EP0896613A1

EP0896613A1 - Process for producing fermentable wort

Info

Publication number: EP0896613A1
Application number: EP97905021A
Authority: EP
Inventors: Marie-Paule Laroye; Jérôme Souppe
Original assignee: Gist Brocades BV
Current assignee: Koninklijke DSM NV
Priority date: 1996-02-12
Filing date: 1997-02-12
Publication date: 1999-02-17
Also published as: HK1018625A1; AU1873197A; BG102684A; YU33998A; BR9707404A; CA2245856A1; KR19990082497A; HUP9901003A3; CN1211275A; SK108698A3; CZ250398A3; PL328304A1; CN1285396A; CN1064398C; UY24458A1; WO1997029179A1; JP2000504571A; HUP9901003A2; AP9801316A0; EA199800711A1

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 α-amylase and/or endoprotease activity, to obtain a liquefied mash; (b) saccharification of the said liquefied mash in the presence of α-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

PROCESS FOR PRODUCING FERMENTABLE WORT

Field of the invention

The present invention is concerned with the preparation of fermentable wort from cereal grains, particularly from unmalted cereal grains. A further aspect of the invention is a wort obtained by carrying out the process according to the invention. The invention is also concerned with fermentation of worts obtained by the process according to the invention, in the production of alcohol, in the distilling industry or in the brewing of beer.

Background art Beer is manufactured by fermentation of either malted grains or unmalted grains.

In the latter instance, grains are liquefied and saccharified with the aid of commercial enzymes, yielding a wort that contains fermentable sugars and amino acids or other forms of nitrogen (collectively referred to as FAN for "Freely Available Nitrogen") necessary for yeast fermentation. For example MacFadden et al. (MacFadden, D.P. and Clayton, M. Brewing and

Beverage Industries International (1989), 1, 77-81) propose the addition of enzymes in the brewing of beer from unmalted sorghum, such as alpha-amylase, protease, beta-glucanase, cellulase, fungal alpha-amylase, amyloglucosidase and the like. MacFadden et al., also recommend to add yeast food for fermentation when unmalted 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. . J.Inst. Brew. (1994) 100, 79-84 3) report brewing of beer from 100% unmalted sorghum grains despite the fact that levels of FAN present in the wort from unmalted sorghum (51 mg/1) is well below that essential for fermentation of wort made from malted barley. In International Patent application WO 92/20777, a process is described for producing ethanol comprising grain liquefaction of unmalted whole corn or milo with amylase, mash saccharification with glucoamylase and acid fungal protease. The process provides for the protease being added during the fermentation step as well as the sacchari¬ fication step. The pH value of the fermentation medium is in the range 4-5 which means that fungal exo-peptidases are not active (Labbe, J.P., Rebeyrotte, P. Biochimie (1974) 56, 839-844, Lehman, K und Uhlig, H. Hoppe Seyler's Z. Physiol. Chem. (1969) 350, 99-104). A disadvantage of the above process is, therefore, that under these conditions, the fungal proteases used perform endoproteolysis only, thereby generating mainly oligopeptides and little free amino-acids.

For a general overview of the field, the review by Palmer should be mentioned (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 provides a process for making wort from unmalted cereal grains with unexpectedly good properties, such as high freely available Nitrogen (hereinafter referred to as FAN), good filterability and yield of the wort. The process may also be employed in the manufacture of worts from malted cereals, but it is particularly advantageous for the production of worts from unmalted grains, such as unmalted sorghum, or mixtures of unmalted sorghum and corn. The advantages are also existent with worts made from malted cereals (such as malted barley) combined with unmalted cereals (such as corn, rice or sorghum), a so-called "mixed brew".

Thus, 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 α-amylase and/or endoprotease activity, to obtain a liquefied mash;

(b) saccharification of the said liquefied mash in the presence of α-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-pepti¬ dases, but also thermostable carboxy-peptidases are useful. Particularly preferred accord¬ ing to the invention are amino-peptidases endogenous to Aspergillus fungi, more in particular A. niger, A. oryzae or A. sojae.

The process is particularly useful with a cereal grain which comprises at least 20% unmalted cereal, preferably more than 50% unmalted cereal, such as sorghum grains supplemented with unmalted corn cobs, rice or other unmalted cereals. It is found to be very advantageous according to the invention when the exopeptidase is present during the liquefaction of the cereal grains.

According to a further aspect of the invention, a fermentable wort is provided which is obtainable by a process according to the invention. Yet another aspect of the invention is in a process for brewing beer, wherein a fermentable wort is used according to the invention.

The invention further provides a process for making an alcoholic beverage, such as beer, comprising the steps of making wort using a process according to the invention and subsequently, or simultaneously, fermenting the said wort, thereby obtaining, e.g. beer. According to another embόclϊment, a process is provided for enzymatically releasing freely available nitrogen from cereals containing high amounts of glutelin and/or prolamin, comprising the step of adding to the grains, or a liquefied mash obtained from said grain, a combination of an endoprotease and an exo-peptidase.

A process for producing alcohol, comprising the step of fermenting the FAN obtained according to the process according to the invention in the presence of a yeast capable of producing alcohol.

The invention is illustrated by the following figures.

Description of the Figures Fig. 1 shows the pH profile of leucine-aminopeptidase from Aspergill s niger

Fig. 2 shows the pH profile of phenylalanine-aminopeptidase from Aspergillus niger Fig. 3 shows the temperature profile both aminopeptidases

The invention will be illustrated in greater detail below.

Detailed description of the invention

A process is provided for the preparation of a fermentable wort from cereals, wherein at least a protein is present having exo-peptidase activity under the conditions used. The aminopeptidases endogenous to fungi, especially the Aspergilli are useful, since they are sufficiently stable and active at pH 5 to 8, which is about the range wherein the pH falls during the liquefaction step. At this temperature and pH, carboxypeptidases are less useful. During the saccharification step slightly more acidic conditions prevail than during liquefaction, and carboxy-peptidase may be used advantageously in this step, provided they are sufficiently stable in the temperature range 50 - 60°C, preferably 50 - 70°C.

The use of exopeptidases according to the present invention does not only lead to an increase of the freely available nitrogen (FAN) of the wort, but also to an improve¬ ment of the filterability and the yield of the wort, compared to the process wherein no exopeptidases are used.

The process according to the invention is particularly advantageous for the preparation of fermentable wort from unmalted cereals, more in particular unmalted sorghum, optionally supplemented with other cereal material, such as corn, wheat, oat or rice. Cereals, in the context of this invention includes sorghum, wheat, barley, oat, rice and corn, and the like.

The use of exopeptidases according to the invention is also advantageous in the case of mixed brews, wherein as a rule malted cereal material as well as unmalted raw cereal material is used, (for example having up to 80%, or even up to 90% of malted cereals, the remainder consisting of unmalted cereals), as it was found that exopeptidases positively influence the organoleptic properties (taste and/or smell). It is envisaged, that these advantages are common to brewing from unmalted as well as malted cereals, as well as mixtures thereof.

Cereals wherein the use of exopeptidases is particularly advantageous in terms of

FAN, filterability, yield, and organoleptic properties, are those having relatively high prolamin and glutelin protein fractions. In addition to sorghum, also rice (about 80% glutelin) belongs to this category. When sorghum is used, attention should be paid that varieties are selected which are relatively low in polyphenol content.

Apart from the addition of exopeptidases, the preparation of the wort, for example for brewing beer, may be carried out as usual. Generally, it comprises the liquefaction of the cereal raw material to obtain a mash, followed by saccharification of the mash to obtain a wort. Filtration prior to fermentation is important.

The liquefaction step usually comprises grinding of the cereal raw material to obtain a flour of suitable particle size, hydrating with from about 1 to about 4, preferably about 3 parts of water, and optionally, depending on the endoprotease used, from about 50 to about 300 ppm of calcium, preferably 200 ppm Ca²⁺. Enzymes from Bacillus stearot- hermophilus appear to be less Calcium-dependent. Consequently, no Ca²⁺ supplementation is required in that case. The particle size of the ground cereals should not exceed about 3 mm; not more than 3,5% should exceed 1,3 mm; not more than 1,5% should be smaller than 0.25 mm. Enzymes that may be used in addition are cellulases, β-glucanases, and or other plant cell wall degrading enzymes.

The liquefaction medium is usually adjusted to a pH of between about 5 and 8, preferably between about 6 and 7, using, for example, calcium hydroxide. It is important to add α-amylase, preferably a thermostable α-amylase to the liquefaction medium as well as an endoprotease in a dosage sufficient to at least partially liquefy the cereal starch, and to at least partially degrade protein. Suitable dosages of α-amylase are from about 0,5 to about 2,0, preferably about 1 - 1,5 kg per Ton, when B.A.T.S. is used. Suitable dosages of proteases are, in the case of Brewers protease 2000, more than 0.5 kg/Ton grains (kg/T), preferably more than 1 kg/T. In the case of Panstimase 400 more than 2 kg/T, preferably more than 5, more preferably more than 10 kg/T should be used.

In the liquefaction process a number of steps are usually carried out at elevated temperature: after adding α-amylase and protease the mixture is maintained at a tempera¬ ture between about 40°C and 65°C, preferably between about 45 and 55°C, most prefe- rably 50°C, until a sufficient liquefaction is obtained. The time needed depends on the cereal or mixture of cereals used, but usually from about 30 minutes till about 2 hours is satisfactory. Subsequently, the temperature is raised gradually, the rate not being critical, till about 90-95°C and left at that temperature for about 30 minutes to about 1 hour. Then, the mixture is cooled to a temperature at which saccharification takes place: usually at about 50°C to about 70°C, preferably between about 55°C and 65°C, most preferably about 60°C. Slightly higher temperatures than 70°C should be possible, depending on the thermostability of the enzymes used in the saccharification step. When the preferred temperature is reached saccharifying enzymes are added, such as Brewers Fermex (α- amylase) or Novamyl (recombinant β-amylase) in amounts usually ranging from about 400 g/T to about 1 kg/T for Brewers Fermex. Also glucoamylases are frequently used. The saccharification takes from about 30 minutes to about 2 hours, whereafter the temperature is raised to about 75°C to about 85°C, inter alia to inactivate enzymes and unwanted microorganisms, and kept at the preferred elevated temperature for about 10 minutes; the period is not very critical. The mash so obtained is subsequently filtered using equipment well known in the art; a funnel with Schleicher & Schuell paper filter works satisfactorily. After filtration, the wort is fermented by a suitable yeast, under conditions depending on the strain used, and the final purpose; in addition to brewing beer, production of alcohol as biofuel or as alcoholic beverage are envisaged by the instant invention. Suitable strains, and suitable conditions are well known to the person skilled in the art.

It was found that the use of exo-peptidase during the preparation of wort, for example for brewing beer, is especially advantageous during the liquefaction step of the cereal raw material. However, compared to the absence of exo-peptidase, the addition of exo-peptidase during the saccharification step also leads to the mentioned advantages, such as higher FAN-levels, improved filterability and higher yield of the wort.

The process according to the invention should allow a phase wherein the pH and temperature conditions allow the fungal exo-peptidases to be active. Suitable exo-peptida- ses are those endogenous to fungi in general, more in particular those of Aspergillus. It is found that aminopeptidases of Aspergillus species, including A. niger, A. sojae and A. oryzae are especially useful.

As will be clear from the above description, the addition of enzymes which show exo-peptidase activity during the liquefaction and or the saccharification step in wort preparation produces worts with good filterability, high yield and high FAN. This makes it more attractive to brew beer, manufacture alcoholic beverages (liquors) and produce alcohol (as biofuel) using a significant percentage of unmalted cereals, or even exclusively unmalted cereals. The latter is very advantageous for brewers in countries where importa¬ tion of malts is restricted, or economically less attractive. Especially in Africa, beer is produced from (mixtures of) cereal raw material comprising a large percentage of sorghum.

Moreover, it is found that organoleptic properties (taste and smell) of beers produced from wort wherein exo-peptidases have been used in the liquefaction step or the saccharification step (or both), are improved. It is envisaged that this advantage is obtained when exo-peptidases is used in beer manufacture from malted cereals, such as traditional barley malts, or mixed brew beers (i.e. from a combination of malted and unmalted cereals). Experimental

A. Thermostable α-amylase (liquefaction)

The α-amylase used in the liquefaction step of the process according to the invention is generally an enzyme which cleaves α-1,4 -glucose-glucose bonds in starch. It is chosen amongst thermostable α-amylases. Very good results may be obtained with the α-amylase from Bacillus licheniformis commercially available from Gist-Brocades under the trademark Brewers Amyliq Thermo Stable (B.A.T.S.).

B. Endo protease (liquefaction)

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

C Exo-peptidases (liquefaction and/or saccharification) The exo-peptidases used in the liquefaction step of the process according to the invention are generally enzymes which cleave N-terminal bonds of peptides or proteins. Very good results may be obtained with preparations from Aspergillus species. A method for obtaining aminopeptidases from Aspergillus niger is disclosed below.

C.1 Determination of enzymatic activities

Exo-peptidase activity is expressed as Leucine aminopeptidase unit or as Phenylalanine aminopeptidase unit:

1 Leu-AP unit is the amount of enzyme needed to produce 1 μmole p-nitroaniline per minute at pH 7,2 and 20°C from L-leucine-p-nitroanilide * Phenylalanine aminopeptidase unit

1 Phe-AP unit is the amount of enzyme needed to produce 1 μmole p-nitroaniline per minute at pH 7,2 and 20°C from L-phenylalanine-p-nitroanilide. C.1.1 - Phenylalanine-aminopeptidase (Phe-AP)

Phenylalanine paranitroanilid was dissolved in 7.5 mM HCl at a concentration of 0.9 mM. 1 ml of that substrate solution was mixed with 1.5 ml 0.1M phosphate buffer pH 7.2. At t=0, 0.5 ml enzyme was introduced and left for reaction at 20°C. 1 ml IN HCl was added 15 minutes later. A blank was run with IN HCl being introduced at t=0. Optical density was determined for the blank (OD_btank) and for the assay (OD^_y) at 400 nm. Activity was calculated as follows:

(OD^ - OD_blank) 4 A = x — Phe-AP/ml

9.8 x 15 0.5

C.1.2 - Leucine-aminopeptidase (Leu-AP)

Leucine paranitroanilid was dissolved in water at a concentration of 9 mM. 1 ml of that substrate solution was mixed with 1.5 ml 0.1M phosphate buffer pH 7.2. At t=0, 0.5 ml enzyme was introduced and left for reaction at 20°C. 1 ml IN HCl was added 15 minutes later. A blank was run with IN HCl being introduced at t=0. Optical density was determined for the blank (OD_blank) and for the assay (OD^_y) at 400 nm. Activity was calculated as follows:

(OD^_y - OD_blank) 4

A = • x — Leu-AP/ml

9.8 x 15 0.5

C.1.3 - Endoprotease (PU)

This activity is measured by the hydrolysis of casein at pH 6.0, 40°C for lh. One PU is the amount of enzyme needed to liberate the equivalent of 1 μmole tyrosine per minute after precipitation of the remaining proteins with trichloracetic acid.

C.2 Screening of Aspergillus niser strains

200 Aspergillus niger strains isolated from different sources or obtained from culture collections, were grown in a medium containing 15 g/1 potato flour, 20 g/1 bactopeptone, 7 g/1 yeast extract, 4 g/1 potassium dihydrogenophosphate, 0.5 g/1 magnesi- um sulphate, 0.5 g/1 calcium chloride, 0.5 g/1 zinc chloride. pH was 4.8. After 24 h preculture at 240 rpm 30°C and 96 h culture at 275 rpm 30°C, supematants were collected and assayed for leucine-, phenylalanine- and valine-aminopeptidase activity as described above. Several Aspergillus niger strains showed high production potentials for at least one of these enzymatic activities, as shown in Table 1 (each value is a mean value from four individual results):

Table 1

From the above strains, strains 1108 and 1502 have been obtained from a culture collection and were deposited under the accession numbers NRRL 3112 and CBS 115.39, respectively. Strain NRRL 3112 has been used for the production of amyloglucosidase, α- amylase and glucoamylase. Strain CBS 115.39 has been used for the production of amylase.

C.3 Production of exopeptidase at laboratory scale

Some strains from the screening described in Example 1 have been fermented in laboratory fermenters (10 liters). Results obtained with strain 1502 are presented in this Example.

Spores of Aspergillus niger strains No 1502 were collected on PDA-plates after 7- 10 days of incubation at 30°C. An inoculum step was performed on shake flask in a medium composed of glucose (20 g/1) and corn steep (20 g/1) at pH 4.8 during 24 h. The main fermentation was performed according to a batch process. The following nutrients used were: 100 g/1 maltodextrins, 40 g/1 soy bean flour, 40 g/1 hydrolysed casein, 5 g/1 corn steep, 2 g/1 gelatin, 2 g/1 potassium dihydrogenophosphate, 1.3 g/1 sodium nitrate, 1 g/1 ammonium chloride, 0.01 g/1 iron sulphate and 0.5 g/1 antifoaming agent. All nutrients were firstly mixed together except maltodextrin. pH was adjusted at

4.8 ± 0.1. The fermenter was then sterilized at 125°C for 40 minutes. The maltodextrin solution was sterilized separately and added to the sterile but cooled fermentation medium.

The main fermentation was run in a laboratory fermenter which was filled with 6 liters of the medium described above and inoculated with the inoculum flask. Stirring and air providing were adjusted to maintain dissolved oxygen concentration as high as possible. The temperature was maintained at 30°C. The fermentation was stopped when all nutrients had been consumed, i.e. after about 130 hours.

The fermentation broth was filtrated to remove all microorganisms. Aminopeptida- se and endoprotease activities were measured in the filtrate: 0.15 Leu-AP/ml 1.0 Phe-AP/ml < 0.05 Val-AP/ml < 0.1 PU/ml UF concentration was then performed to formulate liquid aminopeptidase, glycerol (50%) being the stabilizing agent. The obtained solution called 'Peptidase L2' had the following activities:

0.5 . Leu-AP/ml 3.2 Phe-AP/ml < 0.05 Val-AP/ml

< 0.1 PU/ml

These results show that the selected Aspergillus niger strain grown under our selected conditions produces aminopeptidases without substantial amounts of endopro¬ tease.

C.4 pH profiles of enzymatic activities

Leu-AP and Phe-AP activities were determined from peptidase L2 (see Example 2) but using different buffers to screen a pH range from 2.5 to 9.0. pH profile of leucine-aminopeptidase from Aspergillus niger is shown in Fig. 1. pH profile of phenylalanine-aminopeptidase from Aspergillus niger is shown in Fig. 2.

The Figures show that Leu-AP is active in the pH range from 5 to 8.5, whereas the Phe-AP is active in the pH range from 5.5 to 9 which is similar to aminopeptidases from other Aspergillus species.

C.5 Temperature profiles of enzymatic activities

Leu-AP and Phe-AP activities were determined from peptidase L2 but using different incubation temperatures to screen a temperature range from 5 to 70°C. Temperature profiles are shown in Fig. 3.

Results show that each enzyme has a different optimal temperature, i.e. 50°C for Leu-AP and 60°C for Phe-AP.

A method for producing aminopeptidases from a culture of Aspergillus niger is disclosed. These aminopeptidases have an optimal activity in a range of pH 6-8 and in a range of temperature of 50-60°C; moreover, under the culture conditions, aminopeptidases can be produced without detectable or substantial amounts of endoprotease. Advantageously more activity of aminopeptidase is present than exoprotease, preferably 10 times more, more preferably 30 times more.

D, Maltogenic amylase (saccharification)

The amylase used in the saccharification step of the process according to the invention is an enzyme which cleaves α-1,4 glucose-glucose bonds in dextrins or starch to yield maltose and/or glucose as the major products. Very good results may be obtained with the α-amylase from Aspergillus oryzae commercially available from Gist-Brocades under the trademark Brewers' Fermex or with the recombinant β-amylase from Bacillus amyloliquefaciens commercially available from Novo under the trademark Novamyl. Exa ple 1

Sorghum (var. FAFA FARA) and co cobs are ground according to standard specifications for beer production. One part grains (60% sorghum + 40% com cobs) is hydrated with 3 parts water. Calcium chloride is added in order to warrant 200 ppm total Ca2+ in the liquefaction medium. pH is adjusted to 6,5 with calcium hydroxide. B.A.T.S. is added at a dose of 1,5 kg per Ton grains. Other proteolytic enzymes are added in amounts shown in Table 2:

Table 2

The mixture is maintained at 50°C for lh ; the temperature is then raised up to 95°C (rate l°C/min) and maintained at 95°C for 45 minutes. It is then cooled down to 60°C within 5 minutes. Brewers Fermex is then added (600g/T). The mash is then saccharified at 60°C for 45 minutes. The temperature is then raised up to 76°C and maintained at that temperature for 10 minutes. The mash is poured into a funnel contai¬ ning Schleicher and Schuell paper filter. The volume of filtered wort is then measured and its specific gravity is also determined. This allows to calculate extract and yield. Amino-acids are measured with the ninhydrin reagent using glycine as standard. The ami¬ no-acids concentrations obtained are corrected for comparison between worts at the same sugar content (12° Plato). Results are presented in Table 3:

Table 3

These results show that using endoprotease in combination with exo-peptidase in the process according to the invention enables to increase not only the amount of amino-acids in the wort but also yield and filterability.

Example 2

In that series, the same brews as in example 1 are carried out but using the combination of neutral protease from Bacillus amyloliquefaciens (1,8 kg Brewers Protease 2000 per T grains) with different exo-peptidases from Aspergillus species, as presented in Table 4:

Table 4

Results are shown in Table 5: Table 5

All combinations of endoprotease + exo-peptidase perform better than endoprotea- se alone, whatever the biological origin of the exo-peptidase is. Here too, yield and filterability is improved besides amino-acids' content.

Example 3

In order to control the ability of the obtained worts for beer production, sorghum and corn cobs are brewed as described in Example 1 ; simultaneously another brew is carried out in which no enzyme is used but malt replaces sorghum completely. See Table 6:

Table 6

Results are shown in Table 7: Table 7

Detailed amino-acids composition of each brew has been determined by HPLC. Amino-acids are classified according to the rate of assimilation by Saccharomyces sp. :

Group A : quick assimilation Group B : middle assimilation Group C : slow assimilation

Results are shown in Table 8:

(N.B. values in mg/1 do not fit with values of total amino-acids given in Table 6 because glycine was used as standard in Table 7 whereas HPLC calibration is quite different.)

Table 8

Each wort has been boiled for 45 minutes ; boiled and distilled water has been added aseptically to each wort in order to adjust the sugar content at 12° Plato. 350 ml of each standardised wort was poured aseptically into sterile flasks ; brewers' yeast was inoculated in each flask (5 g/1). Fermentations have been run for 8 days at 11 °C. Apparent attenuati¬ on of each fermented wort was determined from the density after 8 days fermentation.

Results are presented in Table 9 :

Table 9

Test No. Apparent attenuation at 8 days (%)

1 82,0

2 81,0 3 80,4 4 not determined

The combination of endoprotease + exopeptidase applied to sorghum in the process according to the invention enables to produce worts with satisfactory ability for beer fermentation when compared to worts obtained with malt. Surprisingly the combination of endoprotease from Bacillus amyloliquefaciens + exo-pep¬ tidase from Aspergillus sojae yields an important improvement in Group A + Group B amino-acids content.

Example 4 This example shows the advantages of introducing exo-peptidases at the liquefac¬ tion step rather than at the saccharification step in the process according to the invention. Sorghum + co cobs are brewed as described in example 1. Brewers Protease 2000 is added at the liquefaction step (1,8 kg/T) in all brews. Exo-peptidase from Aspergillus sojae (40000 Leu-AP/T) is added either at the liquefaction step (tests n°l-2) or at the saccharification step (tests n°3-4).

Results are presented in Table 10 : Table 10

Standard deviations for volume filtered, yield and amino-acids content have been determined by repeating identical tests. Estimations are : 10 ml, 0,5% and 0,9 mg/1 respectively.

Consequently, above results show that the introduction of exo-peptidases at the liquefaction step brings a significant positive advantage for amino-acids production and filterability mainly.

Example 5 This Example illustrates the effect of increasing doses of exopeptidases from A. oryzae in the brews.

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

Table 11

Results are shown in Table 12:

Table 12

The more exopeptidase, the higher yields and the higher amounts of free amino acids. Conversely filtration effect reaches optimal value even at low exopeptidase level.

Example 6 This Example illustrates the effect of increasing doses of exopeptidases from A. niger in the brews.

Sorghum + corn cobs are brewed as described in Example 1. Brewers Protease 2000 and exopeptidase are added at the liquefaction step.

Table 13

Results are shown in Table 14:

Table 14

Here too, the more exopeptidase, the higher yields and the higher amounts of free amino acids. These results illustrate, that the A. oryzae exopeptidase performs slightly better than the A. niger exopeptidase. Example 7 Barley (var. PLAISANT) was ground into fine flour adapted to filter presses in brewhouse. 57g flour is suspended at 50°C for lh in 300ml water containing: 9mg B.A.T.S.

2mg Filtrase L3000 (+) (β-glucanase from Bacillus amyloliquefaciens commercially available from Gist-brocades) 4mg Brewers Protease 2000 and the relevant amounts of exo-peptidase from Aspergillus sojae. The temperature is then raised up to 63°C (rate l°C/min) and maintained at 63°C for 30 minutes. It is then raised up to 90°C (rate l°C/min) and maintained at 90°C for 20 minutes. The brew is then cooled down at 25°C and centrifuged at 7000g for 15 minutes. The supernatant is finally analysed with regards to soluble proteins and free amino acids.

Results are presented in Table 15:

Table 15

These results show that exo-peptidase from Aspergillus sojae enables to reach a level of free amino acids in a barley brew as high as that reached in a malt brew, when applying the enzyme during the liquefaction steps. Example 8

Barley was brewed as described in Example 7 but replacing the exo-peptidase from Aspergillus sojae by the exo-peptidase from Aspergillus niger.

Results are given in Table 16:

Table 16

Even though the exo-peptidase from Aspergillus niger seems to be less efficient than that from Aspergillus sojae, it also enables to reach 100 mg/I amino acids in a 12° Plato barley wort, which is a satisfactory value for beer fermentation.

Deposits of microorganisms

The strains used for the production of the exopeptidases have been deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baam, The Netherlands under deposition number 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); date of deposit: 12 february 1996). MICROORGANISMS

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Centraalbureau voor Schimmelcultures (CBS)

Addraae pf dtαβaitan institution (Including poatal coda and country) '

P.O.Box 273

3740 AG BAARN, The Netherlands

Oata ot aeoo.it ' , Accaaalon NufflBtir *

12 February 1996 j CBS 209 . 96

«. ADDITIONAL INOICATIOMS ^' Heave Diana il not 100110*01*). Thie inlormation i* continue_d on a jaoarai* artacneα eneet —

We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT, shall be effected only by issue of a sample to an expert nomina¬ ted by the requester until the publication of the menti¬ on of grant of the national patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

C. OCSIβMATCD STATCS FOR WHICH IMOICATtONS AMI MAOC > (It th* indications it* not tor all designated StatMl

O. SCFARATX FURNISHING OF INDICATIONS • Ue«v* blank il not apollcaβi*)

The indication* liated αeiow will be luomitted to the International Bureau later * (Søociry tne general nature ot the indication! e α.. " Accession Nurnoer at Oeoosit '",

This sneet was received with tne international application wnen filed (to be cneoed by Ihe receiving Office)

X — — fcXer) 2 ^r^ — — -=---

(Authonied Ofi ^"1 * »-^tn ρrτiJ> β^r The data at receipt llrom tne tooiicsnt) by tne International Bureau "

was

(Autnoriied Offlcar)

Form PCTιRO'134 (January IM1) - 2b -

MICROORGANISMS

Optional She*! In connection with the microorganism r*t*rτ*d to on page 23 , Una 25 of tit* deecrlptJon '

A. lOiNTIFICATION OF DCPOSIT >

Further deposlta ar* Idantinad on an additional sh*«t Q] >

Nam* ol d*poaιtary institution «

Centraalbureau voor Schimmelcultures (CBS)

Address ot deooeltary inatltulion (including postal cod* and country) >

Oosterstraat 1

P.O.Box 273

3740 AG BAARN, The Netherlands

Data ot daooalt * Acceeaion Number *

12 February 1996 CBS 210.96 a. ADDITIONAL INDICATIONS ^' (leave blank it not applicable). ThI* Information la continued on a separate atuchad sheet

C. OCSIβNATCO STATCS FOR WHICH INDICATIONS AM MAOC > (It the indications are not tor all deslgnsted SUMS)

O. StRARATI FURNISHING. OF INDICATIONS • (leave Blank it not applicable)

The indications listed below will b* submitted to the International Bureeu later • (Specify ihe general nature ot the Indleatlona a 0-. '* Accession Number ot Deooeit ")

E. fj This sneet toes received with tne international application whan filed (to be checked by the receiving Office)

(Author ed Officer) teπiiάm&iter-- j I The data ol receipt (from the applicant) By the International Buraau "

waa

(Authorized Officer)

Form PCT/RO/134 (January 1M1 I

Claims

1. A process for the production of a fermentable wort, comprising the steps of:

(a) liquefaction of cereal material, with the aid of α-amylase and/or endoprotease activity, to obtain a liquefied mash, and

(b) saccharification of the said liquefied mash in the presence of α-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.

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 of claims 1 to 4, wherein the exopeptidase is present during the liquefaction of the cereal material.

6. A process according to any of claims 1 to 5, wherein the exopeptidase is a fungal exopeptidase.

7. A process according to claim 6, wherein the fungus is an Aspergillus species.

8. A process according to claim 7, wherein the fungus is Aspergillus sojae.

9. A process according to claim 6, wherein the exo-peptidase is an amino-peptidase.

10. A process according to claim 8, wherein the endoprotease is obtainable from Bacillus amyloliquefaciens.

11. A process for brewing beer, comprising the steps of making wort using a process according to any of claims 1 to 10 and fermenting the said wort to obtain beer.

12. A process for enzymatically releasing freely available nitrogen from cereals containing high amounts of glutelin and/or prolamin, comprising the step of adding to the grains, or a liquefied mash obtained from said grain, a combination of an endoprotease and an exo-peptidase.

13. A process for producing alcohol, or an alcohol containing beverage, comprising the step of fermenting a wort obtainable according to a process of claim 1 in the presence of a yeast capable of producing alcohol.

14. Use of an exopeptidase in a process of making a fermentable wort.

15. Use according to claim 14, wherein said exopeptidase is a fungal exopeptidase.

16. Use according to claim 14 or 15, wherein said exopeptidase is from a fungus selected from the group consisting of Aspergillus niger, Aspergillus oryzae or Aspergillus sojae.

17. Use according to any one of the preceding claims, wherein the wort is made from at least partially unmalted areal material.

18. Use according to any one of the preceding claims, wherein the wort is made from unmalted sorghum or unmalted barley.

19. Use of exopeptidases to improve the organoleptic properties of beer.

20. Use of exopeptidase to improve the filterability of wort.