeptidases are deposited with the Central Bureau voor Schimmelcultures, Oosterstraat i 1, Baarn, The Nederlands under deposit numbers CBS 115.39 (public collection), CBS 209.96 (A.sojae (DS 8351); date of deposit: 12 February 1996) and CBS 210.96 (A.oryzae (DS 23617); with deposit date: 12 February 1996).

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Claims (20)

patent claims' A process for producing fermented malt, comprising the steps of: (a) liquefying the cereal material with an auxiliary agent having an α-amylase and / or endoprotease activity to obtain liquefied mash, and (b) saccharifying said liquefied mash in the presence of α-amylase, wherein at least one of the above steps (a) and (b) is carried out in the presence of an enzyme having exo-peptidase activity. The process of claim 1, wherein the cereal material comprises at least 20% non-malt I cereals. The process of claim 2, wherein the cereal material comprises at least 20% non-malt sorghum. The method of claim 3, wherein the cereal material comprises at least 50% non-malt sorghum. A process according to any one of claims 1 to 4, characterized in that the exopeptidase is present during the liquefaction of the cereal material. A process according to characterized by a fungal exopeptidase. any one of claims 1 to 8, wherein the exopeptidase 5, is 7. The method of claim 6, wherein the fungus is Aspergillus species. 8. The method of claim 7, wherein the fungus is Aspergillus sojae. 9. The process of claim 6, wherein the exo-peptidase is an amino-peptidase. Process according to claim 8, characterized in that the endopeptidase is obtained from Bacillus amyloliquefanciens. A brewing process comprising the steps of producing malt using the process of any one of claims 1 to 10 and fermenting said malt to obtain a beer. A process for enzymatically releasing freely available nitrogen from cereals containing a high amount of glutelin and / or prolamine, comprising the step of adding a combination of endoprotease and exo-peptidase to the grains or liquefied mash obtained from said grains. A process for producing an alcohol or alcohol-containing beverage, comprising the step of fermenting the malt obtained by the process of claim 1 in the presence of yeast capable of forming alcohol. Use of exopeptidase in a process for producing fermentable malt. Use according to claim 14, characterized in that said exopeptidase is a fungal exopeptidase. Use according to claim 14 or 15, characterized in that said exopeptidase is from a fungus selected from the group consisting of Aspergillus niger, Aspergillus oryzae or Aspergillus sojae. Use according to any one of the preceding claims, characterized in that the malt is made from at least partially non-malt regional material. Use according to any one of the preceding claims, characterized in that the malt is made from malt sorghum or malt barley. Use of exopeptidases to improve the organoleptic properties of beer. Use of exopeptidases to improve malt filterability.