Field of the invention

The present invention provides a process for making wort having improved filterability and/or showing increased brewing yield The invention also provides for methods of brewing beer, making wine and potabie alcohol from wort made according to the present invention The invention also concerns the use of enzymes in improving filterability and/or yield of wort made from malted and/or unmalted cereals, as well as compositions comprising mixtures of enzyme activities according to the invention

Background of the invention

The use of enzymes in making fermentable wort is known for a long time For example, for brewing beer grams and/or malted grains are liquefied and saccharified in order to yield fermentable sugars Liquefaction steps may be improved by the use of thermostable α-amylases as described for instance in US 4,285,975 or US 5, 180,669 Also proteases are used to increase the amount of freely available nitrogen m the wort to improve fermentation Alter filtration of the liquefied and saccharified mash, the obtained wort is inoculated with special strains of yeast (Saccharomyces sp ) which convert sugars into ethanol and characteristic flavour compounds

However, apart from starch other polysaccharides are present in cereal grains For example β-glucans are present (R J HENRY J Sci Food Agπc (1985) 36, 1243- 1253) as listed below for various cereals Cereal % β-glucans

wheat 0 5 - 8 0 barley 4 0 - 8 0 rye 5 0 - 10 0 0 oat 5 0 - 10 0

These ^β -glucans consist in β-l ,3/ ^β -l ,4 linkages between β-D-glucopyranose moieties (M J EDNEY, B.A MARCH YLO and A W Mac GREGOR J Inst. Brew ( 1991 ) 97, 39-44) β-glucans are highly viscous (N WAGNER et al Monatschπft fur Brauwissenschaft ( 1988) Heft 10, 384-395) and bring wort and beer filtration problems (S.AASTRUP Carlsberg Res Commun (1979) 44, 289-304) if they are not hydrolysed during the liquefaction step This is the reason why ^β -glucanases from Bacillus subtilis (R.BORRISS Zeitschr fur allgem Mikrobiol (1981) 21(1), 7-17), Pentcilhum emersonii (A P MOLONEY, et al Enzym Microb Technol (1983) 5, 260-264), Mucor miehei (F BRANISLAV European Patent 0 504 947 A2, 1992) or Tnchoderma sp (S P SHOEMAKER and R D BROWN Jr Biochim Biophys Acta (1978) 523, 133- 146) are widely used at industrial scale, in addition to the ^β -glucanase present in the malt, the latter is not sufficiently thermostable to be active

durmg the brewing diagramme processing (M HRMOVA and G B FINCHER Biochem J (1993) 289,

453-461 )

Non-starch polysaccharides also include pentosans, the structure of which have been widely studied recently (H GRUPPEN et al Carbohydr Res (1992) 233, 45-64, G ANN I SON et al , Carbohydr

Polym (1992) 19, 151-159, T ITO et al , J Carbohydr Chem ( 1994) 13(3) 491 -498) in particular those of barley and malt (R J VIETOR et al , Carbohydr Res ( 1994) 254, 245-255,

R J VIETOR et al , Carbohydr Polym (1994) 24, 1 13- 1 18) British patent 2, 150,933 shows the interest for a pentosanase from Pemctlhum emersonii to improve the production and extraction of fermentable sugars in brewing US Patent 4,746,517 demonstrates the high efficiency of the xylanolytic system from

Disporotnchum dimorphosporum to improve wort and beer filterability

The use of xylanase B to improve filterability of wort has also been mentioned in W094/ 14965 This application is herein incoφorated by reference

Applications which may be mentioned in conjunction with the use of enzymes in making wort for fermentation are

W094/21785

Despite the advance which has been made in this area, there is still a need for methods of preparing wort with further improved filterability and/or higher yields, and enzyme preparations for use therein

Summary of the invention

The present invention provides worts with increased yield and improved filterability as well as a process for preparing said wort comprising the steps of

(a) preparing mash from malted or unmalted cereals, or a mixture of malted and unmalted cereals, in the presence of a mixture of enzyme activities,

(b) filtering the mash so obtained to obtain the wort, wherein said mixture of enzyme activities is selected from a mixture comprising at least β-glucanase activity and α-L-arabmofuranose releasing activity or a mixture comprising at least β-glucanase activity endo-xylanase activity and α-arabmofuranose releasing activity Preferably, according to the process of the invention, arabinofurnaose releasing activity is provided by an enzyme selected from α-L-arabmofur- anosidase (EC 3 2 1 55) and (l ->4)-β-D-arabmoxylan arabinofuranohydrolase (AXH), or a mixture of the said enzymes The use of α-L-arabinofuranosidase or ( l ->4)-β-D-arabmoxylan arabinofuranohydrolase (AXH) obtainable from Aspergillus strain is still further preferred According to one embodiment α-L- arabinofuranosidase A from Aspergillus niger is used

According to another embodiment the invention provides a process for making beer, and/or potable alcohol, and other alcoholic beverages, wherein a wort is fermented obtainable according to the invention

According to a further embodiment the invention envisages the use of α-L-arabinofuranosidase B from Aspergillus niger m a process of improving filterability of wort

According to another embodiment the invention provides for the use of α-L-arabinofuranosidase A in a process of increasing yield of wort

The invention also provides an enzyme preparation suitable for use in a process of making wort, said composition comprising a mixture of enzyme acitivities selected from a mixture comprising at least β-glucanase activity and α-arabinofuranose releasing activity or a mixture comprising at least β- glucanase activity endo-xylanase activity and α-arabinofuranose releasing activity

Preferred according to the invention is an enzyme preparation, wherem said arabinofuranosyl releasing activity is provided for by an enzyme selected from α-L-arabinofuranosidase (EC 3 2 1 55) and ( l ->4)-β-D-arabιnoxylan arabinofuranohydrolase (AXH), or a mixture thereof Preferred according to the invention is an enzyme preparation wherein said α-L-arabinofuranosidase or said (l ->4)-β-D-arabιnoxv- lan arabinoturanohydrolase (AXH) is obtainable from Aspergillus strain

Description of the invention

We have now surprisingly found that when wort is made in the presence of a mixture of enzvme activities comprising β-glucanase activity, α-L-arabinofuranosidase activity and preferably endo-β- 1 4- -xylanase, a liquefied and saccharified mash is obtained which can be filtered much easier than mash obtained using β-glucanases alone or mixtures of β-glucanases and endo-xylanase Moreover, the wort obtained after filtration of the mash shows higher yield Yield in this regard refers to the amount of fermentable sugars in the wort, expressed as a percentage of sugars present in the raw materials The yield improvement of the wort is dependent on the malt chosen The malts exemplified herein show already good yields without the enzymes activities according to the invention It is envisaged that more dramatic improvements may be obtained with poorer quality malts, with unmalted cereals or with mixed brews

The manufacturing of the wort may be performed conventionally involving liquefaction and sacchaπfication of cereal material, usually with the aid of α-amylases and proteases, to obtain a liquefied and saccharified mash

Suitable starting materials are cereals, either so-called raw or unmalted mateπal or malted, or mixtures thereof (mixed brew) For example, cereals such as barley, wheat, corn, sorghum, oat and rice can be used, either malted or raw, or mixed Preferably, the method according to the invention is used in 100% malt brew

In the case of raw cereals, the liquefaction step usually compπses grinding of the cereal raw mateπal 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 Ca ^:* Enzymes from Bacillus stearothermophilus appear to be less Calcium-dependent Consequently, no Ca ^:+ 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, m 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 temperature between about 40°C and 65 ^C C, preferably between about 45 and 55°C, most preferably 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 sacchaπfication 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 sacchaπfication 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 sacchaπfication 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

Other enzymes in the prepration of the wort that are conventionally used are β-glucanases The β-glucanase is preferably thermostable enough to be active during the first step of a standard brewing diagramme (typically several minutes at 50°C and pH 5 6) Several microbial enzymes can fullfil this requirement e g β-glucanase from Pemctllium emersonii, Trtchoderma longtbrachiatum. Bacillus amyloliquefaciens Excellent results may be obtained with β-glucanase from Bacillus amyloliquefaciens commercially available from Gist-Brocades under the trademark Filtrase L (+), having an activity of 600 BGR/g Preferably, β-glucanase is obtained from a recombinant strain of Bacillus amyloliquefaciens Usually also proteases may be used, such as Brewer's Protease and the like

Endo-xylanases that may be used have been described in the section background art The patent specifications mentioned there are herein incoφorated by reference

Endo-β- 1 ,4-xylanase (preferably isoenzyme endo I (R A FOURNIER Biotechnology and Bιoeng ( 1985) XXVII, 539-546) and α-L-arabino-furanosidase (preferably isoenzyme A (F J M KORMELINK et al , Carborhydr Res (1993) 24, 345-353)) may be obtained from wild-type, mutated or recombinant strains of Aspergillus niger s As regards the α-L-arabinose releasing activities, several enzymes may be envisaged We have shown that AXH leads to better filterability Two α-L-arabmosidases from Aspergillus niger appeared to lead to significantly higher filtration rates As the mode of action of AXH, α-L-arabinosidase A and B all differ, it may be expected, that a mixture of the three arabinose releasing activities may yield to still better filtration rates io Using the process according to the invention substantially improved filtration rates may be obtained of the liquefied and saccharified mash This brings advantages in terms of a speedier process, less clogging of filters, and larger wort volumes

Also the improved yield leads to a more economic brewing process The wort according to the invention can be used in brewing beer, or making potable alcohol or biofiiel, or in a process of making other i s alcoholic beverages The practicing of the invention and the associated advantages are illustrated in greater detail in the following non-limitative Examples

Experimental part 1/β-glucanase

20 In the Examples β-glucanase was used from Bacillus amyloliquefaciens commercially available from Gist-Brocades under the trademark Filtrase L (+), having an activity of 600 BGR/g

2/Endo-β-l,4-xylanase

Endoxylanase was obtained from a pure culture of A spergillus niger in a sterile tank and medium The 2s culture medium contains appropriate carbon and nitrogen sources just as mineral salts The fermentation is carried out at a constant temperature between 30-40°C and pH is maintained within the range 3-5 The activity of the enzyme is measured by the hydrolysis of xylan from oat spelts suspended (35g/l) in I M glycine buffer pH 2,75 the viscosity of this solution is determined by using a capillary viscometer (Ubbelhode type) at 47°C The time dt needed for the upper menisk of the liquid to fall down between io two reference points is measured within time T The slope of the plot T versus 1/dt yields an apparent kinetic constant 1 Lyx unit is the amount of enzyme needed to reach a value of 1 mιn-1 for that kinetic constant

3/a-L-arabιnofuranosιdase j ₅ Isoenzyme A or isoenzyme B (F J M KORMELINK et al , Carborhydr Res (1993) 24, 345-353) or Arabinoxylanhydrolase (F J M KORMELINK et at , Appl Microbiol Biotechnol (1991) 35, 753-758) have been obtained from a culture of recombinant Aspergillus niger or Aspergillus mdulans strains The ammo acid sequences, their encoding DNA, as well as the recombinant production of arabinofura- nosidase A and B is described in detail in European patent application

0 506 190 A l , published on September 30, 1992, particularly in the Examples and Figures and the Sequence Listing, which relevant parts are herein incoφorated by reference

Activity of isoenzymes A and B is measured by the hydrolysis of p-nitrophenyl-α-L-arabinofuranoside 1 ARF unit is the amount of enzyme needed to liberate 1 μmole p-nitrophenol per minute under the conditions of the test described in (Z GUNATA et al , J Agπc Food Chem (1989) 38, 772)

Example 1

Wort was prepared from malt ground according to standard specifications for lauter tun filtration Malt was ground with the EBC MIAG mill according to standard specifications, l e yielding a difference in fine ( 1 mm) to coarse (2 5 mm) extract in the range 1 5-2 % One part malt is hydrated with 3 parts water or aqueous solution of enzyme at 50°C This temperature is maintained during 20 minutes, it is then raised up to 63°C ( l °C/mιn) and maintained at that temperature during 30 minutes The medium is then heated at 72°C ( l °C/mιn) and maintained at this temperature during 20 minutes It is finally heated at 76°C and maintained at that temperature for 5 minutes Water is added to compensate for water evaporation

The mash is poured into a funnel containing Schleicher and Schuell paper filter The volume of filtered wort is measured after 15 minutes Specific gravity is determined at the end of the filtration This value allows to calculate extract and yield 1st serie (British malt) 5 brews have been carried out with different enzymes as shown in Table 1

Brew n° Enzymatic units/kg malt

BGR LYX ARF-A AXH ARF-B

1 0 0 0 0 0

2 90 1200 0 0 0

3 90 1200 1500 0 0

4 90 1200 - 0 1500

5 90 1200 0 1500 0

In brew n°3, ARF is from α-L-arabinofuranosidase isoenzyme A

In brew n°4, ART is from α-L-arabinofuranosidase isoenzyme B

In brew n°5, the arabinoxylanhydrolase AXH (F.J.M KORMELINK et al Appl Microbiol Biotechnol

( 1991 ) 35, 753-758) is used to hydrolyse the arabmose moieties of arabinoxylans The dose was 0, 15 mg pure AXH per kg malt (AXH is not active on p-nitrophenyl-α-L-arabinofuranoside, therefore AXH has no ARF activity)

Results are presented in Table 2

Brew n° Volume filtered Yield after 15 minutes (%)

(ml)

62 83,04

88 83,69

102 83,64

106 83,24

100 83,44

2nd seπe (French malt)

The same brews as described in the 1 st series have been produced from a french malt Results are presented in Table 3

Brew n° Volume filtered Yield after 15 minutes (%) (ml)

1 54 82,35

2 82 82,50

3 88 83,71

4 92 82,60

5 88 82,50

From these 2 series, the combination ^β -giucanase + endoxylanase + α-L-arabinofuranosidase isoenzyme B performs best to improve wort filtration, whereas the same combination in which α-L-arabino-fura- nosidase isoenzyme A replaces α-L-arabinofuranosidase isoenzyme B performs best to improve the yield, the filtration being also highly improved in comparison with the blank (brew 1)

Example 2 Worts were produced the same way as in example 1 , always with the same batch of malt but varying the composition of the enzymes' mixture 12 brews were carried out according to an experimental design in order to determine the role of each component of the mixture with regards to yield and filtration improvement In all trials, α-L-arabinofuranosidase was isoenzyme A from A niger

All runs performed are presented in Table 4

Brew n° Enzymatic units/kg malt

BGR LYX ARF (isoenzyme A)

1 30 300 300

2 90 300 300

3 30 1200 300

4 90 1200 300

5 30 300 1500

6 90 300 1500

7 30 1200 1500

8 90 1200 1500

9 60 750 900

10 60 750 900

II 60 750 900

12 0 0 0

Results are shown in Table 5

Brew n° Volume filtered Yield after 15 minutes (%)

(ml)

1 115 80,16

2 100 80,21

3 116 80,01

4 110 80,81

5 116 80,11

6 128 80,26

7 122 80,01

8 142 80,56

9 116 80,06

10 120 80,11

11 117 80,11

12 89 80,01

From Brews n°9-10-l 1 standard deviations may be determined

2,1 ml for volume filtered after 15 minutes 0,03% for the vield

From Brews n°l to 8, the effect of each component just as those of interactions between components may be determined

Cause E ffect on

Filterabil «y Yield

BGR +2,75 +0,39

LYX +7,75 +0,16

ARF +16,75 -0,06

BGR* ^: LYX +4,25 +0,29

BGR ⁴ ARF + 13,25 -0,04

LYX" 'ARF +2,75 -0,06

Only figures in bold type characters may be considered as significant (> 95%) α-L-arabinofuranosidase A alone and in combination with β-giucanase brings the strongest effect in filtration improvement, i5 whereas β-glucanase, endoxylanase alone and in combinations contribute significantly to the yield improvement.

Example 3

2o In that serie, the same combination of enzymes is used

60 BGR/kg malt + 750 LYX/kg malt + 900 ARF/kg malt

but different malt were brewed with and without this enzyme combination Worts were produced according to the same procedure as in Example 1.

Results are presented in Table 5

Malt's Volume filtered Yie Id origin after 15 minutes (%)

(mi; 1 without with without with enzyme enzyme enzyme enzyme

USA ( 1 ) 13 1 171 79,75 79,77

USA (2) 1 14 164 83,74 83,97

UK 1 17 167 81 ,08 83,21

Canada ( I ) 106 153 81 ,42 81 ,52

Canada (2) 99 137 81 ,30 81 ,86

France 89 1 18 80.18 80,09

Thus, the mixture of enzyme activities is efficient for filtration improvement, whatever the malt brewed The effect on yield is low but this is mainly because the malts used are already of a very high quality

Example 4

Wheat is used as crude adjunct in several breweries This cereal contains a high amount of pentosans This is one reason to test the above mentioned combination of enzymes in a mixed brew Worts were produced according to the same procedure as in example 1 but 20% malt was replaced by 20% wheat

Table 6 describes all runs performed

Brew n° Enzvmatic units/kg malt

BGR LYX ARf

1 0 0 0

2 90 0 0

3 0 120 0

4 0 120 300

5 0 120 1500

6 90 120 1500

Results are given in Table 7

Brew n' ³ Volume filtered Yieid after 15 i minutes (%) (ml)

1 66 81,01

2 89 81,52

* j> 62 80,81

4 69 80,91

5 80 80,91

6 99 81 ,52

D These results confirm the interest for the combination descπbed in the invention in the case of brews involving wheat adjuncts

Example 5 In this example, wort was prepared from crude barley grains, variety PLAISANT Barley grains were ground with the EBC MIAG mill in order to make filter press type barley flour 57g barley flour are added in 300ml water or aqueous solution of enzymes at 50°C This temperature is maintained for lh, it is then heated up to 63°C ( l°C/mιn) and maintained at that temperature during 30 minutes The medium is then heated up to 90°C ( l°C/mιn) and maintained at that temperature during 20

ι: minutes Water is added to compensate for water evaporation The mash is then poured into a funnel containing Schleicher and Schuell paper filter

6 brews have been carried out with different enzymes as shown in Table 8 (ARP when mentioned is from Ara A)

(*) according to the invention

(**) commercially available from Gist-brocades under the trade name Filtrase® NL

(***) commercially available from Gist-brocades under the trade name Filtrase® Br (****) commercially available from Gist-brocades under the trade name Natugrain®

Results are presented in Table 9

These results show that the combination described in the invention is at least as good or even better performing than existing preparations in the case of brews involving 100% unmalted barley