Highly attenuated beer production

Abstract

The present invention relates to a method for increasing the degree of fermentation in a fermented beverage, more specifically, the method relates to the addition of transglucosidase to a maltose-depleted fermentation broth to convert non-fermentable dextrins to maltose.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 282,709, filed November 24, 2021, which is incorporated by reference in its entirety herein.

[0002] Sequence Listing The Sequence Listing filed herewith, entitled "NB42015-WO-PCT_SequenceListing.xml", was created on November 21, 2022 and is 3KB in size. The Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] In beer production, during the mashing process, fermentable sugars are produced from starch, which are then converted by yeast into ethanol. However, not all starch is converted into fermentable sugars. Short glucose oligomers remain after fermentation and cannot be converted into ethanol by brewer's yeast. These oligomers, also called dextrins, usually have four or more sugar units (DP4+). As the dextrins are not converted into ethanol, these sugars are present in the resulting beer and increase the calorie content of the beer. Many consumers consider high-calorie beer to be undesirable.

[0004] There remains a need in the art for methods to produce beer with less non-fermentable sugars and a lower caloric content. Summary of the Invention [Means for solving the problem]

[0005] According to an aspect of the present invention, a method is presented for increasing the attenuation in a fermented beverage by adding transglucosidase to a maltose-depleted fermentation broth.

[0006] Optionally, the fermented beverage is beer.

[0007] Optionally, the fermentation broth has a concentration of maltose less than 0.01% (w / w).

[0008] Optionally, less than 3 U / ml, 1 U / ml, or 0.5 U / ml of transglucosidase is added to the fermentation broth.

[0009] Optionally, the transglucosidase is added after more than 50% of the total fermentation time.

[0010] Optionally, the beer has an RDF of at least 85, 86, 87, 88, or 89%.

[0011] Optionally, the beer has an apparent extract level of -0.20% by weight or less.

[0012] Optionally, the transglucosidase is added to the fermentation broth after the fourth day from the start of fermentation.

[0013] Optionally, a glucoamylase is also added to the fermentation broth. Optionally, the glucoamylase is added at the beginning of the fermentation.

[0014] Optionally, the transglucosidase is a polypeptide having 70% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0015] Optionally, the polypeptide has 75% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0016] Optionally, the polypeptide has 80% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0017] Optionally, the polypeptide has 85% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0018] Optionally, the polypeptide has 90% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0019] Optionally, the polypeptide has 95% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0020] Optionally, the polypeptide has 99% or greater sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0021] Optionally, the polypeptide has 100% sequence identity to SEQ ID NO:1, or a transglucosidase-active fragment thereof.

[0022] A brief description of the sequence number SEQ ID NO:1 shows the mature amino acid sequence of alpha-glucosidase (transglucosidase) from Aspergillus niger.

[0023] Definitions and Abbreviations 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, 2 nded., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary Of Biology, Harper Perennial, NY (1991), provide one of skill in the art with the general meaning of many of the terms used herein. Nonetheless, for clarity and ease of reference, certain terms are defined below.

[0024] As used herein, the term "glucoamylase (EC 3.2.1.3)" refers to an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides.

[0025] A "variant" or "variants" refers to either a polypeptide or a nucleic acid. The term "variant" may be used interchangeably with the term "mutant". Variants include insertions, substitutions, transversions, truncations and / or inversions at one or more locations in the amino acid or nucleotide sequence, respectively. The terms "variant polypeptide", "polypeptide variant", "polypeptide", "variant" and "variant enzyme" refer to a polypeptide / protein having an amino acid sequence that either has a selected amino acid sequence, such as, for example, SEQ ID NO: 1, 2, 3, 4 or 5, or that is modified compared to a selected amino acid sequence, such as, for example, SEQ ID NO: 1, 2, 3, 4 or 5.

[0026] As used herein, "homologous sequence" and "sequence identity" with respect to a nucleic acid or polypeptide sequence means having approximately 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, where the function of the candidate nucleic acid or polypeptide sequence is essentially the same as the nucleic acid or polypeptide sequence to which the candidate homologous sequence is being compared. In some embodiments, a homologous sequence has at least about 85%-100% sequence identity, while in other embodiments, it has about 90%-100% sequence identity, and in other embodiments, it has at least about 95%-100% sequence identity.

[0027] 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 el al., Nucleic Acid Res., 12:387-395 (1984)).

[0028] "Percent (%) nucleic acid sequence identity" or "percent (%) amino acid sequence identity" is defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical with the nucleotide or amino acid residues of the starting sequence. Sequence identity can be measured over the entire length of the starting sequence.

[0029] Homologous sequences are determined by known sequence alignment methods. A commonly used alignment method is BLAST, 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, e.g., Altschul et al., Meth. Enzymol. 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the 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, set by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched. However, these values ​​can be adjusted to increase sensitivity. The % amino acid sequence identity value is determined by dividing the number of matching identical residues by the total number of residues of the "longer" sequence in the alignment region. The "longer" sequence is the sequence that has the most residues actually present in the alignment region (ignoring gaps introduced by WU-BLAST-2 to maximize the alignment score).

[0030] There are other methods for use in sequence alignment. One 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 plot 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. 35:351-360 (1987)). The method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151153 (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.

[0031] As used herein, the term "beer" refers to a traditionally malted alcoholic beverage, derived from barley, optionally from adjuncts such as cereal 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, with the glucose residues linked by either alpha-1,4- or alpha-1,6-linkages, with the former predominating. The process of making fermented malt beverages is commonly called brewing. The main raw materials used in these beverages are water, hops, and malt. In addition, for example, common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehulled barley, wheat, wheat starch, roasted grains, grain flakes, rye, oats, potato, tapioca, and additives such as syrup, cane syrup, invert sugar syrup, barley and / or wheat syrup, can be used as starch sources. The starch is ultimately converted into dextrins and fermentable sugars. For a number of reasons, malt, which is mainly produced from selected barley varieties, has the greatest effect on all the characteristics and quality of the beer. First, malt is the main flavoring agent in beer. Second, malt provides the major portion of the fermentable sugars. Third, malt provides proteins that will contribute to the body and foam characteristics of the beer. Fourth, malt provides the enzyme activity required during mashing.

[0032] As used herein, the term "hops" refers to its use as a major contributor to beer quality, including flavorings. In particular, hops (or hop components) add desirable bitter substances to beer. Additionally, hops act as protein precipitants, establish preservatives, and aid in foam formation and stabilization.

[0033] As used herein, a "process for making beer" is a process well known in the art, but briefly it involves five steps: (a) mashing and / or subcooking, (b) wort separation and extraction, (c) wort boiling and hopping, (d) cooling, fermentation, and storage, and (e) maturation, processing, and packaging. In the first step, milled or crushed malt is mixed with water and held for a period of time under a controlled temperature 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 "grain residue".

[0034] The mash is typically subjected to extraction, which includes adding water to the mash to recover residual soluble extract from the grain waste. In a third step, the wort is vigorously boiled. This sterilizes the wort and aids in the development of color, flavor and odor. Hops are added at some point during the boil. In a fourth step, the wort is cooled and transferred to a fermenter, which either contains yeast or has yeast added to it. After the addition of yeast, the liquid is called fermentation liquid. The yeast converts sugars to alcohol and carbon dioxide by fermentation, and the fermenter is cooled at the end of fermentation or may be cooled to stop fermentation. The yeast is flocculated and removed. At the end of the process, the beer is stored in the cool for a period of time during which the beer clarifies and develops its aroma, and any substances that may impair the appearance, aroma and shelf life of the beer are precipitated. Before packaging, the beer is saturated with carbon dioxide and optionally filtered and pasteurized. After fermentation, a beverage is obtained that typically contains about 2% to about 10% by weight alcohol. Non-fermentable carbohydrates are not converted during fermentation and form the majority of the dissolved solids in the final beer. This residue remains due to the inability of malt amylase to hydrolyze the α-1,6-bonds in starch. Non-fermentable carbohydrates contribute about 50 calories per 12 ounces of beer.

[0035] The term "fermentation", in the context of brewing, means the conversion of sugars in malt by enzymes in brewer's yeast to ethanol and carbon dioxide, with the formation of other fermentation by-products.

[0036] As used herein, a "fermentation liquor" is a liquid solution that undergoes a fermentation process to effect a chemical change to the food, beer or beverage by the action of yeast or bacteria, which produces carbon dioxide and converts the carbohydrates therein into alcohol.

[0037] As used herein, "transglucosidase" is synonymous with the term α-glucosidase, which has the enzyme nomenclature EC 3.2.1.20, predominantly transglucosylating activity, and the systematic name α-D-glucoside glucohydrolase.

[0038] As used herein, an "alpha-glucosidase" hydrolyzes or transglucosylates terminal non-reducing (1→4) linked alpha-glucose residues to produce glucose or IMO. Exo-type enzymes have the systematic name α-D-(1→4)-glucan glucanohydrolase and the enzyme code designation EC 3.2.1.1.

[0039] As used herein, "isomaltooligosaccharides (IMOs)" generally refer to oligosaccharides of glucose containing α-D-1,6 linkages. Exemplary isomaltooligosaccharides and their condensed IUPAC names (Id.) include, but are not limited to, isomaltose (Glc(α-1,6)Glc), isomaltotriose (Glc(α-1,6)Glc(α-1,6)Glc), and isomaltotetraose (Glc(α-1,6)Glc(α-1,6)Glc(α-1,6)Glc). Branched oligosaccharides with both α-D-1,4 and α-D-1,6 linkages, such as panose (Glc(α-1,6)Glc(α-1,4)Glc), are often also considered IMOs.

[0040] The term "malt" as used herein is understood to mean any cereal grain, for example barley.

[0041] As used herein, the term "wort" refers to the unfermented liquid that overflows after extraction of the grist during mashing.

[0042] As used herein, the term "meal" refers to the discarded solids remaining when the flour is extracted and the wort is separated from the mash.

[0043] As used herein, the term "beer" refers to an alcoholic beverage brewed from fermented wort, such as barley malt, and optionally adjuncts and hops.

[0044] As used herein, the term "extract recovery" in wort is defined as the sum of soluble material extracted from the grain (malt and adjuncts) expressed as a percentage on a dry matter basis.

[0045] As used herein, the term "pasteurization" means killing microorganisms in an aqueous solution by heating. The practice of pasteurization in the brewing process is typically done through the use of flash pasteurizers or tunnel pasteurizers. As used herein, the term "pasteurization unit or PU" refers to a quantitative measure of pasteurization. One pasteurization unit (1 PU) for beer is defined as one minute at 60 degrees Celsius. It is calculated as follows: PU=t×1.393^(T-60), where: t = time in minutes at pasteurizer temperature T = temperature in the pasteurizer in degrees Celsius [^(T-60) represents the exponent of (T-60)] Depending on the type of beer, the raw materials and the microbial contamination, the brewer and the perceived effect on the beer flavor, different minimum PUs may be used. Typically, 14-15 PUs are required for beer pasteurization. Depending on the pasteurization equipment, the pasteurization temperature typically ranges from 64 to 72 degrees Celsius and the pasteurization time is calculated accordingly. Further information can be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3-921690-49-8.

[0046] As used herein, the term "DP1" (degree of polymerization 1) refers to glucose or fructose. "DP2" refers to maltose and / or isomaltose. "DP3" refers to maltotriose, panose and isopanose. "DP4 / 4+" refers to dextrins or maltooligosaccharides with a degree of polymerization of 4 or higher that are non-fermentable.

[0047] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range is understood to be specifically disclosed as well, to the first decimal place of the unit of the lower limit. Each narrower range between any stated value or intervening value in a stated range, and any other stated or intervening value within that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included or excluded within the range, and each range in which either or both limits are included within the narrower range, or both limits are not included, is also encompassed within the disclosure, provided that any limits are specifically excluded in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0048] Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are now described.

[0049] As used in this specification and 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, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0050] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] According to an aspect of the present invention, it has been discovered that transglucosidase can be utilized in brewing to produce highly fermented beer. Without being bound by theory, according to the present invention, it has been discovered that transglucosidase can be added to the fermentation liquor to convert non-fermentable dextrins to maltose, which is converted to ethanol. However, transglucosidase can be added to the fermentation liquor only when the maltose carried from the wort is sufficiently depleted. When the maltose is sufficiently depleted, the transglucosidase will convert the non-fermentable dextrins to maltose. However, if the transglucosidase is added to the fermentation liquor (or even to the wort as taught by the prior art) before sufficient maltose depletion, the transglucosidase will carry out the reverse reaction, converting maltose to non-fermentable dextrins.

[0052] In recent years, due to changes in consumer health consciousness and preferences, low sugar beer-flavored beverages have become more popular among consumers. In the case of beers or beverages obtained by fermentation of various malt-based raw materials, the final sugar content in the corresponding beer or beverage can be reduced by increasing the amount of fermentable sugars in the fermentation liquid.

[0053] Transglucosidases (also known as a-glucosidases) can carry out transglucosylation in the presence of maltose as a substrate, for example during mashing or in the final wort. Transglucosidases produce isomaltooligosaccharides (IMOs) from maltose. These IMOs are generally known to be non-fermentable. Maltose is the donor molecule in the transglucosylation reaction, which hydrolyzes maltose, releasing one free glucose molecule and transferring another glucose molecule to an acceptor. The acceptor can be another maltose molecule resulting in a trisaccharide. The most abundant trisaccharide formed is panose. Glucose can also be transferred to higher sugars, resulting in longer chains of isomaltooligosaccharides, which are transferred to glucose, leading to the formation of isomaltose, or to water, releasing it as another free glucose molecule. The rate at which the different oligosaccharides are formed depends on the concentration of the different acceptors.

[0054] Most importantly, under conditions in which maltose is absent, transglucosidase can hydrolyze these non-fermentable IMO sugars, such as pannose and isomaltose, and thus these sugars can be converted from non-fermentable to fermentable sugars, but only when transglucosidase is applied in the absence of mannose, resulting in an increase in IMO.

[0055] Transglucosidase produces glucose through hydrolysis reactions, but when the substrate concentration is high, it catalyzes transglucosylation reactions. For example, malt beverages using transglucosidase in the mash prior to heat treatment produce high concentrations of isomaltooligosaccharides, such as the non-fermentable sugars isomaltose and panose.

[0056] Similarly, adding transglucosidase to the final wort before the fourth day of fermentation leads to a very high production of isomaltooligosaccharides: isomaltose, nigerose, gentiobiose and cellobiose. For brewing low carbohydrate or light beers, this is clearly undesirable.

[0057] An embodiment of the present invention relates to the efficient addition of very low doses (less than 3 U / mL) of transglucosidase after at least 4 days of fermentation to convert non-fermentable sugars in the absence of maltose and without producing isomaltooligosaccharides.

[0058] According to an aspect of the present invention, a method is provided for increasing the fermentation degree in a fermented beverage by adding transglucosidase to a maltose-depleted fermentation broth.

[0059] Preferably, the fermented beverage is beer.

[0060] Preferably, the fermentation broth has a maltose concentration of 0.01% (w / w).

[0061] Preferably, less than 3 U / ml, 1 U / ml or 0.5 U / ml of transglucosidase is added to the fermentation broth.

[0062] Preferably, the transglucosidase is added after more than 50% of the total fermentation time.

[0063] Preferably the beer has an RDF of at least 85, 86, 87, 88 or 89%.

[0064] Preferably, the beer has an apparent extract concentration of −0.20% by mass or less.

[0065] Preferably, the transglucosidase is added to the fermentation broth 4 days after the start of the fermentation.

[0066] Preferably, a glucoamylase is also added to the fermentation broth. Preferably, the glucoamylase is added at the beginning of the fermentation.

[0067] Preferably, the transglucosidase is a polypeptide having 70% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0068] Preferably, the polypeptide has 75% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0069] Preferably, the polypeptide has 80% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0070] Preferably, the polypeptide has 85% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0071] Preferably, the polypeptide has 90% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0072] Preferably, the polypeptide has 95% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0073] Preferably, the polypeptide has 99% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0074] Preferably, the polypeptide has 100% sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

[0075] The disclosure is further described in the following examples, which are not intended to limit the scope of the claimed disclosure in any way. The accompanying drawings are intended to be considered incorporated into and part of the specification and description of the disclosure. The following examples are provided to illustrate, but are not intended to limit the scope of the claimed disclosure. They are presented to illustrate the disclosure and are not intended to be limitations of the disclosure. EXAMPLES

[0076] Example 1 - HPLC analysis of DP sugars in malt-based worts supplemented with alpha-glucosidase All standards: glucose, maltose, maltotriose and maltotetraose were prepared in double distilled water (ddH20) and filtered through a 0.45 μm syringe filter. A set of each standard was prepared in a concentration range of 10-100,000 ppm. All wort samples containing active enzymes were inactivated by heating the samples to 95°C for 10 min. Subsequently, the wort samples were prepared in 96-well MTP plates (Corning, NY, USA), diluted a minimum of 4-fold in ddH20 and filtered through a 0.20 μm 96-well plate filter (Corning filter plate, PVDF hydrophilic membrane, NY, USA) before analysis. All samples were analyzed in duplicate. Quantification of sugars: DP1, DP2, DP3, DP4 and DP5+ was performed by HPLC. Sample analysis was performed on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a DGP-3600SD Dual-Gradient analytical pump, a WPS-3000TSL thermostatic autosampler, a TCC-3000SD thermostatic column oven, and a RI-101 refractive index detector (Shodex, JM Science). Chromeleon data system software (Version 6.80, DU10A Build 2826, 171948) was used for data acquisition and analysis.

[0077] Chromatographic conditions Samples were analyzed using an Ag+4% cross-linked (Phenomenex, Netherlands) RSO oligosaccharide column equipped with an analytical guard column (Carbo-Ag+neutral, AJ0-4491, Phenomenex, Netherlands) operated at 70°C. The column was eluted with double distilled water (filtered through a 0.45 μm regenerated cellulose membrane and purged with helium gas) at a flow rate of 0.3 ml / min. An isocratic flow of 0.3 ml / min was maintained throughout the analysis with a total run time of 45 min, and the injection volume was set to 10 μL. Samples were maintained at 20°C in a thermostatic autosampler compartment. The eluent was monitored using a refractive index detector (RI-101, Shodex, JM Science) and quantification was performed by peak area relative to the peak area of ​​a given standard (DP1: glucose; DP2: maltose; DP3: maltotriose and maltotetraose peaks ≥4 were used as standards).

[0078] A 0.5M MES pH 5.5 stock was prepared as follows: 4.881 g MES powder (Sigma Aldrich, M8250) was dissolved in 40 ml MilliQ., the pH was adjusted using 10% w / w NaOH, and the volume was made up to 50.0 ml. Diluted Muntons malt extract (Munton's Light Malt Extract, batch XB 35189) was prepared by dissolving 25 g Muntons malt extract in 25 g of 0.5M MES buffer pH 5.5. A 100-fold stock dilution of alpha-glucosidase was prepared by diluting 0.2 g of enzyme (with an activity of 2000 U / g, FoodPro® TGO, Dupont Nutrition Bioscience) in 20 ml MES pH 5.5. Wort samples were prepared by mixing 600 μL of diluted Muntons extract, 100 μL of 0.5M MES pH 5.5, 262.5 μL of MilliQ water and 37.5 μL of diluted alpha-glucosidase (or water as a no enzyme control). Samples were incubated in a thermomixer at 60° C. and 750 rpm to assess accelerated DP sugar conversion. Samples were taken at 30 minute intervals and final enzyme activity was stopped by incubating at 95C for 15 minutes in a thermomixer at 750 rpm, then frozen for HPLC sugar DP analysis.

[0079] The relative distribution of sugars from the HPLC analysis of the wort with alpha-glucosidase is shown in Table 1 below. It can be clearly seen that maltose (DP2) is the major sugar in the malt-based wort. The accelerated conversion of DP sugars in the presence of alpha-glucosidase in the wort (with maltose) clearly demonstrates the production of non-fermentable IMOs (DP3+DP4+) throughout the reaction time (4 hours). The production of IMOs in the malt or during fermentation is not favorable to obtain a high degree of fermentation. Therefore, alpha-glucosidase should not be added in the presence of maltose.

[0080] [Table 1]

[0081] Example 2 - DP sugar HPLC analysis of fermentation broth samples All standards: glucose, maltose, maltotriose and maltotetraose were prepared in double distilled water (ddH20) and filtered through a 0.45 μm syringe filter. A set of each standard was prepared in a concentration range of 10-100,000 ppm. All wort samples containing active enzymes were inactivated by heating the samples to 95°C for 10 min. Subsequently, the wort samples were prepared in 96-well MTP plates (Corning, NY, USA), diluted a minimum of 4-fold in ddH20 and filtered through a 0.20 μm 96-well plate filter (Corning filter plate, PVDF hydrophilic membrane, NY, USA) before analysis. All samples were analyzed in duplicate.

[0082] Quantification of sugars: DP1, DP2, DP3 and DP4+ was performed by HPLC. Sample analysis was performed on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a WPS-3000TSL thermostatic autosampler, a TCC-3000SD thermostatic column oven and a RI-101 refractive index detector (Shodex, JM Science). Chromeleon data system software (Version 6.80, DU10A Build 2826, 171948) was used for data acquisition and analysis.

[0083] Chromatographic conditions Samples were analyzed using an Ag+4% cross-linked (Phenomenex, Netherlands) RSO oligosaccharide column equipped with an analytical guard column (Carbo-Ag+neutral, AJ0-4491, Phenomenex, Netherlands) operated at 70°C. The column was eluted with double distilled water (filtered through a 0.45 μm regenerated cellulose membrane and purged with helium gas) at a flow rate of 0.3 ml / min. An isocratic flow of 0.3 ml / min was maintained throughout the analysis with a total run time of 45 min, and the injection volume was set to 10 μL. Samples were maintained at 20°C in a thermostatic autosampler compartment. The eluent was monitored using a refractive index detector (RI-101, Shodex, JM Science) and quantification was performed by peak area relative to the peak area of ​​a given standard (DP1: glucose; DP2: maltose; DP3: maltotriose and maltotetraose peaks ≥4 were used as standards).

[0084] Example 3 - Reduction in carbohydrate content of beer by conversion of non-fermentable sugars with alpha-glucosidase during beer fermentation The objective of this analysis was to test whether the addition of transglucosidase during fermentation, specifically at low maltose concentrations, could hydrolyze non-fermentable sugars in the wort to fermentable sugars in parallel with ethanol formation by the yeast.

[0085] Wort was prepared from a mashing operation using a water to flour ratio of 3:1 with 50% pilsner malt (Pilsner Malt; Fuglsang Denmark, batch number: 10.12.2019) and 50% corn grits (Nordgetreide GmBH Luebec, Germany, batch number: 02.05.2016.) The pilsner malt was milled in a Buhler Miag mill (0.5 mm setting).

[0086] The corn adjunct was liquefied in the following manner: corn grits (35.0 g), malt (milled Pilsner malt, 5.5 g) and tap water (105 g) were mixed in a mashing bath (Lockner, LG-electronics) cup and adjusted to pH 5.5 with 2.5 M sulfuric acid. AMYLEX® 5T (Dupont Nutrition Bioscience, Denmark) was added at a dosage of 0.25 kg / t flour to facilitate liquefaction.

[0087] The adjuncts were mashed using the following program: heated to 60°C, held at 60°C for 1 minute for mashing, heated to 85°C for 13 minutes by increasing the temperature at 2°C / min, held at 85°C for 30 minutes and mashed off. The adjuncts were then cooled to 64°C and combined with the main mash. LAMINEX® MaxFlow 4G (Dupont Nutrition Bioscience, Denmark) was added at a dose of 0.10 kg / t malt to facilitate filtration. DIAZYME® 87 (Dupont Nutrition Bioscience, Denmark) was added at a dose of 0.0, 1.5 or 3.0 kg / t malt to facilitate saccharification.

[0088] In the main mash, malt (milled Pilsner malt, 35 g) and tap water (105 g) were mixed in a mashing bath (Lochner, LG-electronics) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid. The main mash was heated to 45°C and held at that temperature for 1 minute for mashing, then heated to 63°C and the main mash was combined with the adjuncts (liquefied corn, malt and water); held at 63°C for 120 minutes, heated to 72°C over 9 minutes by increasing the temperature at 1°C / min; held at 72°C for 15 minutes, heated to 78°C over 6 minutes by increasing the temperature at 1°C / min. The mash was finally held at 78°C for 10 minutes and mashed off. Once the temperature reached 72°C, it was tested negative for iodine. The time required to become iodine negative was recorded in minutes.

[0089] At the end of mashing, the mash was built up to 350 g and filtered. After 30 min, the volume of the filtrate was measured. The pH was adjusted to 5.2 with 2.5 M sulfuric acid and one pellet of bitter hops from Hopfenveredlung, St. Johann: 16.0% alpha content (EBC 7.70 specific HPLC analysis, 01.10.2013) was added to each flask (210 g). The wort samples were boiled for 60 min in a boiling bath, the wort was cooled to 17 °C and filtered. 100 g of each wort was weighed into a 500 mL Erlenmeyer flask for fermentation and the wort at 17 °C was added with freshly produced yeast (0.50 g) 0.5% W34 / 70 (Weihenstephan). The remainder of the filtered wort was used for analysis. The wort samples were fermented at 18 °C and 150 rpm after enzyme addition. To ensure a high degree of fermentation and a low content of carbohydrates in the final beer, two different glucoamylases were added at the beginning of the fermentation: DIAZYME® 87 (Dupont Nutrition Bioscience, Denmark) at a volume of 0.5 g / hl and DIAZYME® TGA (Dupont Nutrition Bioscience, Denmark) at a volume of 1.0 g / hl, respectively. The addition of glucoamylase at the beginning of the fermentation was specifically combined with the addition of a low dose of 0.02 U / mL (1.0 g product / hL) of transglucosidase FoodPro® TGO (Dupont Nutrition Bioscience, Denmark) with an activity of 2000 U / g, which was added after 4 days. The enzyme combinations are shown in Table 2 below. All fermentations lasted for 10 days. Analysis was performed when the fermentations were completed.

[0090] [Table 2]

[0091] Wort Analysis: Original Extract (OE), extract in wort samples after mashing was measured using an Anton Paar (Lovis) according to Dupont Standard Instruction Brewing, 23.8580-B28, and fermentable sugars (total %+g / 100ml) which were DP1, DP2, DP3 and DP4+ were determined by HPLC after mashing according to Example 2 above. The relative distribution of sugars in the wort is shown in Table 3 below.

[0092] [Table 3]

[0093] Beer analysis: RDF was measured using an Anton Paar (DMA 5000) according to Standard Instruction Brewing, 23.8580-B28 and alcohol was measured according to Dupont Standard Instruction Brewing, 23.8580-B28. The true degree of fermentation (RDF) can be calculated by the following formula:

number

[0094] Specific gravity and alcohol concentration were determined for fermented samples using a Beer Alcolyzer Plus and a DMA 5000 hydrometer (both from Anton Paar, Gratz, Austria). Based on these measurements, the true degree of fermentation (RDF) was calculated using the following formula:

number

[0095] [Table 4]

[0096] Example 4 - Analysis of non-fermentable sugars in fermentation broth samples by HPLC-PAD The purpose of this analysis was to quantify minor fermentable and non-fermentable sugars upon addition of transglucosidase and glucoamylase during fermentation. Samples were prepared as described in Example 3.

[0097] All standards: glucose, isomaltose, isomaltotriose, maltose, panose, maltotriose, other DP2 and larger sugars DP4+ were prepared in double distilled water (ddH20) and filtered through a 0.45 μm syringe filter. A set of each standard was prepared in the concentration range of 0.5-20 mg / L. All wort samples containing active enzymes were inactivated by heating the samples to 95 °C for 10 min. Samples were then diluted, filtered (0.45 μm Minispike filter) and analyzed. All samples were analyzed in duplicate. Sugar quantification was performed by HPLC-PAD and analysis was performed on a Diones IC system (Thermo Fisher Scientific) equipped with a PAD detector using Chromeleon data system software (version 7.2) for data acquisition and analysis.

[0098] Chromatographic conditions Samples were analyzed using a Carbo PA100 2 mm column (Thermo Fisher Scientific) equipped with a guard column operated at 0.25 mL / min and the elution gradient program shown in Table 5 below.

[0099] [Table 5]

[0100] The injection volume was set to 20 μL and a flow rate of 0.25 ml / min was maintained throughout the analysis (total run time 75 min). Monitoring was performed with a PAD detector (Thermo Fisher Scientific) and quantification was performed by peak area relative to the peak area of ​​a given standard. The concentrations of fermentable and non-fermentable sugars and sugars in the beer samples obtained on the 10th day of fermentation with and without the addition of transglucosidase are shown in Table 6. Surprisingly, on the 4th day of fermentation it could be observed that all non-fermentable sugars, such as isomaltose, isomaltotriose, panose, maltotriose and other DP4+ sugars, in all samples with added transglucosidase (samples 2, 4 and 6) were reduced compared to the corresponding samples without added transglucosidase (samples 1, 3 and 5), respectively. The same was observed for the other DP2 components. The sum of all sugars (fermentable and non-fermentable) was significantly reduced by the addition of transglucosidase added on day 4 of fermentation, e.g., Sample 1 without transglucosidase (1.053%) vs. Sample 2 with transglucosidase (0.253%), Sample 3 without transglucosidase (0.235%) vs. Sample 4 with transglucosidase (0.144%), and Sample 5 without transglucosidase (0.265%) vs. Sample 6 with transglucosidase (0.206%). This is consistent with the increased %RDF and fermentability of the beers produced with transglucosidase, resulting in higher fermentability and lower resulting sugars in the final beer.

[0101] [Table 6]

[0102] Although the above invention has been described in some detail by way of figures and examples for purposes of clarity of understanding, certain changes and modifications can be practiced within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. To the extent the content of any citation, including website or accession number, may change over time, the version in effect on the filing date of this application is intended. Unless otherwise apparent from the context, any step, element, aspect, feature of an embodiment can be used in combination with any other.

[0103] Example 5 - Reduction of beer carbohydrate content by conversion of non-fermentable sugars during beer fermentation with various alpha-glucosidases The objective of this analysis was to test whether the addition of transglucosidase during fermentation, specifically at low maltose concentrations, could hydrolyze non-fermentable sugars in the wort to fermentable sugars in parallel with ethanol formation by the yeast.

[0104] The wort was produced as described in Example 3, but in the absence of saccharification enzymes added to the mashing. To ensure a high fermentation degree and a low content of carbohydrates in the final beer, glucoamylase was added to the wort at the beginning of fermentation: DIAZYME® 87 (Dupont Nutrition Bioscience, Denmark) was added at a volume of 3.0 g / hl. The addition of glucoamylase at the beginning of fermentation was combined with two different transglucosidases: FoodPro® TGO (Dupont Nutrition Bioscience, Denmark) with an activity of 2000 U / g at a volume of 0.01, 0.02 or 0.03 U / mL, or transglucosidase L produced by Amano Pharmaceutical Co., Ltd at a volume of 0.5, 1.0 or 1.5 g / hL, respectively. The transglucosidase was specifically added after 4 days to ensure a low maltose concentration. The enzyme combinations are shown below in Table 7. All fermentations lasted for 10 days. When the fermentations were completed, analyses were performed as described in Example 3.

[0105] It can be seen that the addition of transglucosidase at day 4 of fermentation in all combinations further increased the %RDF by conversion of non-fermentable sugars. Thus, with samples 1 and 2-7, it can be observed that the addition of transglucosidase increased the %RDF from 84.39% to over 85.4%, regardless of the transglucosidase used. With the addition of FoodPro TGO, a dose-response effect was observed, increasing the %RDF from 84.39% to 85.76%. In all cases, an increase in the relative high fermentability was further observed with the increase in the % of alcohol (v / v) due to the addition of transglucosidase. Thus, transglucosidase may be added later in the fermentation to increase the amount of fermentable sugars and thus increase the %RDF and alcohol concentration in the final beer.

[0106] [Table 7]

[0107]

Table 8

Claims

1. A method for increasing the degree of fermentation in a fermented beverage, comprising adding transglucosidase to a maltose-depleted fermentation liquor.

2. The method of claim 1 , wherein the fermented beverage is beer.

3. 3. The method of claim 2, wherein the fermentation broth has a maltose concentration of less than 0.01% (w / w).

4. 4. The method of claim 3, wherein less than 3 U / ml of transglucosidase is added to the fermentation broth.

5. 5. The method of claim 4, wherein less than 1 U / ml of transglucosidase is added to the fermentation broth.

6. 6. The method of claim 5, wherein less than 0.5 U / ml of transglucosidase is added to the fermentation broth.

7. 7. The method of claim 6, wherein the transglucosidase is added after more than 50% of the total fermentation time.

8. 8. The method of claim 7, wherein the beer has an RDF of at least 85%.

9. 9. The method of claim 8, wherein the beer has an RDF of at least 86%.

10. 10. The method of claim 9, wherein the beer has an RDF of at least 87%.

11. 11. The method of claim 10, wherein the beer has an RDF of at least 88%.

12. 12. The method of claim 11, wherein the beer has an RDF of at least 89%.

13. The method of claim 12, wherein the beer has an apparent extract concentration of -0.20% by mass or less.

14. 14. The method of claim 13, wherein the transglucosidase is added to the fermentation broth on the fourth day after the start of fermentation.

15. 15. The method of claim 14, further comprising adding a glucoamylase to the fermentation broth.

16. 16. The method of claim 15, wherein the glucoamylase is added at the beginning of fermentation.

17. 17. The method of claim 16, wherein the transglucosidase comprises a polypeptide having 70% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

18. 18. The method of claim 17, wherein the polypeptide has 75% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

19. 19. The method of claim 18, wherein the polypeptide has 80% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

20. 20. The method of claim 19, wherein the polypeptide has 85% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

21. 21. The method of claim 20, wherein the polypeptide has 90% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

22. 22. The method of claim 21, wherein the polypeptide has 95% or more sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

23. 23. The method of claim 22, wherein the polypeptide has 99% or greater sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.

24. 24. The method of claim 23, wherein the polypeptide has 100% sequence identity to SEQ ID NO:1 or a transglucosidase active fragment thereof.