Uninhibited amylase for brewing using high tannin materials

Abstract

The present invention provides a method for saccharifying starch from high-tannin adjuncts. More particularly, high-tannin adjuncts may be insensitive to exogenous enzymes used in barley malt brewing. The present invention provides enzymes that are not inhibited in tannins. In particular, according to the present invention, an α-amylase that degrades raw starch and is not inhibited by tannins is provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an International PCT application that claims the benefit of U.S. Provisional Application No. 63 / 028,042, filed on May 21, 2020, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to methods for mashing high tannin adjuncts. More particularly, the present disclosure provides methods and compositions for mashing raw starch-degrading α-amylases that are not inhibited by tannins to provide wort from high tannin adjuncts. Background Art

[0004] Brewing usually involves three steps: malting, starch saccharification and fermentation. The main purpose of the malting step is to develop enzymes that have subsequent effects on starch and protein degradation during the brewing process. Although beer is traditionally brewed only from barley malt, hops and water; malt is an expensive raw material because it requires high-quality grains, water for germination and energy for kilning. In order to reduce the cost of raw materials, ungerminated grains, also known as adjuncts, such as maize, rice, cassava, wheat, barley, rye, oats, quinoa and sorghum may be included in the brewing process. Adjuncts are mainly used because they are easy to obtain and provide fermentable carbohydrates at a lower cost than barley malt.

[0005] The use of adjuncts in brewing can complicate traditional brewing processes. Numerous enzymes have been developed to improve various aspects of beer production using malted barley as a starch source. However, it is known that high-tannin adjuncts may not be readily processed by these enzymes.

[0006] There is a continuing need for methods by which high tannin adjuncts may be used in beer production. Summary of the Invention

[0007] According to one aspect of the present invention, there is provided a method for producing brewer's wort, the method comprising the steps of mashing grist with a high tannin adjunct in the presence of an exogenously provided enzyme composition comprising a tannin uninhibited enzyme to provide the brewer's wort. Optionally, the tannin uninhibited enzyme is selected from the group consisting of a bacterial α-amylase that degrades raw starch, a glucoamylase, a pullulanase, a fungal α-amylase, and a maltogenic α-amylase.

[0008] Optionally, the grist mill comprises sorghum. Optionally, the grist mill is at least 10% sorghum.

[0009] Optionally, the grist is at least 20% sorghum, at least 30% sorghum, at least 40% sorghum, at least 50% sorghum, at least 60% sorghum, at least 70% sorghum, at least 80% sorghum, at least 90% sorghum, or 100% sorghum.

[0010] In another aspect of the invention, the grist mill optionally further comprises corn, cassava, barley, wheat, rye, millet or rice. In other still preferred embodiments, the grist cereal optionally has at least 10 μM CAE / g grist cereal, at least 20 μM CAE / g grist cereal, at least 30 μM CAE / g grist cereal, at least 40 μM CAE / g grist cereal, at least 50 μM CAE / g grist cereal, at least 60 μM CAE / g grist cereal, at least 70 μM CAE / g grist cereal, at least 80 μM CAE / g grist cereal, at least 90 μM CAE / g grist cereal, at least 100 μM CAE / g grist cereal, at least 110 μM CAE / g grist cereal, at least 120 μM CAE / g grist cereal, at least 130 μM CAE / g grist cereal, at least 140 μM CAE / g grist cereal, or at least 150 μM CAE / g grist cereal.

[0011] Optionally, the enzyme not inhibited by tannins is a raw starch degrading alpha-amylase. Optionally, the raw starch degrading alpha-amylase belongs to class GH13. Optionally, the raw starch degrading alpha-amylase is derived from Cytophagasp. Optionally, the raw starch degrading alpha-amylase has at least 60% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 65% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 70% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 75% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 80% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 85% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 90% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 95% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 98% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 99% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, or has the sequence set forth in SEQ ID NO: 2 or an amylase-active fragment thereof.

[0012] Optionally, the exogenously provided enzyme composition having a raw starch-degrading alpha-amylase that is not inhibited by tannins also has one or more of a protease, a fungal alpha-amylase, a maltogenic alpha-amylase, a glucoamylase, and a lipase.

[0013] In another aspect of the present invention, there is provided a method of fermenting beer wort produced as described above using an enzyme not inhibited by tannins to obtain an alcoholic beverage. Optionally, the alcoholic beverage is beer.

[0014] In another aspect of the present invention, there is provided wort produced using an enzyme not inhibited by tannins as described above. In another aspect of the present invention, there is provided beer produced from the wort as described above.

[0015] In another aspect of the present invention, there is provided a method of determining whether an enzyme is inhibited by tannins, the method comprising the steps of incubating the enzyme in the presence of tannins and detecting cross-linking of the enzyme to the tannins. Optionally, the tannin is catechin.

[0016] Optionally, the enzyme is a brewing enzyme. Optionally, the brewing enzyme is selected from the group consisting of: raw starch-degrading alpha-amylases, proteases, fungal alpha-amylases, glucoamylases, maltogenic alpha-amylases and lipases. Optionally, the enzyme is a raw starch-degrading alpha-amylase.

[0017] Optionally, the alpha-amylase is of class GH 13. Optionally, the raw starch degrading alpha-amylase is derived from Cytophaga sp.

[0018] Optionally, the raw starch degrading α-amylase used in the method of detecting crosslinking has at least 60% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 65% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 70% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 75% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 80% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 85% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 90% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 95% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 98% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 99% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, or has the sequence set forth in SEQ ID NO: 2 or an amylase-active fragment thereof. Optionally, the step of detecting crosslinking is measuring turbidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the liquefaction of red sorghum using the RVA process as described in Example 3 (6.0 g ground red sorghum was mixed with 23.0 g water) with alpha-amylases according to Table 2: 1) GsAA, 2) CsAA, and 3-5) Termamyl SCDS.

[0020] Figure 2 Shown are the optical density / turbidity (OD 600 nm) measured in MTP plates after incubation (30° C. for 30 min followed by 4° C. for 30 min) of catechin with three α-amylases: GsAA, uninhibited CsAA, and Termamyl SCDS in various dilutions.

[0021] Figure 3 Shown are the optical density / turbidity (OD 600nm) measured in MTP plates after incubation (4°C for 30 min, 60°C for 30 min, then 4°C for 23 h) of catechins with the following three α-amylases: GsAA, uninhibited CsAA, and Termamyl SCDS in various dilutions.

[0022] Brief description of SEQ ID NO

[0023] SEQ ID NO: 1 sets forth the mature amino acid sequence of an alpha amylase variant (GsAA) from Geobacillus stearothermophilus.

[0024] SEQ ID NO: 2 sets forth the mature amino acid sequence of a variant alpha amylase from Cytophaga sp. (CsAA).

[0025] Definitions and Abbreviations

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., Dictionary of Microbiology And Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale and Markham, The Harper Collins Dictionary Of Biology, Harper Perennial, New York (1991) provide those of skill with a general meaning for many of the terms used herein. Nevertheless, for clarity and ease of reference, certain terms are defined below.

[0027] "Variant" or "variants" refers to a polypeptide or nucleic acid. The term "variant" can be used interchangeably with the term "mutant". Variants include insertions, substitutions, transversions, truncations and / or inversions at one or more positions in an amino acid or nucleotide sequence, respectively. The phrases "variant polypeptide," "polypeptide variant," "polypeptide," "variant" and "variant enzyme" mean a polypeptide / protein having an amino acid sequence having or comprising, for example, a selected amino acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5 or modified compared to the selected amino acid sequence, for example, SEQ ID NO: 1, 2, 3, 4 or 5.

[0028] As used herein, "homologous sequence" and "sequence identity" with respect to nucleic acid or polypeptide sequences means having about at least 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 88%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, or at least 45% sequence identity to a nucleic acid or polypeptide sequence when optimally aligned for comparison, wherein the candidate nucleic acid or polypeptide sequence functions substantially the same as the nucleic acid or polypeptide sequence to which the candidate homologous sequence is being compared. In some embodiments, homologous sequences have between at least about 85% and 100% sequence identity, while in other embodiments, have between about 90% and 100% sequence identity, and in other embodiments, have at least about 95% and 100% sequence identity.

[0029] Homology is determined using standard techniques known in the art (see, e.g., Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988); programs such as GAP, BESTHT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., Nucleic Acids Res. 12:387-395 (1984)).

[0030] "Percentage (%) nucleic acid sequence identity" or "percentage (%) amino acid sequence identity" is defined as the percentage of nucleotide residues or amino acid residues in the candidate sequence that are identical to the nucleotide residues or amino acid residues in the starting sequence. Sequence identity can be measured over the entire length of the starting sequence.

[0031] Homologous sequences are determined by known sequence alignment methods. A commonly used alignment method is BLAST, which is described by Altschul et al. (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); and Karlin et al. (Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). A particularly useful BLAST program is the WU-BLAST-2 program (see Altschul et al., Meth. Enzymol. 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values ​​and are established by the program itself based on the composition of the specific sequence and the composition of the specific database for which the target sequence is being searched. However, these values ​​can be adjusted to increase sensitivity. The amino acid sequence identity value % is determined by dividing the number of identical residues in the alignment by the total number of residues in the "longer" sequence in the alignment region. The "longer" sequence is the sequence with the most actual residues in the alignment region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

[0032] Other methods can be used to align sequences. An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also draw a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (Feng and Doolittle, J. Mol. Evol. [Journal of Molecular Evolution] 35: 351-360 (1987)). This method is similar to the method described by Higgins and Sharp (Higgins and Sharp, CABIOS [Computers in Biological Sciences] 5: 151-153 (1989)). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. The term "optimal alignment" refers to the alignment that gives the highest percent identity score.

[0033] As used herein, the term "malt beverage" includes, for example, the following fermented malt beverages that form foam: full-malt beer, strong beer, dry beer, thin beer, low-alcohol beer, low-calorie beer, porter, bock, stout, malt liquor, non-alcoholic malt liquor, etc. The term "malt beverage" also includes alternative malt beverages, such as fruit-flavored malt beverages, citrus-flavored malt beverages (e.g., lemon-flavored, orange-flavored, lime-flavored, or berry-flavored), liquor-flavored malt beverages (e.g., vodka-flavored, rum-flavored, or tequila-flavored malt liquor), or coffee-flavored malt beverages (e.g., caffeine-flavored malt liquor), etc.

[0034] As used herein, the term "beer" traditionally refers to an alcoholic beverage derived from malt (which is derived from barley) and optional adjuncts (such as grains) and flavored with hops. Beer can be made from a variety of grains by essentially the same process. All cereal starches are glucose homopolymers, in which glucose residues are connected by α-1,4-links or α-1,6-links, with the former being dominant. The process for making fermented malt beverages is commonly referred to as brewing. The primary raw materials used to make these beverages are water, hops, and malt. In addition, adjuncts such as common corn grits, refined corn grits, ground yeast for beer making, rice, sorghum, refined corn starch, barley, barley starch, shelled barley, wheat, wheat starch, baked cereals, cereal flakes, rye, oats, potatoes, cassava, and syrup, such as corn syrup, cane syrup, invert syrup, barley and / or wheat syrup, etc., can be used as the source of starch. Starch is ultimately converted into dextrin and fermentable sugars. Malt, primarily produced from selected barley varieties, has the greatest impact on the overall character and quality of beer for several reasons. First, malt is the primary flavoring agent in beer. Second, malt provides the majority of fermentable sugars. Third, malt provides protein, which contributes to the beer's body and head. Fourth, malt provides essential enzyme activity during mashing.

[0035] As used herein, the term "tannins" refers to a class of naturally occurring polyphenolic biomolecules found in plants, including sorghum. Tannins are composed of flavonoids, which are 15-carbon compounds with two benzene rings and one heterocyclic ring.

[0036] "Catechin" is a specific flavonoid found in tannins. The chemical name of catechin is (2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol.

[0037] As used herein, the term "CAE" refers to catechin equivalents.

[0038] As used herein, the term "an enzyme not inhibited by tannins" refers to an enzyme that retains substantial activity in the presence of tannins or after exposure to tannins.

[0039] A centipoise is one hundredth of a poise, or one millipascal-second (mPa·s) expressed in SI units (1 cP = 10-3 Pa·s = 1 mPa·s).

[0040] As used herein, the "process of making beer" is a process well known in the art, but in brief, it involves five steps: (a) starch saccharification and / or adjunct cooking, (b) wort separation and extraction, (c) boiling and hopping of the wort, (d) cooling, fermentation and storage, and (e) maturation, processing and packaging. In the first step, the ground or crushed malt is mixed with water and kept at a controlled temperature for a period of time to allow the enzymes present in the malt to convert the starch present in the malt into fermentable sugars. In the second step, the mash is transferred to a "lauter tun" or mash filter, where the liquid is separated from the grain residue. This sweet liquid is called "wort" and the remaining grain residue is called "spent grain".

[0041] The mash is typically extracted, which involves adding water to the mash to recover any remaining soluble extracts from the spent grains. In the third step, the wort is boiled vigorously. This sterilizes the wort and helps develop color, flavor, and aroma. Hops are added at some point during the boil. In the fourth step, the wort is cooled and transferred to a fermenter, which contains yeast or to which yeast is added. The yeast converts sugars into alcohol and carbon dioxide gas through fermentation; at the end of fermentation, the fermenter is cooled, or it can be cooled to stop fermentation. Yeast flocs are removed. In the final step, the beer is cooled and stored for a period of time, during which time the beer becomes clear and develops flavor, and any materials that might impair the beer's appearance, flavor, and shelf life settle. Before packaging, the beer is carbonated and optionally filtered and pasteurized. After fermentation, the resulting beverage typically contains between about 2% and about 10% alcohol by weight. Non-fermentable carbohydrates are not converted during fermentation and constitute the majority of dissolved solids in the final beer. This residue remains because malt amylase is unable to hydrolyze the α-1,6-linkages of starch.Non-fermentable carbohydrates contribute approximately 50 calories per 12 oz of beer.

[0042] As used herein, the "process for making beer" may further be applied to the mashing of any milling grain.

[0043] As used herein, the term "grain for milling" refers to any plant material containing starch and / or sugars that can be derived from any plant and plant part, including tubers, roots, stems, leaves, and seeds. Grain for milling can comprise grains, such as grains from barley, wheat, rye, oats, corn, rice, milo, millet, and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90%, or even 100% (w / w) of the wort is derived from grain. In some embodiments, grain for milling can comprise plant material containing starch and / or sugars obtained from cassava [Manihot esculenta] roots. Grain for milling can comprise germinated grains, such as barley malt. Preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w / w) of the grist for the wort is derived from malted cereals.

[0044] In the context of brewing, the term "fermentation" means the conversion of sugars in the wort into ethanol and carbon dioxide by enzymes in brewing yeast, and the formation of other fermentation by-products.

[0045] As used herein, the term "malt" is understood to mean any germinated cereal grain, such as barley.

[0046] The term "adjunct" is understood to mean the non-barley malt portion of the grain used for milling. Adjuncts can be any carbohydrate-rich material, such as sorghum, corn, cassava, wheat, rye, millet, rice, etc.

[0047] The term "mash" is understood to mean an aqueous starch slurry, eg comprising crushed barley malt, crushed barley, and / or other adjuncts or a combination thereof, which is subsequently mixed with water to separate into wort+spent grains.

[0048] As used herein, the term "wort" refers to the unfermented liquid run-off after grist is extracted during mashing.

[0049] As used herein, the term "spent grains" refers to the dehydrated solids remaining when grist grains have been extracted from the mash and the wort has been separated.

[0050] As used herein, the term "beer" refers to fermented wort, for example, an alcoholic beverage brewed from barley malt, optional adjuncts, and hops.

[0051] As used herein, the term "extract recovery" in wort is defined as the sum of soluble matter extracted from grist grains (malt and adjuncts), expressed as a percentage on a dry matter basis.

[0052] As used herein, the term "pasteurization" means killing microorganisms in an aqueous solution by heating. Typically, pasteurization in the brewing process is performed using a flash pasteurizer or a tunnel pasteurizer. As used herein, the term "pasteurization unit or PU" refers to a quantitative measure of pasteurization. One pasteurization unit (1 PU) of beer is defined as the heat retention of beer at 60 degrees Celsius for one minute. It is calculated as follows:

[0053] PU = tx 1.393^(T-60), where:

[0054] t = time in minutes at pasteurization temperature in the pasteurizer

[0055] T = temperature in the pasteurizer in degrees Celsius

[0056] [^(T-60) represents the index of (T-60)]

[0057] Depending on the type of beer, the raw materials and microbial contamination, the brewer and their perceived impact on the beer flavor, different minimum PU can be used. Typically, for beer pasteurization, 14-15 PU are required. Depending on the pasteurization equipment, the pasteurization temperature is typically in the range of 64 degrees Celsius to 72 degrees Celsius, and the pasteurization time is calculated accordingly. More information can be found in Wolfgang Kunze's Technology Brewing and Malting [Technical Brewing and Malting], Research and Teaching Institute of Brewing, Berlin [Berlin Brewing Research and Teaching Institute] (VLB), 3rd fully updated edition, 2004, ISBN 3-921690-49-8.

[0058] As used herein, the term "DP1 (degree of polymerization 1)" means glucose or fructose. "DP2" means maltose and / or isomaltose. "DP3" means maltotriose, panose, and isopanose. "DP4 / 4+" means non-fermentable dextrins or maltooligosaccharides having a degree of polymerization of 4 or higher.

[0059] Where a range of values ​​is provided, it is understood that each intervening value (to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise) between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any specified value or intervening value in a stated range and any other specified value or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in that range, and each range in which either, neither, or both of the limits are included in the smaller range is also encompassed in this disclosure, subject to any expressly excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also encompassed in this disclosure.

[0060] Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the particular embodiments described, as these embodiments may, of course, vary. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, exemplary methods and materials are now described.

[0061] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" includes a plurality of such candidate agents, and reference to "the cell" includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0062] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention.

[0063] abbreviation

[0064] In this disclosure and in the subsequent experimental section, the following abbreviations are used: GA (glucoamylase); CAE (catechin equivalent); wt% (weight percent); °C (degrees Celsius); rpm (revolutions per minute); H2O (water); dH2O (deionized water); aa (amino acid); AA (alpha amylase); kD (kilodalton); g or gm (gram); μg (microgram); mg (milligram); μL (microliter); ml and mL (milliliter); mm (millimeter); μm (micrometer); M (mole); mM (milliliter); millimole); μM (micromole); U (unit); V (volt); MW (molecular weight); m (mass); sec or s (second / seconds); min or m (minute / minutes); hr or h (hour / hour); cP centipoise; dwb (dry weight basis); (RVA) rapid viscometer; ABS (absorbance); Abs600 (absorbance at 600 nm); OD (optical density); OD 600 (optical density at 600 nm); AU (absorbance units); FAN (free α-amino nitrogen); PCR (polymerase chain reaction); EtOH (ethanol); MTP (microtiter plate); EBC (European Convention for Brewing); (OE) original extract; DP (degree of polymerization); N (normality), and ppm (parts per million). DETAILED DESCRIPTION

[0065] Traditional beer brewing uses only barley, hops, and water. The barley is germinated by soaking it in water and heating it. Malting begins with the process of releasing endogenous enzymes in the barley that can break down starch. The germinated barley is then subjected to saccharification, a process that releases endogenous barley enzymes and converts starch into fermentable sugars to produce wort. The fermentable sugars are then converted into ethanol through fermentation. Because barley can be expensive, brewers strive to use cheaper starch sources in brewing. Such alternative starch sources are called adjuncts and include corn, cassava, wheat, rye, millet, sorghum, and rice, as well as mixtures thereof.

[0066] Barley is a temperate cereal that grows best in cool climates. Growing barley for brewing is impractical in tropical and subtropical regions, and the high cost of importing barley from cooler climates is prohibitive. Crops such as maize, rice, and sorghum are more readily cultivated in such areas. Another consideration is the demand for fuel ethanol, which could divert a significant portion of local maize production.

[0067] If very small amounts of adjuncts are used compared to the amount of barley malt, the endogenous barley maltase may be able to break down the adjunct starch. However, as brewers use larger amounts of adjuncts than when using little or no barley (i.e., 100% adjunct beer), the endogenous barley maltase is insufficient to break down the starch of the added adjuncts into fermentable sugars.

[0068] Although the process of brewing with barley is well developed, brewing with adjuncts has proven to be more challenging, especially considering the cost. For example, there are few endogenous enzymes in germinated sorghum that break down starch. Literature has reported that both the liquefaction and saccharification of sorghum malt are problematic in producing acceptable levels of fermentable sugars from endogenous sorghum starch. In order to make starch easily fermentable by yeast, it must be broken down into fermentable sugars, such as glucose, maltose, and maltotriose. First, the granular structure of starch is broken down via a process called gelatinization. While heating, water is added to the relevant cereal or adjunct. When the gelatinization temperature is reached (which varies depending on the cereal being gelatinized), the starch granules become swollen and leaky (leak), thereby increasing viscosity and losing the granular structure.

[0069] Gelatinization is followed by liquefaction. Once starch is gelatinized, it is easily cut by enzymes. Typically, in commercial brewing, exogenous enzymes are added during the starch saccharification process to increase starch decomposition. For example, alpha-amylases that act endo-wise and degrade raw starch can be used to convert starch into oligosaccharides and reduce the viscosity of the gelatinized starch used in the saccharification step. Other enzymes that can be used for starch saccharification include proteases, lipases, fungal alpha-amylases, and maltogenic alpha-amylases. In the saccharification step, glucoamylases are used to break down oligosaccharides into fermentable sugars.

[0070] However, according to one aspect of the present invention, it has been discovered that adjuvants or adjuvant mixtures with high tannin content may be difficult to enzymatically treat. Tannins are naturally occurring polyphenolic compounds found in a variety of plants, seeds, barks, and peels. Among the cereals used as adjuvants in brewing, sorghum has a particularly high tannin content. While not being bound by any particular theory, applicants have discovered that brewing enzymes may be inhibited and / or inactivated by tannins or compounds within tannins. Tannins can cause enzyme inactivation by reacting with the enzyme and / or by cross-linking the enzyme, thereby inactivating the enzyme. According to one aspect of the present invention, it has been determined that a relatively high percentage of proline residues contributes to enzyme inactivation by tannins, and that enzymes with higher proline content are more susceptible to tannin inactivation than enzymes with lower proline content. By placing proline residues on the surface of the enzyme molecule, thereby increasing their accessibility to interact with tannins, it is possible to increase the reactivity of proline residues to tannin inactivation.

[0071] According to one aspect of the present invention, there is provided a method for producing beer wort, the method comprising the step of mashing grist grains having a high tannin adjunct in the presence of an exogenously provided enzyme composition having enzymes that are not inhibited by tannins to provide the beer wort.

[0072] According to one aspect of the present invention, enzymes that are not inhibited by tannins can be identified by determining the amount of enzyme activity that remains after exposure to tannins. In this regard, a standard assay can be used according to the present invention by including tannins, or more preferably, standard components of tannins (e.g., catechins), in the assay and determining the effect that tannins have on enzyme activity. According to one aspect of the present invention, enzymes that are not inhibited by tannins preferably retain at least 10% activity in a tannin solution, at least 20% activity in a tannin solution, at least 30% activity in a tannin solution, at least 40% activity in a tannin solution, at least 50% activity in a tannin solution, at least 60% activity in a tannin solution, at least 70% activity in a tannin solution, at least 80% activity in a tannin solution, at least 90% activity in a tannin solution, at least 95% activity in a tannin solution, at least 99% activity in a tannin solution, or 100% activity in a tannin solution.

[0073] According to one aspect of the present invention, the tannin solution preferably contains catechins. More preferably, the catechins are present at about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, and about 3 mg / ml. Even more preferably, the catechins are present at about 2 mg / ml and the enzyme retains about 90% activity.

[0074] In another aspect of the invention, the enzyme not inhibited by tannins retains substantial activity in the presence of grist grains containing tannins. According to one aspect of the invention, the enzyme that is not inhibited by tannins preferably retains at least 10% of its activity in milling cereals containing tannins, at least 20% of its activity in milling cereals containing tannins, at least 30% of its activity in milling cereals containing tannins, at least 40% of its activity in milling cereals containing tannins, at least 50% of its activity in milling cereals containing tannins, at least 60% of its activity in milling cereals containing tannins, at least 70% of its activity in milling cereals containing tannins, at least 80% of its activity in milling cereals containing tannins, at least 90% of its activity in milling cereals containing tannins, at least 95% of its activity in milling cereals containing tannins, at least 99% of its activity in milling cereals containing tannins, or 100% of its activity in milling cereals containing tannins.

[0075] According to one aspect of the invention, the grist cereal has at least 10 μM CAE / g grist cereal, at least 20 μM CAE / g grist cereal, at least 30 μM CAE / g grist cereal, at least 40 μM CAE / g grist cereal, at least 50 μM CAE / g grist cereal, at least 60 μM CAE / g grist cereal, at least 70 μM CAE / g grist cereal, at least 80 μM CAE / g grist cereal, at least 90 μM CAE / g grist cereal, at least 100 μM CAE / g grist cereal, at least 110 μM CAE / g grist cereal, at least 120 μM CAE / g grist cereal, at least 130 μM CAE / g grist cereal, at least 140 μM CAE / g grist cereal or at least 150 μM CAE / g grist cereal.

[0076] More preferably, the enzyme not inhibited by tannins retains about 90% of its activity in a grist mill having a CAE / g grist mill.

[0077] Preferably, the enzyme not inhibited by tannins is selected from the group consisting of raw starch degrading alpha-amylases, proteases, fungal alpha-amylases, glucoamylases, maltogenic alpha-amylases and lipases. Still more preferably, the enzyme is a raw starch degrading alpha-amylase.

[0078] Still more preferably, the alpha-amylase is a glucohydrolase of the GH13 class.In an even more preferred embodiment, the raw starch degrading alpha-amylase is derived from a Cytophaga species.

[0079] Still more preferably, the raw starch degrading α-amylase used in the method for detecting cross-linking has at least 60% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 65% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 70% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 75% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 80% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 85% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 90% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 95% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 98% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 99% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, or has the sequence set forth in SEQ ID NO: 2 or an amylase-active fragment thereof.

[0080] Preferably the grist mill comprises sorghum. More preferably the grist mill is at least 10% sorghum.

[0081] More preferably, the grist is at least 20% sorghum, at least 30% sorghum, at least 40% sorghum, at least 50% sorghum, at least 60% sorghum, at least 70% sorghum, at least 80% sorghum, at least 90% sorghum, or 100% sorghum.

[0082] In another aspect of the invention, the grist mill preferably further comprises corn, cassava, barley, wheat, rye, millet or rice.

[0083] In another aspect of the invention, the exogenously provided enzyme composition having a raw starch-degrading alpha-amylase that is not inhibited by tannins further has one or more of a protease, a fungal alpha-amylase, a glucoamylase, a maltogenic alpha-amylase, and a lipase.

[0084] In another aspect of the present invention, there is provided a method of fermenting beer wort produced as described above using an enzyme not inhibited by tannins to obtain an alcoholic beverage. Preferably, the alcoholic beverage is beer.

[0085] In another aspect of the present invention, there is provided wort produced using an enzyme not inhibited by tannins as described above. In another aspect of the present invention, there is provided beer produced from the wort as described above.

[0086] In another aspect of the present invention, a method for determining whether an enzyme is inhibited by tannin is provided, comprising the steps of incubating the enzyme in the presence of tannin and detecting cross-linking between the enzyme and the tannin. This aspect of the invention obviates the need for performing a separate assay for each enzyme. Preferably, the tannin is catechin. Preferably, the step of detecting cross-linking is performed by measuring turbidity.

[0087] Preferably, the enzyme is a brewing enzyme. More preferably, the brewing enzyme is selected from the group consisting of: raw starch-degrading α-amylases, proteases, fungal α-amylases, maltogenic α-amylases, and lipases. Even more preferably, the enzyme is a raw starch-degrading α-amylase.

[0088] Still more preferably, the alpha-amylase is a glucohydrolase of the GH13 class.In an even more preferred embodiment, the raw starch degrading alpha-amylase is derived from a Cytophaga species.

[0089] Still more preferably, the raw starch degrading α-amylase used in the method for detecting crosslinking has at least 60% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 65% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 70% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 75% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 80% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 85% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 90% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 95% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 98% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, at least 99% sequence identity to SEQ ID NO: 2 or an amylase-active fragment thereof, or has the sequence set forth in SEQ ID NO: 2 or an amylase-active fragment thereof. Preferably, the step of detecting crosslinking is measuring turbidity.

[0090] Preferably, the enzyme not inhibited by tannins is selected from the group consisting of raw starch-degrading bacterial alpha-amylases, glucoamylases, pullulanases, fungal alpha-amylases and maltogenic alpha-amylases. Still more preferably, the enzyme not inhibited by tannins is a raw starch-degrading bacterial alpha-amylase.

[0091] Preferably, the enzyme not inhibited by tannins has a specific catechin cross-linking activity of less than 10 AU / μg, less than 9 AU / μg, less than 8 AU / μg, less than 7 AU / μg, less than 6 AU / vg, less than 5 AU / μg, less than 4 AU / vg, less than 3 AU / μg, less than 2 AU / μg or less than 1 AU / μg.

[0092] Examples

[0093] The present disclosure is further described in detail in the following examples, which are not intended to limit the scope of the disclosure claimed in any way. The accompanying drawings are intended to be considered an integral part of the specification and description of the present disclosure. The following examples are provided to illustrate but not to limit the disclosure claimed.

[0094] Example 1 - Enzyme

[0095] GsAA: an α-amylase variant from Geobacillus stearothermophilus having the amino acid sequence shown in SEQ ID NO: 1.

[0096] CsAA: A tannin-uninhibited alpha-amylase variant from Cytophaga sp. having the amino acid sequence shown in SEQ ID NO:2.

[0097] Termamyl SCDS from Novozymes was used as an example of a liquefying alpha-amylase used in brewing.

[0098] Example 2 - Protein determination method

[0099] Protein determination by Stain Free Imager Criterion

[0100] Protein was quantified by SDS-PAGE gel and densitometry using the Gel Doc™ EZ imaging system. Reagents used in the assay: concentrated (2x) Laemmli sample buffer (Bio-Rad, catalog number (Cat. No.) 161-0737); 26-well XT 4-12% Bis-Tris gel (Bio-Rad, catalog number (Cat. No.) 345-0125); Protein marker "Precision Plus Protein Standards" (Bio-Rad, catalog number (Cat. No.) 161-0363); Protein standard BSA (Thermo Scientific, catalog number (Cat. No.) 23208) and SimplyBlue Safestain (Invitrogen, catalog number (Cat. No.) LC 6060). Assay was performed as follows: in a 96-well PCR plate, 50 μL of diluted enzyme sample was mixed with 50 μL of sample buffer containing 2.7 mg of DTT. The plate was sealed with Microseal'B' film from Bio-Rad and placed in a PCR machine and heated to 70°C for 10 minutes. Afterwards, the chamber was filled with running buffer and a gel box was set. 10 μL of each sample and standard (0.125-1.00 mg / mL BSA) were then loaded onto the gel, and 5 μL of markers were added. Afterwards, electrophoresis was run at 200V for 45 min. After electrophoresis, the gel was rinsed 3 times 5 min in water, then stained overnight in Safestain, and finally decolorized in water. The gel was then transferred to an imager. Image Lab software was used to calculate the intensity of each band. BSA (Thermo Fisher Scientific, catalog number 23208) was used to make a calibration curve, and the amount of target protein was determined by band intensity and calibration curve. Protein quantification methods were used to prepare enzyme samples for subsequent examples.

[0101] Example 3 - Viscosity Analysis of Starch Gelatinized Raw Materials by RVA

[0102] Viscosity was analyzed by the RVA method based on AACC (American Association of Cereal Chemists) analysis. The Rapid Visco Analyzer (RVA) is a rotational viscometer that can continuously record the viscosity of a sample under controlled temperature conditions. The RVA can suspend the sample in a solvent, keep it suspended throughout the test, and apply an appropriate degree of shear to match the processing conditions, which makes it particularly valuable in many process and research applications. An RVA from Newport Scientific (making the viscosity between 0 cP and 24,000 cP) was used and controlled by the Thermocline for Windows (TCW3) software program, which enables heating and cooling samples within the range of 0°C-100°C in steps of 0.1°C. The RVA was equipped with a water bath from Thermo Fisher Scientific Accel500LC for temperature control. Initialization and automatic zeroing of the instrument were initially performed with a propeller at 960 rpm before each experiment to preheat the motor, the oil on the RVA, and ensure consistent measurements.

[0103] All raw material samples were prepared in Perten aluminum cans. 6.0 g of raw material was mixed with 24.0 g of preheated tap water (50° C.). The pH was adjusted to 5.6 using acid or sodium hydroxide (2.5 M H2SO4 / 1 N NaOH). This resulted in a water to milling grain ratio of 4:1. 1.0 ml of enzyme solution was applied to the mixture, replacing the 1.0 ml of tap water in the sample. A rubber stopper was placed on top of the can and shaken for approximately 10 seconds to prevent lumps (especially for flour and fine powders).

[0104] The chamber in the RVA was preheated to 50°C and the starch liquefaction was analyzed using the following procedure, as shown in Table 1 below:

[0105] Table 1. Temperature (°C), time (hh:mm:ss), and speed (rpm) settings for the RVA.

[0106] time type value unit 0：00：00 temperature 50 ℃ 0：00：00 speed 500 Rpm 0：00：10 speed 160 Rpm 0：01：00 temperature 50 ℃ 0：23：30 temperature 95 ℃ 0：23：30 Finish 95 ℃

[0107] At the end of the analysis, the RVA was cooled to 50°C in preparation for the next sample to be analyzed. The following properties were measured for each analysis: pasting temperature, peak viscosity, peak time, peak temperature, and final viscosity of the starch.

[0108] Example 4 - Viscosity analysis of high tannin-rich red sorghum after starch gelatinization using α-amylase.

[0109] The viscosity of ground red sorghum (Diago, Kenya, 27.11.2018, ground on a Buhler Miag malt grinder (1.6 mm setting)) with a high content of various tannins (>1 g catechin equivalent / 100 g dwb) was analyzed according to the RVA method described in Example 3. Therefore, 6.0 g of ground red sorghum was mixed with 23.0 g of preheated tap water (50°C). The solution was mixed in a small beaker using a spatula for about 10 sec and the pH was adjusted to 5.6 with 2.5 M H2SO4. 1 mL of enzyme was added to give the given concentrations shown in Table 2 below. After mixing the sample, the rotor was placed in the sample and the measurement was started using RVA.

[0110] Table 2 RVA samples and dosage of liquefaction α-amylase

[0111] serial number raw material GsA CsA Termamyl SCDS ppm (in DS) ppm (in DS) ppm (in DS) 1 Red Sorghum 27 2 Red Sorghum 18 3 Red Sorghum 11 4 Red Sorghum 22 5 Red Sorghum 33

[0112] The RVA analysis of the given samples is shown in Table 3. Surprisingly, CsAA was the only α-amylase that completely reduced the viscosity of the sorghum samples. High enzyme activity was also observed by the following: 18 ppm (in DS) of uninhibited CsAA achieved a significant reduction in peak viscosity, a reduction in peak time, and a complete reduction in final viscosity to 60 Cp, compared to 940 Cp achieved by 27 ppm (in DS) of GsAA and 5080 to 7274 Cp achieved by 11 to 33 ppm (in DS) of Termamyl SCDS. In addition, very effective degradation was also observed by the reduction in peak temperature, time, and viscosity compared to higher doses of GsAA and Termamyl SCDS.

[0113] Photos of processed material taken immediately after RVA processing are Figure 1 Here it can be clearly observed that sample 2 processed with uninhibited CsAA alpha-amylase is further dissolved and has fewer solid particles compared to Termamyl SCDS and a minor extend of GsAA.

[0114] Table 3 RVA analysis: Paste formation temperature, peak viscosity, peak time, peak temperature and final viscosity of the starch

[0115] Sample No. Run Paste forming temperature Peak viscosity Peak time Peak temperature Final viscosity ℃ Cp minute ℃ Cp 1) 72.1 7881 19.3 86.6 940 2) 72.05 7274 17.9 83.6 60 3) 72.1 10228 21.0 89.75 7274 4) 72.05 9810 20.5 88.8 6078 5) 72.1 9181 20.6 89.15 5080

[0116] Example 5 - Leaching Saccharification of Red Sorghum Using Uninhibited α-Amylase

[0117] The purpose of this example is to demonstrate the benefit of an uninhibited α-amylase during the processing of adjuncts with high tannin content in the leaching process. The enzyme was tested in a model mashing operation system using ground red and white sorghum (Kenya Diago, 27.11.2018 and DK18-00735, ground on a Buhler malt grinder (1.6 mm setting)) with high levels of various tannins for wort production.

[0118] Starch saccharification for wort production

[0119] Sorghum milling grain (35.0 g ground white sorghum and 35.0 g ground red sorghum) was mixed in a beaker and mixed with 175 g tap water in a starch saccharification bath (Lockner, LG-electronics) cup and the pH was adjusted to pH 5.4 with 2.5 M sulfuric acid, resulting in a water to milling grain ratio of 2.5:1. Alpha-amylase was added based on mg protein determined according to Example 1 in the following settings: Trial 1, 13.4 μg GsAA / g sorghum; Trial 2, 20.2 μg GsAA / g sorghum; Trial 3, 17.7 μg uninhibited CsAA / g sorghum and 26.5 μg uninhibited CsAA / g sorghum. In addition to achieving filterability, fermentable sugars and appropriate FAN levels, the following enzymes were fixedly added in each trial: 0.250 mg BG2 (Dupont), 0.500 mg NP (DuPont), 0.500mg X4 (DuPont), 3.000mg MA (DuPont) and 1.000mg P10 (DuPont) (all enzymes / g sorghum flour grain). The adjunct was subjected to starch saccharification using the following program: heating to 60°C and holding for 30 minutes to initiate starch saccharification; heating to 70°C for 10.0 minutes by increasing the temperature at 1°C / min; holding at 70°C for 45 minutes; heating to 75°C for 5 minutes by increasing the temperature at 1°C / min; holding at 75°C for 45 minutes; heating to 82°C for 7 minutes by increasing the temperature at 1°C / min; holding at 82°C for 20 minutes to terminate starch saccharification.

[0120] Afterwards, when the temperature reached 82°C and mashing was complete, the test was iodine negative. The time required to become iodine negative was recorded in minutes and the results are given in Table 4. It is clear here that a lower dose of uninhibited CsAA was able to obtain an iodine negative result for the mash in a shorter time than GsAA, indicating further processing of the starch fraction of the sorghum material.

[0121] Table 4. Iodine Test for Sorghum Starch Saccharification. The time (in minutes) required to become iodine negative was recorded as OK for Trials 1-4 with the following α-amylase additions: Trial 1, 13.4 μg GsAA / g sorghum; Trial 2, 20.2 μg GsAA / g sorghum; Trial 3, 17.7 μg uninhibited CsAA / g sorghum and Trial 4, 26.5 μg uninhibited CsAA / g sorghum.

[0122]

[0123] At the end of the starch saccharification, the mash was cooled, made up to 350 g and filtered. After 30 minutes, the filtrate volume was measured. The pH was adjusted to pH 5.2 with 2.5 M sulfuric acid, and a bittering hop particle from St. Johann Hop Processing Company (Hopfenveredlung, St. Johann) with an α content of 16.0% (EBC 7.70 specific HPLC analysis, 01.10.2013) was added to each flask (350 g). The wort sample was boiled in a boiling bath for 60 minutes, and the wort was cooled to 17 ° C and filtered and used for analysis, see below.

[0124] Wort analysis: The raw extract (OE) of the wort samples was measured after mashing using an Anton Paar Lovis according to Standard Instruction Brewing, 23.8580-B28. FAN, the content of free α-amino nitrogen (mg / liter), was measured in the wort using a spectrophotometer Genesys 10S UV-Vis (based on EBC 8.10), according to Standard Instruction Brewing, 23.8580-B15. Fermentable sugars (% total + g / 100 ml) DP1, DP2, DP3, and DP4+ were determined by HPLC after mashing according to Standard Instruction Brewing, 23.8580-B20. The sugar composition of the wort was determined on an HPLC-RI system equipped with an RSO oligosaccharide column, Ag + 4% cross-linked (Phenomenex, The Netherlands) and an analytical guard column (Carbo-Ag + neutral, AJO-4491, Phenomenex, The Netherlands) and operated at 70°C. An isocratic flow rate of 0.3 ml / min was maintained throughout the analysis, with a total run time of 45 min, and an injection volume of 10 μL. Quantification was performed by peak area relative to the peak area of ​​the given standards (DP1: glucose; DP2: maltose; DP3: maltotriose, and peaks with a 4-degree or higher degree of maltotetraose were used as standards).

[0125] The results of post-saccharification HPLC and extract analysis of the wort are shown in Table 5, which includes the relative sugar composition of the wort. As can be seen from the data in Table 5, both high and low doses of uninhibited CsAA delivered more fermentable sugars (as the sum of DP1-DP3 (average 72.5% vs. average 70.8%)) and more extract (13.84-13.90°P vs. 13.49-13.60°P) compared to GsAA. Uninhibited CsAA performed well in liquefying starch and enabling saccharification of the starch in high-tannin materials.

[0126] Table 5 HPLC and raw extract analysis of wort composition using different α-amylases.

[0127]

[0128] Quantitative free α-amino nitrogen (mg / liter) was measured in the wort and is shown in Table 6. There were no differences in the FAN levels measured between trials 1-4 and the variations were within experimental error.

[0129] Table 6. FAN (free α-amino nitrogen (mg / liter)) of sorghum wort in the infusion mash measured using different α-amylases.

[0130]

[0131] Example 6 - Cross-linking activity of active tannins with α-amylase

[0132] The purpose of this example was to demonstrate the preference of various α-amylases for reacting with and cross-linking reactive tannin species, thereby inactivating them. The high polyphenol content found in various sorghum varieties is associated with compromised nutritional quality and reduced brewing value of the grain. In this example, we tested the cross-linking of catechins, one of the most reactive polyphenolic compounds in the tannin fraction, with various exogenously supplemented α-amylases.

[0133] A catechin solution (Sigma Aldrich C1251) was prepared by wetting the material with 70% ethanol and then adding it to 20 mM sodium phosphate / acetate (pH 4.5), 0.2% (v / v) ethanol to a final concentration of 2 mg / ml. The enzymes GsAA, uninhibited CsAA, and Termamyl SCDS were all diluted in 20 mM sodium phosphate / acetate (pH 4.5). As determined according to Example 2, the α-amylase concentrations were 5.4 mg / g GsAA, 7.1 mg / g uninhibited CsAA, and 4.4 mg / g Termamyl SCDS. 125 μL of enzyme was mixed with 125 μL of 20 mM sodium phosphate / acetate (pH 4.5) and 60 μL of catechin solution in a 96-well MTP plate (Corning, New York, USA) sealed with tape and incubated at 30°C for 30 min and then at 4°C for 30 min to promote the cross-linking reaction between the enzyme and the polyphenols. The amount of cross-linking was quantified as turbidity read by a plate reader at OD at 600 nm. In a blank experiment, buffer was used instead of enzyme. For the three enzyme concentrations, the cross-linking or haze / turbidity formed was quantified at 4°C. Figure 2 As shown in Figure 2 , all enzymes in the tested dilutions showed increased cross-linking and a higher response with increasing enzyme concentration compared to the control (buffer, resulting in an OD600 of 0.004). Notably, uninhibited CsAA exhibited the lowest OD of all tested dilutions compared to GsAA and Termamyl SCDS, clearly demonstrating a lower propensity or reactivity to cross-link with catechins and, therefore, less inhibition. Since increasing enzyme concentration significantly enhanced cross-linking, the finding that uninhibited CsAA exhibited the lowest cross-linking when applied at the highest enzyme concentration significantly emphasizes the low reactivity of CsAA with catechins.

[0134] Additional cross-linking reactions were performed with extended incubation times to enhance the resulting turbidity. Similarly, the enzymes GsAA, uninhibited CsAA, and Termamyl SCDS were all diluted in 20 mM sodium phosphate / acetate (pH 4.5), and the α-amylase concentrations were 5.4 mg / g GsAA, 7.1 mg / g uninhibited CsAA, and 4.4 mg / g Termamyl SCDS as determined according to Example 2. The reactions were mixed as described above, and the sealed plates were incubated at 4°C for 30 min, 60°C for 30 min, and 4°C for 23 h. The results were as follows: Figure 3Compared to incubation at 30°C for 30min followed by incubation at 4°C for 30min, combining an elevated temperature of 60°C for 30min with an extended incubation at 4°C also significantly increased catechin reactivity at reduced enzyme dosages. Despite the increased reactivity, it is clear that CsAA showed the lowest OD (cross-linking) compared to GsAA and Termamyl SCDS in all dilutions tested. In addition, the extended catechin experiment emphasized the low reactivity and low inhibitory effect of CsAA with catechins. In this regard, the relative specific cross-linking activity of each enzyme can be calculated using the applied conditions (30min4°C, 30min60°C and 23 hours4°C) and enzyme concentrations between 0.4 and 0.8mg / ml in the assay by the following formula:

[0135]

[0136] Where Abs600(enzyme) is the absorbance measured at 600 nm in MTP after the cross-linking reaction between catechin and enzyme, Abs600(no enzyme) is the absorbance measured at 600 nm in MTP after the cross-linking reaction between catechin and water instead of enzyme, and menzyme is the mass of enzyme used in a given assay. The specific cross-linking activities of the three enzymes were calculated and expressed in AU (absorbance units) / μg and are shown in Table 7.

[0137] Table 7 Specific catechin cross-linking activity of three α-amylases AU (absorbance unit) / μg.

[0138] AU / μg Termamyl SCDC 5.3 GsA 8.9 CsA 1.2

[0139] It can be seen that the specific catechin cross-linking activity of CsAA is very low, at 1.2 AU / μg, compared with GsAA (8.9 AU / μg) and Termamyl SCDS (5.3 AU / vg).

[0140] Example 7 - Amino acid composition of α-amylase

[0141] It has been previously shown that the content of proline residues in proteins is positively correlated with polyphenol interactions, and that the molar percentage of proline in a protein or polypeptide is substantially linearly correlated with the ability of the protein to form a haze or precipitate with catechins (Asano et al., J. Am. Soc. Brew. Chem., 1982), (Siebert et al., Agric. Food Chem., 1996). Therefore, the molar percentage of proline in the polypeptides of the α-amylase variants under investigation was analyzed.

[0142] The amino acid composition of the alpha amylase variant GsAA from Geobacillus stearothermophilus (shown in SEQ ID NO: 1) is given below.

[0143] Amino acid composition of SEQ ID NO: 1:

[0144]

[0145] The amino acid composition of the alpha amylase variant CsAA from Cytophaga sp. (shown as SEQ ID NO: 2) is given below.

[0146] Amino acid composition of SEQ ID NO: 2:

[0147]

[0148]

[0149] It can be observed that the uninhibited CsAA has a low mole percentage of proline in the amylase polypeptide of 4.1%, while GsAA, which shows higher cross-linking reactivity and polyphenol inhibition, has a significantly higher mole percentage of proline in the amylase polypeptide of 4.3%.

[0150] Example 8 - Total flavonoid content of cereals used in brewing

[0151] Total flavonoid content was determined according to a modified version of the method described by Shao et al., J Agr Food Chem. 2014 and expressed or calculated as micromoles of catechin equivalents (CAE) / g of cereal flour (μmol CAE / g). The determined flavonoid content of the cereals used in the brewing is shown in Table 8 below.

[0152] Table 8 Calculated total flavonoid content in cereals, expressed as micromoles of catechin equivalents per gram of cereal

[0153]

[0154]

[0155] 1 Liu et al., J.Agric.Food Chem. 2002

[0156] 2 Taylor et al., J. Inst. Brew. 2013, using catechin with a mw of 290.36 g / mol

[0157] 3Jende-Strid et al., Carlsberg Res. Commun., 1985

[0158] 4 Xiang et al., Food Function, 2019, using catechins with an mw of 290.36 g / mol Sequence Listing <110> DuPont Nurtrition Biosciences <120> Uninhibited amylase for brewing with high-tannin materials <130> NB41775 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 486 <212> PRT <213> Geobacillus stearothermophilus <400> 1 Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Gln Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val 405 410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr 485 <210> 2 <211> 483 <212> PRT <213> Cytophaga sp. <400> 2 Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro 1 5 10 15 Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30 Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95 Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110 Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125 Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140 Gly Arg Gly Thr Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170 175 Phe His Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200 205 Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220 Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270 Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285 His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Ala Tyr Asp Met 290 295 300 Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala 305 310 315 320 Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Ser Gly Gly Tyr Pro Ala Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr Thr Thr Tyr Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380 Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg 385 390 395 400 Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445 Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460 Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Gln Gln

Claims

1. A method for producing wort for brewing beer, the method comprising saccharifying starch of milling grains comprising high tannin adjuncts in the presence of an exogenously provided enzyme composition, the exogenously provided enzyme composition comprising enzymes not inhibited by tannins, wherein the enzymes not inhibited by tannins are α-amylases consisting of the amino acid sequence of SEQ ID NO:

2.

2. The method according to claim 1, wherein the milling grains comprise sorghum.

3. The method according to claim 2, wherein the milling grains comprise at least 10% sorghum.

4. The method according to claim 3, wherein the milling grains comprise at least 20% sorghum.

5. The method according to claim 4, wherein the milling grains comprise at least 30% sorghum.

6. The method according to claim 5, wherein the milling grains comprise at least 40% sorghum.

7. The method according to claim 6, wherein the milling grains comprise at least 50% sorghum.

8. The method according to claim 7, wherein the milling grains comprise at least 60% sorghum.

9. The method according to claim 8, wherein the milling grains comprise at least 70% sorghum.

10. The method according to claim 9, wherein the milling grains comprise at least 80% sorghum.

11. The method according to claim 10, wherein the milling grains comprise at least 90% sorghum.

12. The method according to claim 11, wherein the milling grains comprise 100% sorghum.

13. The method according to any one of claims 1-12, wherein the milling grains further comprise corn, cassava, barley, wheat, rye, millet or rice.

14. The method according to any one of claims 1 to 12, wherein the CAE content in the milling grains is at least 10 μM CAE / g of milling grains.

15. The method according to claim 14, wherein the CAE content in the milling grains is at least 20 μM CAE / g of milling grains.

16. The method according to claim 15, wherein the CAE content in the milling grains is at least 30 μM CAE / g of milling grains.

17. The method according to claim 16, wherein the CAE content in the milling grains is at least 40 μM CAE / g of milling grains.

18. The method according to claim 17, wherein the CAE content in the milling grains is at least 50 μM CAE / g of milling grains.

19. The method according to claim 18, wherein the CAE content in the milling grains is at least 60 μM CAE / g of milling grains.

20. The method according to claim 19, wherein the CAE content in the milling grains is at least 70 μM CAE / g of milling grains.

21. The method according to claim 20, wherein the CAE content in the milling grains is at least 80 μM CAE / g of milling grains.

22. The method according to claim 21, wherein the content of CAE in the milled grain is at least 90 μM CAE / g of milled grain.

23. The method according to claim 22, wherein the content of CAE in the milled grain is at least 100 μM CAE / g of milled grain.

24. The method according to claim 23, wherein the content of CAE in the milled grain is at least 110 μM CAE / g of milled grain.

25. The method according to claim 24, wherein the content of CAE in the milled grain is at least 120 μM CAE / g of milled grain.

26. The method according to claim 25, wherein the content of CAE in the milled grain is at least 130 μM CAE / g of milled grain.

27. The method according to claim 26, wherein the content of CAE in the milled grain is at least 140 μM CAE / g of milled grain.

28. The method according to claim 27, wherein the content of CAE in the milled grain is at least 150 μM CAE / g of milled grain.

29. The method according to any one of claims 1-12 and 15-28, wherein the tannin-uninhibited enzyme is an α-amylase that degrades raw starch, and wherein the α-amylase is a glucanohydrolase of the GH13 class.

30. The method according to claim 29, wherein the α-amylase that degrades raw starch is derived from a Cytophaga sp.

31. The method according to claim 29, wherein the exogenously provided enzyme composition further comprises one or more of a protease, a fungal α-amylase, a raw maltose α-amylase, and a lipase.

32. The method according to any one of claims 1-12, 15-28, and 30-31, further comprising the step of fermenting the wort for brewing beer to obtain an alcoholic beverage.

33. The method according to claim 32, wherein the alcoholic beverage is beer.

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