Brewing method

Abstract

A method of preparing a wort with an increased level of free amino nitrogen (FAN) comprising: a) preparing a mash from a grist comprising malt and/or adjunct; and b) adding a protease having at least 80% sequence identity to the polypeptide of SEQ ID NO: 1.

Claims

1. A method of preparing a wort with an increased level of free amino nitrogen (FAN) comprising: a) preparing a mash from a grist comprising malt and/or adjunct; b) preparing a wort from the mash; and c) adding a protease having at least 80% sequence identity to the polypeptide of SEQ ID NO: 1 to the mash or to the wort in an amount of 5-15mg EP/kg grist, wherein the grist comprises at least 40% malt, and wherein the prepared wort has at least 40% more FAN as compared to the level of FAN in a wort produced in the absence of the protease.

2. The method according to claim 1, wherein the protease comprises or consists of the amino acid sequence of SEQ ID NO: 1.

3. The method according to claim 1, wherein the protease is a variant of the polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or insertion at one or more positions.

4. The method according to claim 1, further comprising adding an alpha amylase to the mash.

5. The method according to claim 1, further comprising adding a beta glucanase to the mash.

6. The method according to claim 1, further comprising adding a pullulanase to the mash.

7. The method according to claim 1, further comprising adding a xylanase to the mash.

8. The method according to claim 1, wherein additionally a lipase is added to the mash.

9. The method according to claim 1, wherein the grist comprises at least 10% (w/w) adjunct.

10. The method according to claim 1, wherein the adjunct is selected from the group consisting of barley, rice, corn, sorghum and cassava.

11. The method according to claim 1, wherein the wort is fermented to obtain a beer.

12. The method according to claim 1, wherein a protein rest step is not included after protease addition.

13. A method of preparing a beer with stable foam, the method comprising: a) preparing a mash from a grist comprising malt and/or adjunct; b) preparing a wort from the mash; c) adding a protease having at least 80% sequence identity to the polypeptide of SEQ ID NO: 1 to the mash or to the wort in an amount of 5-15 mg EP/kg grist, wherein the grist comprises at least 40% malt, and wherein the prepared wort has at least 40% more FAN as compared to the level of FAN in a wort produced in the absence of the protease; and d) fermenting the wort to form a beer, wherein foam stability of the beer is not negatively affected by addition of the protease.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The advantage of the present invention is that it allows the breweries to have a higher level of raw material flexibility with respect to adjunct inclusion and malt quality.

(2) When adjuncts like corn grits, barley or rice are included in the brewing process instead of malt, the level of FAN (free amino nitrogen) will be insufficient to have proper yeast fermentation. The same issue occurs when an under-modified low quality malt is used.

(3) During mashing the endogenous malt proteases are capable of increasing the overall FAN level. This increase occurs mainly during the protein rest (e.g., 20 min, 50° C.).

(4) Adding the protease according to the invention to the mash may allow the breweries to eliminate the protein rest without losing FAN. Eliminating the protein rest will save time and energy in the brewing process and also minimize the lipoxygenase (LOX) catalyzed lipid oxidation leading to off-flavors in the final product.

(5) Wort Production

(6) The present invention relates to a method of producing a wort with an increased level of FAN, wherein a protease that has at least 80% sequence identity to the sequence shown in SEQ ID NO: 1 has been added to the mash or the wort.

(7) The mash is obtainable by grounding a grist comprising malt and/or adjunct. Water may preferably be added to the grist, and is normally preheated in order for the mash to attain the desired mash temperature at the moment of mash forming. If the temperature of the formed mash is below the desired mashing temperature, additional heat is preferably supplied in order to attain the desired process temperature.

(8) The temperature profile of the mashing process may be a profile from a conventional mashing process wherein the temperatures are set to achieve optimal degradation of the grist dry matter by the malt enzymes and the added enzymes.

(9) The malt is preferably derived from one or more of the grains selected from the list consisting of, e.g., corn, barley, wheat, rye, sorghum, millet and rice.

(10) Preferably, the malt is barley malt. The grist preferably comprises from 0.5% to 99% (w/w) malt, preferably from 1% to 95% (w/w) malt, more preferably from 5% to 90% (w/w) malt, even more preferably from 10% to 80% (w/w) malt.

(11) In addition to malted grain, the grist may comprise one or more adjuncts such as unmalted corn, or other unmalted grain, such as barley, wheat, rye, oat, corn, rice, milo, millet and/or sorghum, or raw and/or refined starch and/or sugar containing material derived from plants like wheat, rye, oat, corn, rice, milo, millet, sorghum, pea, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane. According to the present invention, adjuncts may be obtained from tubers, roots, stems, leaves, legumes, cereals and/or whole grain.

(12) Preferred is adjunct obtained from barley, corn, rice, sorghum and/or cassava; e.g., rice starch, corn starch, and/or corn grits.

(13) The grist comprises typically from 1% to 80% (w/w) adjunct, e.g., from 5% to 75% (w/w) adjunct, e.g., from 10% to 70% (w/w) adjunct; in particular the grist comprises at least 10% (w/w) adjunct. In a preferred embodiment, the grist comprises from 30% to 70% (w/w) adjunct.

(14) In one aspect, the protease is introduced at the beginning of mashing. In another aspect, the protease is introduced during mashing. In another aspect, the protease is added to the wort.

(15) The amount of added protease according to the invention generally depends on various factors. For purposes of this invention, the amount of protease used will generally be of from 0.1 mg to 100 mg EP (Enzyme Protein) per kg grist, preferably from 1 mg to 100 mg EP (Enzyme Protein) per kg grist; preferably from 1 mg to 50 mg EP (Enzyme Protein) per kg grist.

(16) In a preferred embodiment, the amount of free amino nitrogen in the wort is increased by at least 20% as compared to a wort produced in the absence of the protease according to the invention, e.g., the amount of free amino nitrogen in the wort is increased by at least 30% as compared to a wort produced in the absence of the protease according to the invention, e.g., the amount of free amino nitrogen in the wort is increased by at least 40% as compared to a wort produced in the absence of the protease according to the invention, e.g., the amount of free amino nitrogen in the wort is increased by at least 50% as compared to a wort produced in the absence of the protease according to the invention.

(17) In another preferred embodiment, a further enzyme(s) is added to the mash, said enzyme(s) including but not limited to alpha amylase, isoamylase, maltogenic amylase, protease, cellulase, beta glucanase, pullulanase, laccase, xylanase, lipase, phospholipase, phytase, and esterase.

(18) In one aspect of the method, the further enzyme added includes a pullulanase.

(19) In one aspect of the method, the further enzyme added includes an amylase, preferably an alpha amylase.

(20) In one aspect of the method, the further enzyme added includes a beta glucanase.

(21) In one aspect of the method, the further enzyme added includes a xylanase.

(22) In one aspect of the method, the further enzyme added includes a lipase.

(23) Following the separation of the wort from the spent grains of the grist, the wort may be used as it is or it may be dewatered to provide a concentrated and/or dried wort. The concentrated and/or dried wort may be used as brewing extract, as malt extract flavoring, for non-alcoholic malt beverages, malt vinegar, breakfast cereals, for confectionary, etc.

(24) In a preferred embodiment, the wort is fermented to produce an alcoholic beverage, preferably a beer, e.g., ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.

(25) Fermentation of the wort may include pitching the wort with a yeast slurry comprising fresh yeast, i.e., yeast not previously used or the yeast may be recycled yeast. The yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Saccharomyces spp. such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms.

(26) It is an advantage that the protease according to the invention may shorten the total fermentation time.

(27) The methods for fermentation of wort for production of beer are well known to the person skilled in the arts.

(28) Proteases According to the Invention

(29) The protease SEQ ID NO:1 is obtainable from Anoxybacillus rupiensis and disclosed in WO2014/194054 for use in detergents.

(30) In one embodiment, the present invention relates to an isolated polypeptide having a sequence identity to the polypeptide of SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84% at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity.

(31) A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof; or is a fragment thereof having protease activity. In another aspect, the polypeptide comprises or consists of the polypeptide of SEQ ID NO: 1.

(32) In another embodiment, the present invention relates to an isolated polypeptide having protease activity encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, or high stringency conditions with the polypeptide coding sequence of SEQ ID NO: 1, or the full-length complement thereof (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

(33) In another embodiment, the present invention relates to variants of the polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

(34) In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the polypeptide of SEQ ID NO: 1 is not more than 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

(35) The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

(36) Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

(37) Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

(38) Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

(39) Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

(40) Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

(41) The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

(42) The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

(43) A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

(44) Enzyme Compositions

(45) The present invention also relates to compositions comprising a polypeptide of the present invention for use in wort production.

(46) The compositions according to the invention may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition.

(47) Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

(48) Preferably, the composition for use in wort production may comprise the protease having at least 80% sequence identity to the polypeptide of SEQ ID NO: 1; or a composition for use in wort production may comprise the protease having at least 80% sequence identity and one or more enzymes selected from the group consisting of alpha amylase, beta glucanase, pullulanase, xylanase, and lipase.

(49) The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.

(50) Examples are given below of preferred uses of the compositions of the present invention. The dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.

(51) The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLE 1

(52) Adding a Protease (SEQ ID NO:1) with Improved Free Amino Nitrogen (FAN) Generation During Small Scale Mashing

(53) The protease (SEQ ID NO:1) was compared to the protease Neutrase™ (Novozymes NS) using the following procedure: 1. Add 5 g corn starch to 100 mL Blue Cap bottles with magnetic stirrer. 2. Grind the malt (from Danish Malting Group (Prod nr 2012-0646)) at gap 0.2 mm (Bühler mill) and weigh out 5 g in weighing plastic cups. 3. Add 25 mL 95° C. H.sub.2O, 300 μL CaCl.sub.2 (0.2 M) and 300 ppm Termamyl™ (Novozymes A/S) to each bottle with 5 g of corn starch 4. Do decoction according to mashing profile (see Table 1 below). 5. Cool down to 50° C. by adding ice or cold water in the water bath, add 5 g of malt, 25 mL 52° C. H.sub.2O, 0.3 mL CaCl.sub.2, and protease according to set-up (5, 10, and 15 mg enzyme protein/kg grist) to each blue cap bottle. 6. Ready for mashing, set time, and do the mashing manually by setting the temperature on the water bath. 7. Cool down to 30° C. and filtrate with small funnels, 50 mL volumetric cylinders and folded filters (Whatman 597½, ø185 mm). 8. Measure the level of Free Amino Nitrogen using NOPA assay and a Gallery Plus. (The NOPA assay was Alpha-Amino Nitrogen (NOPA) test kit from Thermo Fisher Scientific (Cat. No. 984342)).

(54) TABLE-US-00001 TABLE 1 Mashing profiles: Temperature [° C.] Time [min.] Corn Starch - decoction: 95 30 Malt and Corn mashing with a protein rest: 50 20 63 30 72 20 78 15 20 — Malt and Corn mashing without a protein rest: 63 50 72 20 78 15 20 —
Results:

(55) TABLE-US-00002 TABLE 2 FAN results with a protein rest: Protease: 5 mg EP/kg 10 mg EP/kg 15 mg EP/kg grist grist grist FAN results with 24 ppm 34 ppm 40 ppm Neutrase: FAN results with 51 ppm 70 ppm 84 ppm SEQ ID NO: 1:

(56) TABLE-US-00003 TABLE 3 FAN results without a protein rest: Protease: 5 mg EP/kg 10 mg EP/kg 15 mg EP/kg grist grist grist FAN results with  8 ppm 12 ppm 18 ppm Neutrase: FAN results with 44 ppm 64 ppm 73 ppm SEQ ID NO: 1:

(57) It can be seen from Table 2 and Table 3 that the protease SEQ ID NO:1 gives surprisingly more FAN than Neutrase.

EXAMPLE 2

(58) Adding a Protease (SEQ ID NO:1) with Improved Free Amino Nitrogen (FAN) in Lab Fermentation

(59) Mashing:

(60) 1. Grind 1000 g malt (gap 0.2 mm) 2. Add 75 g malt to each of 12 beakers 3. Add 300 mL 52° C. water and 4.5 mL CaCl.sub.2 (0.2 M) solutions 4. Make the following mashing profile:

(61) TABLE-US-00004 Temperature [° C.] Time [min] 50 20 63 30 72 20 78 15 20 — 5. Just after start, add the protease (SEQ ID NO:1 or Neutrase™—10 mg EP/kg grist) and 300 ppm Termamyl™ 6. After mashing, adjust to 450 g with water in each beaker 7. Filter the samples using Falten filer 597½ 8. Mix 500 mL wort in 8 bluecap bottles, according to set up 9. Weigh out 159 mg hops (Hallertau Hallertauer Taurus (Alpha 17%)) in each bottle and boil for 40 min 10. Cool down and adjust the bottles for water loss with sterile water 11. Centrifuge at 8000 rpm for 30 min and transfer the supernatant to sterile bluecaps
Yeast: Weigh out 100 g of YPD (Yeast peptone dextrose media for growing yeast) in a 2000 mL Pyrex flask containing 1 stirring bar—add 1000 mL of MQ water and autoclave the solution. Let it cool to 25° C. “Under sterile condition” add one bag of dry yeast (Saflager w-34/70 (11.5 g; Lesafre)) to the YPD media Place the solution in fume hood and bubble sterile air through the solution, with stirring medium to high. Let it bubble and stir overnight Transfer the yeast to 2×500 mL centrifugation bottles Centrifuge the yeast at 2000 rpm for 3 minutes Discard the supernatant and re-suspend the supernatant in 250 mL sterile water. Transfer all the yeast to one 500 mL centrifugation bottle. Repeat this process 3 times After the final rinse, the yeast pellets are re-suspended in 200 mL sterile water Make a dilution series 1:10, 1:100 and 1:1.000. Count the yeast cells in the 1:1000 dilution Add propagated yeast into the wort to reach 2×10.sup.7 cells/mL and loosely close the lid
Fermentation: Place the bluecaps on shaking table, 145 rpm. at 12° C. for 5 days After 5 days—turn the shaking table down to 120 rpm for the next 2 days Cool the sample down to 0° C. (put it on ice in a styrofoam box with a lid and place the box in the cold-room in the basement 5° C.) Let it stand there for 5 days
Results:

(62) The results confirmed the small scale mashing (Example 1):

(63) The protease (SEQ ID NO:1) released significantly more FAN than Neutrase™ (more than 40%), when 10 mg EP/kg grist was added.

(64) The final beer was analyzed, and no adverse effects were observed (foam damage, etc.).