CA2245856A1

CA2245856A1 - Process for producing fermentable wort

Info

Publication number: CA2245856A1
Application number: CA002245856A
Authority: CA
Inventors: Marie-Paule Laroye; Jerome Souppe
Original assignee: Individual
Current assignee: DSM Delft BV
Priority date: 1996-02-12
Filing date: 1997-02-12
Publication date: 1997-08-14
Also published as: HK1018625A1; YU33998A; EE9800240A; CN1211275A; CZ250398A3; AP9801316A0; CN1064398C; BG102684A; SK108698A3; EA001075B1; EA199800711A1; KR19990082497A; WO1997029179A1; BR9707404A; UY24458A1; CN1285396A; JP2000504571A; HUP9901003A2; PL328304A1; EP0896613A1

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

CA 0224~8~6 1998-08-07 WO 97/29179 PCTll~:P97/00686 - - S PROCESS FOR PRODUCING FERMENTABLE WORT

Field of the invention The present invention is concerned with the ple~ation of ferment~}e wort from cereal grains, particularly from lmm~lte-l 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 ferment~tion 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 m~mlf~ctllred by ferment~tion of either malted grains or Immalted grains.
In the latter instance, grains are liquefied and saccharified with the aid of commercial enzymes, yielding a wort that contains ferment~hle sugars and amino acids or other forms of nitrogen (collectively referred to as FAN for "Freely Available Nitrogen") necessary for yeast fçrment~tion.
For example MacFadden et al. (MacFadden, D.P. and Clayton, M. Brewing and Beverage Industries Tnt~rn~tional (1989), 1, 77-81) propose the addition of enzymes in the brewing of beer from lmm~lted SOl~hl~ll, such as alpha-amylase, protease, beta-glnf~n~e7 cellulase, fungal alpha-amylase, amyloglucosidase and the like. MacFadden et al., also recommend to add yeast food for ferment~tion when unmalted sorghurn is used.
B~ajomo 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) report brewing of beer from 100% nnm~lte~l sorghum grains despite the fact that levels of FAN present in the wort from unmalted sorghum (51 mg/l) is well below that essential for ferment~tion of wort made from malted barley.
In Tnttorn~tional 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 CA 02245856 l998-08-07 _ ~ _ 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 5 oIigopeptides 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 Uni~el~iLy Press, Aberdeen, Scotland, 61-242).

Summa~ of the il.v~ ~ion The present invention provides a process for making wort from llnm~ltç-l 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 aIso be employed in the m~nllf~ch~re of worts firom malted cereals, but it is particularly 15 advantageous for the production of worts from llnm~ited grains, such as unmalted , or mixtures of unmalted sorghum and corn. The advantages are also f~ t~nt with worts made from malted cereals (such as malted barley) combined with unmalted cereals (such as corn, rice or ~ l), a so-called "mixed brew".
Thus, the invention provides a process for the production of a ferment~ble wort 20 from cereal grains, Colll~l;si.lg the steps of:
(a) liquefaction of cereal m~t~ri~l, with the aid of a-amylase and/or endoprotease activity, to obtain a liquefied mash;
(b) s~crh~rilFic~tion of the said liquefied mash in the presence of oc-amylase, (c~ filtration of the liquefied and saccharified mash to obtain a f~rm~nt~ble 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 arnino-peptidases endogenous to Aspergillus fungi, more in particular A. niger, A. ory2ae or A. sojae.
The process is particularly useful with a cereal grain which comprises at least 20%
mm~lted cereal, preferably more than 50% llnm~ltçd cereal, such as sorghum grains suppl~n~ntPd with lmm~ltçcl corn cobs, rice or other unmalted cereals. It is found to be CA 0224~8~6 1998-08-07 wo 97/29179 PCT/EPg7/00686 .
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 ferment~hle wort is provided which is obtainable by a process according to the invention. Yet another aspect of the S invention is in a process for brewing beer, wherein a ferment~ble wort is used according to the invention.
The invention further provides a process for m~king an alcoholic beverage, such as beer, comprising the steps of making wort using a process according to the invention and subsequently, or ~imult~neously, fçrrnentin~ the said wort, thereby obtaining, e.g. beer.
According to another emb~nnent a process is provided for enzymatically releasing freely available nitrogen from cereals co~ g high arnounts 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
~5 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.

D~ ,lion of the Figures 20 Fig. 1 shows the pH profile of leucine-aminopeptidase from Aspergillus niger Fig. 2 shows the pH profile of phenyl~l~nine-aminopeptidase from ~spergiUus niger Fig. 3 shows the t~ eldlu~c; profile both aminopeptidases The invention will be illustrated in greater detail below.

Detailed des~ tion of the invention A process is provided for the pL~dlion of a ferment~hle 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 lelllpeldlul~ 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 S0 - 60~C, preferably 50 -WO 97/29179 PCT~EP97/00686 .
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 5 exopeptidases are used.
The process according to the invention is particularly advantageous for the pre~udLion of fermentable wort from llnm~ltorl cereals, more in particular llnm~ltecl sorghum, optionally supplemented with other cereal material, such as corn, wheat, oat or rice. Cereals, in the context of this invention includes sol~ ~ll, wheat, barley, oat, rice 10 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 rem~in~ r con~i.C~ing of lmm~ltPd cereals), as it was found that exopeptidases 15 positively influence the organoleptic plo~.Lies (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 ~ P~L Lies, are those having relatively high 20 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 plel.~udlion of the wort, for example for brewing beer, may be carried out as usual. Generally, it comprises the liquefaction of 25 the cereal raw material to obtain a mash, followed by saccharification of the mash to obtain a wort. Filtration prior to f~rm~nt~ti~-n is important.
The liquefaction step usually compri~es grin-linp 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 30 to about 300 ppm of calcium, preferably 200 ppm Ca~+. Enzymes from Bacillus stearot-hermop~2ilus appear to be less Calcium-dependent. Consequently, no Ca2+ suppl~ment~tion 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 -CA 0224~8~6 1998-08-07 WO 97/29179 PCTtEPg7/00686 than 0.25 mm. Enzymes that may be used in addition are cellulases, i3-gll~c~n~ces, 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 in~oll~ll to add a-amylase, preferably a thermostable a-amylase to the liquefaction meflillm 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 a-arnylase 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 ~ protease 2000, more than 0.5 kg/Ton grains (kg/T), preferably more than 1 kg/T. In the case of p~n~tim~e 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 a-amylase and protease the mixture is m~int~ined 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 mimltes till about 2 hours is satisfactory. Subsequently, the ~elllpe~Lule is raised gradually, the rate not being critical, till about 90-95~C and left at that temp~ld~uLe for about 30 minllfPs to about 1 hour.
Then, the llli~l~e 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 tempclaLules than 70~C should be possible, depending on the thermostability of the enzymes used in the saccharification step. When the preferred ttlll~ldLul~ is reached saccharifying enzymes are added, such as Brewers Fermex (a-amylase) or Novamyl (recombinant ~3-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 t~lllp~dlwC is raised to about 75~C to about 85~C, inter alia to inactivate enzymes and unwanted microorg~ni~m~, and kept at the preferred elevated temperature for about 10 miml~es, 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 fltration, the wort is fermente~l 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 ~ aiion of wort, for example for brewing beer, is especially advantageous during the liquefaction step of the S cereal raw m~t~ri~ql 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-10 ses are those endogenous to fungi in general, more in particular those of Aspergillus. It isfound 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 15 ~le~ lion produces worts with good filterability, high yield and high FAN. This makes it more attractive to brew beer, m~mlf~rtnre alcoholic beverages (liquors) and produce alcohol (as biofuel) using a significant percentage of nnm~lte~:l cereals, or even exclusively nm~ltet1 cereals. The latter is very advantageous for b.c~e.~ in countries where importa-tion of malts is restricted, or economically less attractive. Especially in Africa, beer is 20 produced from ~ es of~ cereal raw m~t~ri~l comprising a large pc.ce~ ge 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 sacch~rific~tion step (or both), are improved. It is envisaged that this advantage is 25 obtained when exo-peptidases is used in beer m:~m~ lre from malted cereals, such as traditional barley malts, or mixed brew beers (i.e. from a combination of malted and llnms-lte-l cereals).

CA 0224~8~6 1998-08-07 WO 97/:~9179 PCT/EP97/00686 Experimental -- - A. Thermostable ~c-amylase ~liquefaction) The oc-amylase used in the liquefaction step of the process according to the invention is generally an enzyme which cleaves a-1,4 -glucose-glucose bonds in starch. It is chosen amongst thermostable o~-amylases. Very good results may be obtained with the a-amylase from Bacillus licheniformis commercially available from Gist-Brocades under the tr~(lem~rk Brewers Amyliq Thermo Stable (B.A.T.~.).

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 beginnin~ 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 fr~ m~rk Blcwcl'S Protease 2000.
Also the proteolytic enzymes from Streptomyces fradiae may be used, which are commer-cially available from P~n.etim~.ee SARL under the tr~lem~rk p~netim~ee 400.

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

C.1 D~t~i in~fi~ of enzymatic activities Exo-peptidase activity is expressed as Leucine aminopeptidase unit or as Phenyl~l~nine aminopeptidase unit:
1 Leu-AP unit is the amount of enzyme needed to produce 1 ,umole p-nitroaniline per minute at pH 7,2 and 20~C from L-leucine-p-nitro~nili~le * Phenyl~l~nine aminopeptidase unit 1 Phe-AP unit is the amount of enzyme needed to produce 1 ,umole p-nitroaniline per minute at pH 7,2 and 20~C from L-phenyl~l~nine-p-nitro~nilide.

CA 02245856 l998-08-07 W O 97/29179 PCT~EP97/00686 . ~ 8 C.1.1 - PhenYI~l~nine-aminopeptidase (Phe-AP) Phenyl~l~nine ~ ~ilr~anilid 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 ph(~sI~h~tf? buffer pH
7.2. At t=0, 0.5 ml enzyme was introduced and left for reaction at 20~C. 1 ml lN HCl 5 was added 15 minllte~ later. A blank was run with lN HCl being introduced at t=0.
Optical density was tlt-termined for the blank (ODblank) and for the assay (ODassay) at 400 nm. Activity was calculated as follows:

(ODassay ~ ODblanl~) A = - x Phe-AP/ml 9.8 x 15 o.5 C.1.2 - Leucine-aminopeptidase (Leu-AP) Leucine pa~ oanilid was dissolved in water at a concentration of 9 mM. 1 rnl 15 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 lN HCI was added 15 minutes later. A blank was run with lN HCl being introduced at t=0. Optical density was ~1~t~rmined for the blank (ODbla~k) and for the assay (OD~ssay) at 400 nm. Activity was calculated as follows:

(OD~ssay ~ ~Dbtank) A = x -- Leu-AP/ml 9.8x 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 arnount of enzyme needed to liberate the equivalent of 1 ,umole tyrosine per minute after precipitation of the rem~ining proteins with trichloracetic acid.
C.2 Screenin~ of Aspergillus niger strains 200 AspergiUus niger strains isolated from different sources or obtained from culture collections, were grown in a medium co~ 15 g/l potato flour, 20 g/lbactopeptone, 7 g/l yeast extract, 4 g/l potassium dihydrogenophosphate, 0.5 g/l m~gn~si-CA 0224~8~6 l998-08-07 WO 97/29179 PCT/E:P97/00686 _ g _ um sulphate, 0.5 g/l calcium chloride, 0.5 g/l zinc chloride. pH was 4.8. After 24 h preculture at 240 rpm 30~C and g6 h culture at 275 rpm 30~C, supern~t:~ntc were - - collected and assayed for leucine-, phenyl~l~nine- and valine-aminopeptidase activity as described above. Several Aspergillus niger strains showed high production potentials for at S least one of these enzymatic activities, as shown in Table 1 (each value is a mean value from four individual results):

Table 1 Skain number aminopeptidase activities in sup~ t~nt~ endo-peptidase Leu-AP/l Phe-AP/l Val-AP/l 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 have been obtained from a culture 20 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, a-amylase and glucoamylase. Strain CBS 115.39 has been used for the production of amylase.

25 C 3 Production of exopeptidase at laboratorY scale Some strains from the sclet;~ g 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 7-30 10 days of in~ lh~tion at 30~C. An inoculum step was perfor~ned on shake flask in amedium composed of glucose (20 g/l) and corn steep (20 g/l) at pH 4.8 during 24 h.

The main fermentation was p~;.r~ ed according to a batch process. The following nutrients used were: 100 g/l malto-le~t~n~, 40 gll soy bean flour, 40 g11 hydrolysed casein, - - 5 g/l corn steep, 2 g/l gelatin, 2 g/l potassium dihydrogenophosphate, 1.3 g/l sodiurn nitrate, 1 gll ammonium chloride, 0.0~ gA iron sulphate and 0.5 g/l antifoaming agent.
All nutrients were firstlv mixed together except maltodextrin. p~ was adjusted at 4.8 + 0.1. The f~l...r.,.ler was then sterilized at 125~C for 40 mimltes The maltodextrin solution was s~rili7~ paldlely and added to the sterile but cooled fçrment~tion medium.
The main ferment~tion was run in a laboratory fermenter which was filled with 6 10 liters of the medium described above and inoculated with the inoculum flask. Stirring and air providing were adjusted to m~int~in dissolved oxygen concentration as high as possible. The t~ ldLul~; was ..~i."~ ed at 30~C. The fermentation was stopped when all nutrients had been consumed, i.e. after about 130 hours.
The ferment~tion broth was filtrated to remove all microorg~ni.cm~ 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 perforrned to form~ t~ liquid arninopeptidase, glycerol (50%) being the stabilizing agent. The obtained solution called 'Peptidase L2' had the following activities:
0.5 ~eu-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 arninopeptidases without substantial amounts of endopro-tease.
C.4 pH profiles of enzvmatic activities Leu-AP and Phe-AP activities were det~rmin~l from peptidase L2 ~see Exarnple 2) but using different buffers to screen a pH range from 2 5 to 9Ø

CA 0224~8~6 1998-08-07 WO 97/29179 PCT/I~P97/00686 pH profile of leucine-aminopeptidase from Aspergillus niger is shown in Fig. 1.
pH profile of phenyl~l~nin~-aminopeptidase from Aspergillus niger is shown in - - Fig. 2.
The Figures show that Leu-AP is active in the pH range from S to 8.5, whereas 5 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 10 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 arninopeptidases from a culture of Aspergillus niger is disclosed.
15 These aminopeptidases have an optimal activity in a range of pH 6-8 and in a range of telllpeLalule 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 ex-~plol~ase, preferably 10 times more, more preferably 30 times more.
D. Malto~enic amYlase (saccharification) The amylase used in the saccharification step of the process according to the invention is an enzyme which cleaves a-1,4 glucose-glucose bonds in ~lextrin~ or starch to yield maltose and/or glucose as the major products. Very good results may be obtained 25 with the a-amylase from Aspergillus oryzae commercially available from Gist-Brocades under the trademark Brewers' Fermex or with the recombinant ~3-amylase from Bacillus amyloliquefaciens commercially available from Novo under the trademark Novamyl.

W O 97/29179 PCT~EP97/00686 Example 1 - - Sorghum (var. FAFA FARA) and corn cobs are ground according to standard specifications for beer production. One part grains (60% sorghum ~ 40% corn cobs) is 5 hydrated with 3 parts water. Calcium chloride is added in order to W~ldlll 200 ppm total Ca2+ in the liquefaction medium. pH is ad~usted to 6,5 with calcium l~dl~xide. 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:

10Table 2 Test No. Enzymes Dose per T grains none 0 2 I3~ Protease 2000 1,2 kg 3 Brewers Protease 2000 1,2 kg Exo-peptidase from A.sojae 73000 Leu-AP

4 p~n~tim~e 400 10 kg p~n~timz~e 400 10 kg Exo-peptidase from~4.sojae 73000 Leu-AP

The l~ {LUL~ is m~int~ined at 50~C for lh; the temperature is then raised up to 95~C (rate 1~C/min) and m~;nt~ined at 95~C for 45 minllt~s It is then cooled down to 20 60~C within 5 minlltes ~ we:ls Fermex is then added (600g/T~. The mash is then saccharified at 60~C for 45 mimltes The t~nl~c~dlule is then raised up to 76~C and m~int~in~d at that ~elll~eldlule for 10 min~1tes 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 ~ termin~l This allows to calculate extract and yield.
25 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 ~ Test No.Volume filtered Yield Amino-acids at 12~ Plato (ml) (%) (mg/l) 1 310 85,4 26,9 2 370 86,9 54,7 3 382 90,2 119,5 4 365 86,9 76,2 372 91,0 102,3 These results show that using endo~l~otease in combination with exo-peptidase in the process according to the invention enables to increase not only the arnount 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 difr~lGlll exo-peptidases from Aspergillus species, as plc3el.lGd in Table 4:
Table 4 Test No. Exo-peptidases involved Biological origin Leu-AP/T Phe-AP/T
none 0 0 2Aspergillus sojae 73000 49000 3Aspergillus oryzae 73000 53000 4Aspergillus niger 25000 100000 .
- Results are shown in Table 5:

Table 5 .
Test No. Volume filtered Yield Amino-acids at 12~ Plato (ml) (%) (mgA) 340 86,1 61,4 2 400 90,0 1 16,0 3 385 87,9 95,7 4 382 89,8 99,8 A11 combinations of endoprotease + exo-peptidase perform better than endoprotea-10 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 15 and corn cobs are brewed as described in Example 1; ~imllltzmeously another brew is carried out in which no enzyme is used but malt replaces sorghum completely. See Table 6.

Table 6 20Test No. Enzymes Dose Malt replacing sorghum 2 13~ Protease 2000 1,8 kg/T
Exo-peptidase from A.sojae 42000 Leu/AP/T
3 Brewers protease 2000 1,8 kg/T
Exo-peptidase from A. oryzae 40000 Leu-AP/T
4 Brewers Protease 2000 1,8 kg/T
Exo-peptidase from A.niger 25000 Leu-AP/T

30 Results are shown in Table 7:

_ Table 7 Test No.Volume filtered YieldAmino-acids at 12~ Plato (ml) (%) (mg/l) 400 82,3 222,0 2 395 89,5 127,0 3 390 87,9 109,0 4 375 91,1 1 13,1 Detailed amino-acids composition of each brew has been determined by HPLC.
10 Amino-acids are classified according to the rate of ~simil~tion by Saccharomyces sp.:

Group A: quick ~imil~tion Group B: middle ~imilAtion Group C: slow ~ imil:~tion Results are shown in Table 8:
(N.B. values in mg/l 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 dirrc.~ L.) Table 8 Amino-acids Test No.
(mg/l) 1 2 3 4 5Group A
Glu 36 38 30 39 Asp 53 29 26 29 Ser 143 65 51 56 Thr 54 28 21 22 10 Lys 95 65 45 47 Arg 133 114 87 74 Total 513 340 260 268 Group B
15 Val 102 36 32 28 Met 26 37 30 28 Leu 158 216 205 166 Ile 75 41 42 28 His 58 26 16 15 20Total 418 356 325 265 Group C
~ly 29 19 13 17 Phe 134 89 85 95 25 Tyr 107 67 58 57 Trp 55 11 11 12 Ala 97 80 66 73 Total 421 265 233 253 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 rnl of each standardised wort was poured aseptically into sterile flasks; blC;W~ yeast was inoculated 5 in each flask (5 g/l). Ferment~tions have been run for 8 days at 11~C. A~ n~ e~
on of each fçrmente~l wort was ~let~nined from the density after 8 days f~rment~tion.

Results are presented in Table 9:

10 Table 9 Test No. Apparent attenuation at 8 days (%) 82,0 2 81,0 3 80,4 4 not ~letermin~

The combination of endoprotease + exopeptidase applied to sol~hulll in the process according to the invention enables to produce worts with s~tiA~ctory abilit,v for beer fermentation when conl~ ,d to worts obtained with malt.
20 SurpriAin~ly the comhin~tion of endoprotease from Bacillus amy~oliquefaciens + exo-pep-tidase from ~spergillus sojae yields an illll~ol~l~ hllplov~ment in Group A + Group B
arnino-acids content.

Example 4 Th;s ex~.. ple shows the adv~tageA of ir,troducirlg exo-peptidases at ~he liquefac-tion step rather than at the saccharification step in the process according to the invention.
Solghulll + corn cobs are brewed as described in example 1. ~l~vv~ 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~1-2) or at the 30 A~crh~rification step (tests n~3-4).

~esults are plcse~ d in Table 10:

Table 10 Test No.Volume filtered Yield Amino-acids at 12~ Plato (mg/l) (%) (mg/l) 380 89,3 1 15,1 2 385 89,5 1 19,8 3 370 89,4 109,1 4 370 89,8 1 12,0 .
Standard deviations for volurne filtered, yield and amino-acids content have been 10 determined by repeating identical tests. F~tim~tions are : 10 ml, 0,5% and 0,9 mg/l respectively.
Consequently, above results show that the introduction of exo-peptidases at the liquefaction step brings a ~ignific~nt positive advantage for atnino-acids production and filterability mainly.
~ys~ml~le 5 This Example illu~ Les the effect of increasing doses of exopeptidases from A.
o~yzae in the brews.
Sorghum + corn cobs are brewed as described in Example 1. Brewers Protease 20 2000 and exopeptidase are added at the licluefaction step (Table 11).

W O 97/29179 PCT~EP97/00686 .

Table 11 Test No. Enzymes Dose none 0 2 Brewers Protease 2000 1,0 kg/T
3 Brewers Protease 2000 1,0 kg/T
Exopeptidase from A. oryzae 62000 Leu-AP/T
4 Brewers Protease 2000 1,0 kg/T
Exopeptidase from A. oryzae 109000 Leu-AP/T
Brewers Protease 2000 1,0 kg/T
Exopeptidase from A. oryzae 155000 Leu-AP/T

6 13~ Protease 2000 1,0 kg/T
Exopeptidase from A. oryzae 234000 Leu-AP/T

7 Brewers Protease 2000 1,0 l~g/T
Exopeptidase from A. oryzae 313000 Leu-AP/T

Results are shown in Table 12:

WO 97/29179 PCT/EP97/001i86 Table 12 Test No.Volume filtered YieldAmino-acids at 12~ Plato (ml) (%) (mg/l) 188 88.4 39.1 2 275 88.9 59.8 3 280 89.1 81.5 4 280 89.6 90.5 280 89.7 95.3 6 280 90.4 101.0 7 280 89.5 114.0 The more ~op~lidase, the higher yields and the higher amounts of free amino acids. Conversely filtration effect reaches optimal value even at low exopeptidase level.

Example 6 15 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 Test N~ Enzymes Dose none 0 2 Brewers Protease 2000 1,0 kg/T
3 Brewers Protease 2000 1,0 kglT
Exopeptidase from A. niger 51000 Phe-AP/T
4 Brewers Protease 2000 1,0 kg/T
Exopeptidase from A. niger 90000 Phe-AP/T
Brewers Protease 2000 1,0 kg/T
Exopeptidase from A. niger 127000 Phe-AP/T

10 Results are shown in Table 14:

Table 14 Test N~ Volume filtered Yield Amino-acids at 12~ Plato (ml) (%) (mg/l) 188 88.4 39.1 2 240 88.6 60.1 3 2~6 89.7 63.7 4 258 89.5 64.2 232 89.7 65.7 -20 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 cont~inin~:
9mg B.A.T.S.
2mg Filkase L3000 (+) (~ gl~lc~nzlce from Bacillus amyloliquefaciens commercially available from Gist-brocades) 4mg Brewers Protease 2000 and the relevant amounts of exo-peptidase from ~Ispergillus sojae. The temperature is then raised up to 63~C (rate 1~C/min) and m~int~ined at 63~C for 30 minllte~ It is then raised up to 90~C (rate 1~C/min) and ~ ed at 90~C for 20 minlltes The brew is then cooled down at 25~C and centrifuged at 7000g for 15 minutes. The SUpt;lllak~ iS finally analysed with regards to soluble proteins and free amino acids.

Results are presented in Table 15:

Table 15 Test No.Dose exo-peptidaseAmino-acids at 12~ PlatoSoluble proteins (Leu-AP/T) (mg/l (% of dry matter) 0 77.9 4.21 2 25000 95.3 4.75 3 50000 118.4 4.99 4 100000 133.2 5.42 200000 203.6 6.63 6 400000 295.0 7.55 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 Test No.Dose exo-peptidaseAmino-acids at 12~ Plato Solu~le protei~s (Leu-AP/T) (mg/l) (% of dly matter 1 0 68.2 4.37 2 100~0 69.8 4.20 3 50000 75.2 4.25 4 200000 78.0 4.16 500000 89.1 4.50 6 1000000 101.0 4.18 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/l almino acids in a 12~ Plato barley wort, which is a s~t;.~fastory value for beer ferrnentation.
Del~osits of microor~ni~m~
The skains used for the production of the exopeptidases have been deposited at the Cenkaal Bureau voor Schirnmelcultures, Oosterskaat 1, Baarn, The Netherlands under deposition number CBS 115.39 (public collection), CBS 209.g6 (A. sojae (DS 8351): date 25 of deposit: 12 February 1996) and CBS 210.96 (A. oryzae (DS 23617), date of deposit: 12 february 1996).

=
WO97/29179 PCT~P97/00686 MICROOIRGANISMS
- ODuon-l Sh--~ in conn~on wltb tn- _ r t rr d to on _ 23, 24 Ot th- d--erlDDon ' ~ no~orocros~T~
Furth-r a-Do~ r- Id-ntlll d on ~n ~ddiUon-l ~n- t It-m- ot a-ao-~Ury In~tltuaon ~
Centraalbureau voor S~-hi ?lcultures (CBS) Ad~'~ 'OaS~~ët~s'~rtala't~'n'~U~'n~ oo-l l coa- ~nd countr~ ~
P.O.Box 273 3740 AG BAARN, The Netherlands O-~- ot d-Oo-lt ' I Acc---lon Nunno~ s 12 February 1996 I CBS 209.96 A~L~ tOlC ~TtO~ v- Dl-nll It not ~oollc~ol-) Th~- Inform~lon 1- conbnu a on ~ ~o~t- ~n cn o ~n We inform you that the availability o~ 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 reguester 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 D~~1C~l~T~t 5,TAT'~5 FOR Wl~tlC~t l~tDlC ~TIQ- 5 AR~ ~AD~ ~il ~n- nalc~tlon~ ~r- not tor ~11 d--~on~t d 5Ut--l D 5Yr~l~.~T~ r ~ II~C OF It~t~lC ~TtO t5 ' (1--~- ol-nll d nr~t ~DDlic~bl~
Th~ ~nalc~ on~ t~d D- OW w~ll r,- ~uDm tt-a lo ~n- ! -' Etut--u l-t-r ~So-Clty ~n~ o-n-~-l n~ur~ ol h- ~nalc tlon- ~ o Acc~lon ~tumo~r rJI D-oo-d E. --' Th.~ ~n-~t _A~ -c- ~-a w ~n ~n- ~Dol c~lon wn-n fil-d Ito b- cn-cri~d by In~ r~c~ n~ Clnc-) ~_~., .0 D~u~hor~2-d OlllC-r) ' ' ~ ~ ~
. Th~ a~t~ ot r--C--ID~ ~Rom ~n~ ~ODIIC--nt~ D~ ~n-- Bu~----u ' ' ~Autho~12-d ~ C-r~
fo~m PCTiRO/tl~ nu-rY tWt) WO97/29179 . PCT~P97/00686 MICROORGANISMS ~ -OpUon-l Sht~ t In connt ctlon wlth th~ I _ r t nt~d ~o on 1~ 23 ,. 25 of th- d--ertptton ' A. ,..ir~no~o~oEro~lT~
Furth-~ d--Po-lt- ~r Id-ntln d on ~n ~ddltlond ~ho t O ' Nttm- ot d-po~lt~ry In~ltutlon ' Centraa}bUreau voor Schimmelcultures (CBS) oosterstraat 1 P.O.Box 2~3 3740 AG BAARN, '~he Netherlands D-t- ot d-Po-lt ' ¦ Acc---lon ~ Umb-- -12 February 1996 ! CBS 210.96 ~ AISL O 11 IttDlCAT10~ - bl-nlt it not ~POlic-hl-~ Th'- Intorm-tlon 1- ~ontlnu d on ~ ~-p-ml- ~tUtCh~d ~n t G
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 ~ ?~ to be withdrawn.

C DC51CttATED 5TATE~ FOllt W~IC~ I~DIC ~T10- ~ AlltT I~AD~ ' (It th- mdlc-tlon- ~r- not tor ~11 d--i~n-tt~ t St-tt -\

Il ~ErAlltATE _ 1 3 0~ ItiDlCATlOlt5 ' (I ~- bt-nlt d not ~pollc-bl-) Th~ ndic~tion~ -d b-low will b- ~ubmltt-d to th-; ~ Bur--u l-t-r ' (Sp~clty th- ~I-n~l n-~ur- ot th- Indlc-~lon- ~ p ~cc---~on l~uma-r ot D-po-d ) E O ThlJ ~n--~ ~-- r-c- v~d w~n tn- ~prJllc-non wn-n fil-rJ (to b- cn-ct -d b~ th- r~c-~ nt~ Orfic~) (Autnor~-d O~i;C r) O Th- d-t- ol r-c- ~t (trom tn~ ~ppl~c-nt) ~Y th- nur--u '~

(Authori~-d O~IC-r) Form RCTIRO/13~ nu-ry 1Ul)

Claims

1. A process for the production of a fermentable wort, comprising the steps of:(a) liquefaction of cereal material, with the aid of .alpha.-amylase and/or endoprotease activity, to obtain a liquefied mash, and (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.

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 cerealscontaining 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.