PRODUCTION OF BREWER'S WORT HAVING INCREASED FERMENTABLE SUGARS

BACKGROUND OF THE INVENTION

In the production of beer, during the mashing step, fermentable sugars are generated from starch which are subsequently converted into ethanol by yeast. However, not all starch is converted into fermentable sugars. Short glucose oligomers remain after mashing which cannot be converted into ethanol by brewer's yeast. These oligomers, also called dextrins, are usually 4 sugar units and above (DP4+). After lautering of the mash, the wort typically contains about 65 to 80% fermentable sugar and 20 to 35% soluble dextrin which is not fermentable, i.e., will not be converted into ethanol. The presence of non-fermentable sugars in a beer causes a beer to have more calories than a beer fermented from a wort having more fermentable sugars.

There remains a need in the art for methods to produce worts having less non-fermentable sugars and more fermentable sugars to produce beers with a lower calorie content.

SUMMARY OF THE INVENTION

In an aspect of the present invention, a method for producing a brewer's wort having increased DPI sugar is presented having the step of adding to a mash having a grist a first glucoamylase and a second glucoamylase wherein the first glucoamylase is not the same as the second glucoamylase to produce the brewer's wort.

Optionally, the first and said second glucoamylase are obtained from a filamentous fungus.

Optionally, the filamentous fungus is selected from the group consisting of Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,

Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,

Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, and Xylaria.

Optionally, the first and second glucoamylases are from different filamentous fungi.

Optionally, the filamentous fungus is selected from the group consisting of Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi , Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum,

Phanerochaete chrysosporium, Penicillium thomii, Penicillium oxalicum, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

Optionally, the filamentous fungus is selected from the group consisting of Aspergillus fumigatus, Fusarium venenatum, and Trichoderma reesei.

Optionally, the first and/or said second glucoamylases are variants of a glucoamylase from Aspergillus fumigatus, Fusarium venenatum, and Trichoderma reesei.

Optionally, the first and second glucoamylase have 60% or less sequence identity.

Optionally, the first and second glucoamylase have 58% or less sequence identity.

Optionally, the first and second glucoamylase have 56% or less sequence identity.

Optionally, the first and second glucoamylase have 54% or less sequence identity.

Optionally, the first and second glucoamylase have 52% or less sequence identity.

Optionally, the first and said glucoamylase have 50% or less sequence identity. Optionally, the first and second glucoamylases are selected from the group consisting of a Fusarium venenatum glycoamylase as set forth in SEQ ID NO: 1 (FvGA) or a sequence having at least 80% identity thereto, a Aspergillus fumigatus glucoamylase as set forth in SEQ ID NO: 2 (AfuGA) or a sequence having at least 80% identity thereto, a Trichoderma reesei glucoamylase as set forth in SEQ ID NO: 3 (TrGA) or a sequence having at least 80% identity thereto, a first Trichoderma reesei variant glucoamylase as set forth in SEQ ID NO: 4 (TrGA varl) or a sequence having at least 80% identity thereto and a second Trichoderma reesei variant glucoamylase as set forth in SEQ ID NO: 5 TrGA_var2 or a sequence having at least 80% identity thereto.

Optionally, the first and second glucoamylases are selected from the group consisting of

FvGA as set forth in SEQ ID NO: 1 or a sequence having at least 90% identity thereto, AfuGA as set forth in SEQ ID NO: 2 or a sequence having at least 90% identity thereto, TrGA as set forth in SEQ ID NO: 3 or a sequence having at least 90% identity thereto, TrGA varl as set forth in SEQ ID NO: 4 or a sequence having at least 90% identity thereto and TrGA_var2 as set forth in SEQ ID NO: 5 or a sequence having at least 90% identity thereto.

Optionally, the first and second glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1 or a sequence having at least 95% identity thereto, AfuGA as set forth in SEQ ID NO: 2 or a sequence having at least 95% identity thereto, TrGA as set forth in SEQ ID NO: 3 or a sequence having at least 95% identity thereto, TrGA varl as set forth in SEQ ID NO: 4 or a sequence having at least 95% identity thereto and TrGA_var2 as set forth in SEQ ID NO: 5 or a sequence having at least 95% identity thereto.

Optionally, the first and second glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1 or a sequence having at least 99% identity thereto, AfuGA as set forth in SEQ ID NO: 2 or a sequence having at least 99% identity thereto, TrGA as set forth in SEQ ID NO: 3 or a sequence having at least 99% identity thereto, TrGA varl as set forth in SEQ ID NO: 4 or a sequence having at least 99% identity thereto and TrGA_var2 as set forth in SEQ ID NO: 5 or a sequence having at least 99% identity thereto

Optionally, the first and second glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1, AfuGA as set forth in SEQ ID NO: 2, TrGA as set forth in SEQ ID NO: 3, TrGA varl as set forth in SEQ ID NO: 4 and TrGA_var2 as set forth in SEQ ID NO: 5. Optionally, the first and said second glucoamylases are other than TrGA and TrGA varl, TrGA varl and TrGA_var2 or TrGA and TrGA_var2.

Optionally, the first and said second glucoamylases comprise TrGA and FvGA, AFuGA and FvGA, TrGA and AFuGA or TrGA_var2 and FvGA.

Optionally, the first and said second glucoamylases are TrGA and FvGA, AFuGA and FvGA, TrGA and AFuGA or TrGA_var2 and FvGA.

Optionally, the first and second glucoamylases have a predetermined ratio.

Optionally, the first and second glucoamylases are TrGA and FvGA having a mass ratio of about 3 :2 or greater. Optionally, the mass ratio is about 4: 1.

Optionally, the first and second glucoamylases are AfuGA and FvGA having a mass ratio of about 3 :2 or greater. Optionally, the mass ratio is about 4: 1.

Optionally, the first and second glucoamylases are TrGA_var2 and FvGA having a mass ratio of about 3 :2 or greater. Optionally, the mass ratio is about 4: 1.

Optionally, the first and second glucoamylases are TrGA and AFuGA having a mass ratio of about 1 : 1 or less. Optionally, the mass ratio is about 2:3.

Optionally, the method of producing a brewer's wort with higher DPI has the further step of adding an enzyme selected from the group consisting of a pullulanase, a protease, a xylanase, a lipase, a cellulase, an amylase, a glucoamylase and a beta glucanase.

Optionally, the wort has less than about 4% DP4/4+, less than about 3.8% DP4/4+, less than about 3.5% DP4/4+ or less than about 3% DP4/4+.

Optionally, the grist contains malt.

Optionally, the grist further contains corn, rice, sorghum, barley, wheat, rye, oats or tapioca or mixtures thereof.

Optionally, the brewer' s wort has about 93%> or greater DP 1.

Optionally, the brewer's wort has about 94%> or greater DPI .

Optionally, the brewer's wort is fermented to produce a beer. Optionally, the beer has an RDF of greater than 84%>. Optionally, the beer has an RDF of greater than 85%>. Optionally, the beer has an RDF of greater than 86%>. Optionally, the beer has an RDF of greater than 87%>. Optionally, the beer has an RDF of greater than 88%>. Optionally, the beer has an RDF of greater than 89%. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a graph with the concentration of DPI sugars obtained in the wort (in % of all sugar) as function of the glucoamylase dosage in ppm. Wort was analyzed after mashing 240 minutes at 64°C. The relative concentration of DPI sugars in the wort is shown in the region from 90-97%. Three glucoamylase preparations were applied: AfuGA (filled triangles), FvGA (filled circles) and 1 : 1 AfuGA:FvGA (filled squares).

BRIEF DESCRIPTION OF SEQ ID NOs

SEQ ID NO: 1 sets forth the mature amino acid sequence of the glucoamylase from Fusarium verticillioides (FvGA).

SEQ ID NO: 2 sets forth the mature amino acid sequence of the glucoamylase from Aspergillus fumigatus (AfuGA).

SEQ ID NO: 3 sets forth the mature amino acid sequence of the glucoamylase from Trichoderma reesie (TrGA).

SEQ ID NO: 4 sets forth the mature amino acid sequence of the glucoamylase variant from Trichoderma reesie including the mutations L417V, T430A, Q511H, A539R and N563I (TrGA varl).

SEQ ID NO: 5 sets forth the mature amino acid sequence of the glucoamylase variant from Trichoderma reesie including the mutations D44R and A539R (TrGA_var2).

DEFINITIONS and ABBREVIATIONS

Unless defined otherwise, 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 ^nd ed., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS

DICTIONARY OF BIOLOGY, Harper Perennial, N. Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference. 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.

A "variant" or "variants" refers to either polypeptides or nucleic acids. 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 phrases "variant polypeptide", "polypeptide variant", "polypeptide", "variant" and "variant enzyme" mean a polypeptide/protein that has an amino acid sequence that either has or comprises a selected amino acid sequence of or is modified compared to the selected amino acid sequence, such as SEQ ID NO: 1, 2, 3, 4 or 5.

As used herein, a "homologous sequence" and "sequence identity" with regard to a nucleic acid or polypeptide sequence 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 sequence or polypeptide sequence when optimally aligned for comparison, wherein the function of the candidate nucleic acid sequence or polypeptide sequence is essentially the same as the nucleic acid sequence or polypeptide sequence the candidate homologous sequence is being compared with. In some embodiments, homologous sequences have between at least about 85% and 100%) sequence identity, while in other embodiments there is between about 90% and 100% sequence identity, and in other embodiments, there is at least about 95% and 100% sequence identity.

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)).

The "percent (%) nucleic acid sequence identity" or "percent (%) amino acid sequence identity" is defined as the percentage of nucleotide residues or amino acid residues in a candidate sequence that is identical with the nucleotide residues or amino acid residues of the starting sequence. The sequence identity can be measured over the entire length of the starting sequence Homologous sequences are determined by known methods of sequence alignment. 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 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. The adjustable parameters are set with 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 depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

Other methods find use in aligning sequences. One example of a useful algorithm is

PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise 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: 151-153 (1989)). Useful PILEUP parameters including 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 giving the highest percent identity score.

As used herein, the term "malt beverage" includes such foam forming fermented malt beverages as full malted beer, ale, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic malt liquor and the like. The term "malt beverages" also includes alternative malt beverages such as fruit flavored malt beverages, e. g. , citrus flavored, such as lemon-, orange-, lime-, or berry-flavored malt beverages, liquor flavored malt beverages, e. g. , vodka-, rum-, or tequila-flavored malt liquor, or coffee flavored malt beverages, such as caffeine-flavored malt liquor, and the like. As used herein, the term "beer" traditionally refers to an alcoholic beverage derived from malt, which is derived from barley, and optionally adjuncts, such as cereal grains, and flavored with hops. Beer can be made from a variety of grains by essentially the same process. All grain starches are glucose homopolymers in which the glucose residues are linked by either alpha- 1, 4- or alpha-l,6-bonds, with the former predominating. The process of making fermented malt beverages is commonly referred to as brewing. The principal raw materials used in making these beverages are water, hops and malt. In addition, adjuncts such as common corn grits, refined corn grits, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like may be used as a source of starch or fermentable sugar types. The starch will eventually be converted into dextrins and fermentable sugars. For a number of reasons, the malt, which is produced principally from selected varieties of barley, has the greatest effect on the overall character and quality of the beer. First, the malt is the primary flavoring agent in beer. Second, the malt provides the major portion of the fermentable sugar. Third, the malt provides the proteins, which will contribute to the body and foam character of the beer. Fourth, the malt provides the necessary enzymatic activity during mashing.

As used herein, the term "Hops" refers to it use in contributing significantly to beer quality, including flavoring. In particular, hops (or hops constituents) add desirable bittering substances to the beer. In addition, the hops act as protein precipitants, establish preservative agents and aid in foam formation and stabilization.

As used herein, the "process for making beer" is one that is well known in the art, but briefly, it involves five steps: (a) adjunct cooking and/or mashing (b) wort separation and extraction (c) boiling and hopping of wort (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 controlled temperatures to permit the enzymes present in the malt to e.g. 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 left over grain residue is called "spent grain". The mash is typically subjected to an extraction during mash separation, which involves adding water to the mash in order to recover the residual soluble extract from the spent grain. In the third step, the wort is boiled vigorously. This sterilizes the wort and helps to develop the colour, flavour and odour. Hops are added at some point during the boiling. In the fourth step, the wort is cooled and transferred to a fermentor, which either contains the yeast or to which yeast is added. The yeast converts the sugars by fermentation into alcohol and carbon dioxide gas; at the end of fermentation the fermentor is chilled or the fermentor may be chilled to stop fermentation. The yeast flocculates and is removed. In the last step, the beer is cooled and stored for a period of time, during which the beer clarifies and its flavor develops, and any material that might impair the appearance, flavor and shelf life of the beer settles out. Prior to packaging, the beer is carbonated and, optionally, filtered and pasteurized. After fermentation, a beverage is obtained which usually contains from about 2% to about 10% alcohol by weight. The non- fermentable carbohydrates are not converted during fermentation and form the majority of the dissolved solids in the final beer. This residue remains because of the inability of malt enzymes to hydrolyze the alpha- 1,6-linkages of the starch and fully degrade the non-starch

polysaccharides. The non-fermentable carbohydrates contribute with less than 50 kilocalories per 12 ounces of a lager beer.

As used herein, the term "light beers, reduced calorie beers or low calorie beers", refers to the recent, widespread popularization of brewed beverages, particularly in the U. S.

market.FDA defines "light" foods as having 1/3 fewer calories than a standard reference food/drink, according to this definition these highly attenuated beers have approximately 30% fewer calories than a manufacturer's "normal" beer." Further information on conventional brewing processes may 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."

As used herein, the "process for making beer" may further be applied in the mashing of any grist.

As used herein, the term "grist" refers to any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers, roots, stems, leaves and seeds. The grist may comprise grain, such as grain from barley, wheat, rye, oat, 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 grist of the wort is derived from grain. In some

embodiments the grist may comprise the starch and/or sugar containing plant material obtained from cassava [Manihot esculenta] roots. The grist may comprise malted grain, 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 of the wort is derived from malted grain.

The term "fermentation" means, in the context of brewing, the transformation of sugars in the wort, by enzymes in the brewing yeast, into ethanol and carbon dioxide with the formation of other fermentation by-products.

As used herein the term "malt" is understood as any malted cereal grain, such as barley.

The term "adjunct" is understood as the part of the grist which is not barley malt. The adjunct may be any carbohydrate rich material. In term "adjunct" includes the starch and/or sugar containing plant material obtained from cassava [Manihot esculenta] roots.

The term "mash" is understood as aqueous starch slurry, e. g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort + spent grains.

As used herein, the term "wort" refers to the unfermented liquor run-off following extracting the grist during mashing.

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

As used herein, the term "beer" refers to fermented wort, e.g. an alcoholic beverage brewed from barley malt, optionally adjunct and hops.

As used herein, the term "extract recovery" in the wort is defined as the sum of soluble substances extracted from the grist (malt and adjuncts) expressed in percentage based on dry matter.

As used herein, the term "pasteurisation" means the killing of micro-organisms in aqueous solution by heating. Implementation of pasteurisation in the brewing process is typically through the use of a flash pasteuriser or tunnel pasteuriser. As used herein, the term

"pasteurisation units or PU" refers to a quantitative measure of pasteurisation. One pasteurisation unit (1 PU) for beer is defined as a heat retention of one minute at 60 degrees Celsius. One calculates that:

PU = t x 1.393^(T - 60), where:

t = time, in minutes, at the pasteurisation temperature in the pasteuriser

T = temperature, in degrees Celsius, in the pasteuriser

[^(T -60) represents the exponent of (T-60)]

Different minimum PU may be used depending on beer type, raw materials and microbial contamination, brewer and perceived effect on beer flavor. Typically, for beer pasteurisation, of a lager beer 15-25 PU are required. Depending on the pasteurising equipment, pasteurisation temperatures are typically in the range of 64 - 72 degrees Celsius with a pasteurisation time calculated accordingly. Further information may 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.

As used herein, the term "DPI" (degree of polymerization 1) means glucose or fructose. "DP2" denotes maltose and/or isomaltose. "DP3" means maltotriose, panose and isopanose. "DP4/4+" means dextrin or maltooligosaccharides of a polymerization degree of 4 or higher which are unfermentable.

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 stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

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.

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.

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.

As used herein, " Trichoderma reesef refers to a filamentous fungus of the phylum Ascomycota, subphylum Pezizomycotina. This organism was previously classified as

Trichoderma longibrachiatum, and also as Hypocrea jecorina

Abbreviations

In the disclosure and experimental section which follows, the following abbreviations apply: GA (glucoamylase); GAU (glucoamylase unit); wt % (weight percent); °C (degrees Centigrade); rpm (revolutions per minute); H2O (water); dH ₂ 0 (deionized water); aa or AA (amino acid); kD (kilodaltons); g or gm (grams); μg (micrograms); mg (milligrams);

\ L (microliters); ml and mL (milliliters); mm (millimeters); μm (micrometer); M (molar); mM (millimolar); μΜ (micromolar); U (units); V (volts); MW (molecular weight); sec(s) or s(s) (second/seconds); min(s) or m(s) (minute/minutes); hr(s) or h(s) (hour/hours); ABS

(Absorbance); EtOH (ethanol); and MTP (microtiter plate); N (Normal); ppm (parts per million).

DETAILED DESCRIPTION OF THE INENTION

General

A look-up in the UniProt (http ://w\vw.uniprot. org/) database revealed only a single annotated glucoamylase [alpha-(l,4)-D-glucan glucohydrolase, EC 3.2.1.3, GA] (Coutinho and Reilly, 1997) gene entry for the following fungi: Fusarium verticillioides, Aspergillus fumigatus and Trichoderma reesei (see table 1).

Table 1 List of fungal glucoamylases

The major commercial application of glucoamylase is to catalyze starch saccharification to yield glucose for use in food and fermentation industries. Glucose production from starch, along with glucoamylase requires the synergistic action of a series of amylases. Additionally, debranching enzymes (pullulanase or isoamylase) are used to hasten starch processing by cleaving a- 1,6 glycosidic bonds, which allows to attain an early peak in glucose yield with less byproduct formation (Reilly, 2006).

Industrial use of GAs confronts a major problem at high dissolved solids concentrations, here it does not yield all glucose, but instead condenses some of the glucose to various di-, tri-, and tetra-saccharides, where isomaltose [a-D-glucopyranosyl-(l→6)-D-glucose] previously was speculated to be the most important of them (Reilly, 1999). Previously also Ford et al. (1998) successfully showed increased GA selectivity and glucose yield by a series of single and multiple site-directed mutations in the GA catalytic domain. The aim was to modify the dimensions of the active site just enough to make it difficult to bind isomaltose, the speculated most prevalent by product, while not affecting the binding of maltose. This, however also resulted in lowering the specific catalytic efficiency for a-1,6 glyosidic bonds important for the starch processing.

Besides iso-maltose many other non-fermentable smaller saccharides may be formed by enzymatic reversion such as: panose, nigerose, kojibiose, isomaltotriose, isomaltotetraose, isomaltopentaose, In the present application inventors propose the use of several GA's with inherent different specificity to obtain synergistically high catalytic efficiency and low level of residual non-fermentable sugars. Glucoamylases from different sources is known to have various degree of condensation and specificity. As shown by present inventors the right combination of glucoamylase with different specificity may lower the total sum of non-fermentable sugars, including enzymatically produced condensation products such as, iso-maltose, panose, nigerose, kojibiose, isomaltotriose and so forth. Lowering the amount of non-fermentable sugars may result in production of even higher attenuated beers. In brewing, attenuation is the percentage that measures the conversion of sugars into alcohol and carbon dioxide by the fermentation process; a more attenuated beer will generally be drier and more alcoholic than a less attenuated beer made from the same wort. This may also be expressed in the brewing term RDF, real degree of fermentation. RDF measures the degree to which sugar in wort has been fermented into alcohol in beer, defined as attenuation. The higher the RDF percentage, the lighter and drier the beer. As seen from examples 6 in the present application there is a very good correlation between the obtained DP1% and resulting RDF.

Recently, there has been a widespread popularization of brewed beverages called light beers, reduced calorie beers or low-calorie beers, particularly in the U.S. market. As defined in the U.S., these beers have 50% or fewer calories than a manufacturer's "normal" beer.

As used herein, the term "light beers, reduced calorie beers or low-calorie beers", refers to the recent, widespread popularization of brewed beverages, particularly in the U.S. market. As defined in the U.S., these highly attenuated beers have approximately 50% fewer calories than a manufacturer's "normal" beer." Further information on conventional brewing processes may 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.

Several types of ultra-light beers have less than 80 kilocalories (per 12 oz.), such as Budweiser Select 55, Miller Genuine Draft 64. Reducing the non-fermentable sugars further in mashing, will enable fermentations to higher RDF and production of even higher attenuated beer, with less calories.

In accordance with an aspect of the present invention, it has been discovered that a brewer's wort with high percentage of fermentable DPI sugar can be produced using

combination of glucoamylases. In accordance with an aspect of the present invention, a method is presented for producing a brewer's wort having increased DPI sugar having the steps of adding to a mash comprising a grist a first glucoamylase and a second glucoamylase wherein the first glucoamylase is not the same as the second glucoamylase to produce the brewer's wort. Preferably, the first and the second glucoamylase are obtained from a filamentous fungus. More preferably, the filamentous fungus is selected from the group consisting of Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,

Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,

Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,

Trichoderma, Trichophaea, Verticillium, Volvariella, and Xylaria.

In still more preferred aspects of the present invention, the first and second

glucoamylases are from different filamentous fungi. Preferably, the filamentous fungus is selected from the group consisting of Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium,

Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum,

Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi , Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,

Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea,

Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum,

Phanerochaete chrysosporium, Penicillium thomii, Penicillium oxalicum, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride. Still more preferably, the filamentous fungus is selected from the group consisting of Aspergillus fumigatus, Fusarium venenatum, and Trichoderma reesei. In yet another aspect of the present invention, the first and/or the second glucoamylases are variants of a glucoamylase derived from a filamentous fungus. Preferably, the variant glucoamylases are derived from Aspergillus fumigatus, Fusarium venenatum, and Trichoderma reesei. Still more preferably, the variant glucoamylase is derived from Trichoderma reesei.

In another preferred aspect of the present invention, the first and second glucoamylase have 60% or less sequence identity. More preferably, the first and second glucoamylase have 58%) or less sequence identity. Still more preferably, the first and second glucoamylase have 56%> or less sequence identity. Still more preferably, the first and second glucoamylase have 54% or less sequence identity. Still more preferably, the first and said second glucoamylase have 52% or less sequence identity. In a still more preferred aspect of the present invention the first and second glucoamylase have 50% or less sequence identity.

In another aspect of the present invention, the first and second glucoamylases are selected from the group consisting of a Fusarium venenatum glycoamylase as set forth in SEQ ID NO: 1 (FvGA) or a sequence having at least 80%> identity thereto, a Aspergillus fumigatus

glucoamylase as set forth in SEQ ID NO: 2 (AfuGA) or a sequence having at least 80%> identity thereto, a Trichoderma reesei glucoamylase as set forth in SEQ ID NO: 3 (TrGA) or a sequence having at least 80%> identity thereto, a first Trichoderma reesei variant glucoamylase as set forth in SEQ ID NO: 4 (TrGA varl) or a sequence having at least 80%> identity thereto and a second Trichoderma reesei variant glucoamylase as set forth in SEQ ID NO: 5 TrGA_var2 or a sequence having at least 80%> identity thereto.

More preferably, the first and second glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1 or a sequence having at least 90% identity thereto, AfuGA as set forth in SEQ ID NO: 2 or a sequence having at least 90% identity thereto, TrGA as set forth in SEQ ID NO: 3 or a sequence having at least 90% identity thereto,

TrGA varl as set forth in SEQ ID NO: 4 or a sequence having at least 90% identity thereto and TrGA_var2 as set forth in SEQ ID NO: 5 or a sequence having at least 90% identity thereto.

Till more preferably, the first and second glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1 or a sequence having at least 95% identity thereto, AfuGA as set forth in SEQ ID NO: 2 or a sequence having at least 95% identity thereto, TrGA as set forth in SEQ ID NO: 3 or a sequence having at least 95% identity thereto, TrGA varl as set forth in SEQ ID NO: 4 or a sequence having at least 95% identity thereto and TrGA_var2 as set forth in SEQ ID NO: 5 or a sequence having at least 95% identity thereto. 17. The method of claim 16 wherein said first and second glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1 or a sequence having at least 99% identity thereto, AfuGA as set forth in SEQ ID NO: 2 or a sequence having at least 99% identity thereto, TrGA as set forth in SEQ ID NO: 3 or a sequence having at least 99% identity thereto, TrGA varl as set forth in SEQ ID NO: 4 or a sequence having at least 99% identity thereto and TrGA_var2 as set forth in SEQ ID NO: 5 or a sequence having at least 99% identity thereto In still more preferred aspects of the present invention, the first and second

glucoamylases are selected from the group consisting of FvGA as set forth in SEQ ID NO: 1, AfuGA as set forth in SEQ ID NO: 2, TrGA as set forth in SEQ ID NO: 3, TrGA varl as set forth in SEQ ID NO: 4 and TrGA_var2 as set forth in SEQ ID NO: 5. In yet more preferred aspects of the present invention, the first and said second glucoamylases are other than TrGA and TrGA varl, TrGA varl and TrGA_var2 or TrGA and TrGA_var2. In still a more preferred aspect of the present invention, the first and said second glucoamylases comprise TrGA and FvGA, AFuGA and FvGA, TrGA and AFuGA or TrGA_var2 and FvGA.

In another aspect of the present invention, the first and said second glucoamylases have a predetermined ratio. Preferably, the first and second glucoamylases are TrGA and FvGA having a mass ratio of about 3 :2 or greater. More preferably, the mass ratio is about 4: 1. In another preferred embodiment, the first and second glucoamylases are AfuGA and FvGA having a mass ratio of about 3 :2 or greater. More preferably, the mass ratio is about 4: 1. In another preferred embodiment, the first and second glucoamylases are TrGA_var2 and FvGA having a mass ratio of about 3 :2 or greater. More preferably, the mass ratio is about 4: 1. In yet another preferred embodiment, the first and second glucoamylases are TrGA and AFuGA having a mass ratio of about 1 : 1 or less. More preferably, the mass ratio is about 2:3.

In another preferred aspect of the present invention, the method for producing a brewer's wort having increase DPI, further comprises adding an enzyme selected from the group consisting of a pullulanase, a protease, a xylanase, a lipase, a cellulase, an amylase, a third glucoamylase and a beta glucanase. Preferably, the third glucoamylase is different than the first and second glucoamylases described herein. More preferably, the first glycoamylase is a

Fusarium venenatum glycoamylase as set forth in SEQ ID NO: 1 (FvGA) or a sequence having at least 80% identity thereto, the second glucoamylase is an Aspergillus fumigatus glucoamylase as set forth in SEQ ID NO: 2 (AfuGA) or a sequence having at least 80% identity thereto, and the third glycoamylase is a Trichoderma reesei variant glucoamylase as set forth in SEQ ID NO: 4 (TrGA varl) or a sequence having at least 80% identity thereto. Still more preferably, the first glycoamylase is FvGa, the second glucoamylase is AfuGA and the third glucoamylase is TrGA varl

In another aspect of the present invention, the brewer's wort has less than about 4% DP4/4+. More preferably, the brewer's wort has less than about 3.8% DP4/4+. Still more preferably, the brewer's wort has less than about 3.5% DP4/4+. In the most preferred aspect of the present invention, the brewer' s wort has about 3% DP4/4+.

In still another aspect of the present invention, the grist contains malt. More preferably, the grist further comprises corn, rice, sorghum, barley, wheat, rye, oats or tapioca or mixtures thereof.

In still another aspect of the present invention, the brewer's wort has about 93% or greater DP 1. More preferably, the brewer' s wort has about 94% or greater DP 1.

In still another aspect of the present invention, the method further comprises fermenting the brewer's wort to produce a beer. Preferably, the beer has an RDF of greater than 84%. Still more preferably, the beer has an RDF of greater than 85%. Still more preferably, the beer has an RDF of greater than 86%. Still more preferably, the beer has an RDF of greater than 87%. Still more preferably, the beer has an RDF of greater than 88%. Still more preferably, the beer has an RDF of greater than 89%.

To be effective in the instantly claimed invention, the glucoamylase should be chosen such that it retains sufficient activity at the process temperatures of the claimed process and retain sufficient activity under the moderately acidic pH conditions of a typical mash. In addition, the glucoamylases must be employed in sufficient quantity to be effective in accordance with the present invention.

In accordance with the present invention, glucoamylases can be produced directly from the organism giving in which they naturally reside. Alternatively, common techniques of molecular biology may be employed to clone the gene sequence corresponding to the

glucoamylase and then to inserted into an organism for heterologous expression. Glucoamylases (GAs) are produced by numerous strains of bacteria, fungi, yeast and plants. Many fungal glucoamylases are secreted from the cell, for example from strains of Aspergillus (Svensson et al., Carlsberg Res. Commun. 48: 529-544 (1983); Boel et al., EMBO J. 3 : 1097-1 102 (1984); Hayashida et al., Agric. Biol. Chem. 53 : 923-929 (1989); U.S. Patent No. 5,024,941 ; U.S. Patent No. 4,794,175 and WO 88/09795); Talaromyces (U.S. Patent No. 4,247,637; U.S. Patent No. 6,255,084; and U.S. Patent No. 6,620,924); Rhizopus (Ashikari et al., Agric. Biol. Chem. 50: 957-964 (1986); Ashikari et al., App. Microbio. Biotech. 32: 129-133 (1989) and U.S. Patent No. 4,863,864); Humicola (WO 05/052148 and U.S. Patent No. The isolation, cloning and expression of the TrGA are described in WO 2006/060062 and U.S. Patent No. 7,413,887, similar procedures may be used to construct heterologous GA expression hosts of other fungal origin.

In an aspect of the present invention, the concentration of glucoamylase added is between about 0.01 mg to about 2.0 mg per gram of total grist. More preferably, the concentration of glucoamylase added is between about 0.1 mg to about 0.5mg per gram of total grist.

The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.

Example 1 - Production of glucoamylases in Trichoderma reesie

Production of TrGA and variants hereof in Trichoderma reesie may essential be carried out described previously in WO2014029808. Heterologous expression of FvGA and AfuGA may be carried as previously described for MkGA in WO2013092840. Culturing Trichoderma reesie for glucoamylase expression may be carried essential as described below:

400x Trace element solution: Dilute in 1000 ml of demi water: Anhydrous Citric Acid (175 g), FeS0 ₄ *7 H ₂ 0 (200g), ZnS0 ₄ *7 H2O (16g), CuS0 ₄ *5 H2O (3.2g), MnS0 ₄ *H ₂ 0 (1.4g), H3BO3 (0.8g). It may be helpful to acidify this to get all components into solution. The solution was filtered and sterilized. LD-medium: Add to ~ 800 ml of demi water: Casamino acids (9g), MgS04*7H20 (lg),

( H ₄ ) ₂ S04 (5g), KH2PO4 (4.5g), CaCl ₂ *2H ₂ 0 (lg), Piperazine-l,4-bis-propanesulfonic acid (PIPPS) buffer (33g), 400x T. reesei trace elements (2.5ml), Adjust pH to 5.5 with NaOH 4N. Adjust final volume to 920 ml. 2xAmdSBase ager (1 litre): Mix KH2PO4 (30 g), 1M Acetamide (20 ml), 1M CsCl (20 ml), 20% MgS04.7H ₂ 0 (6 ml), 20% CaCl ₂ .2H ₂ 0 (6 ml), T. reesei spore elements 400x (2 ml), 50% glucose.H ₂ 0 (80 ml). Adjust pH to 4.5 with 4N NaOH Make up to 1 L and filter sterilize. Store at 4°C.

Initial culture: Trichoderma reesei strains were grown on AmdS-Base agar plates. To produce agar plates minimal media agar was boiled and after cooling down to app. 50°C it was diluted with 2x AmdS Base 1 : 1 and poured on petri dishes. After sporulation (app. 6-7 days) the plates were scraped with 2 ml saline 0.015% Tween 80. Approx 1 ml was added to glycerol tubes containing 500-600 μΐ 35% glycerol and stored at -80°C. The pre-culture fermentations were started directly from this spore suspension. Pre culture: The medium is made by adding 2.5% glucose to the LD-medium, which is subsequently made up to 1 L. To produce biomass 50 μΐ spore suspension is added to 100 ml medium (sterilised in 500 ml shake flask). The flasks are incubated on a rotary shaker at 30°C, 180 rpm for 2 days, then 10 ml suspension is used to inoculate a new baffled shake flask, which is incubated under similar conditions for 1 day. The content of this flask is used to inoculate a fermentor. Alternatively, fermentation of the pre-culture was initiated by a piece (~1 cm ² ) of a fresh PDA plate with T. reesei.

Main culture : To make 1 L of medium, 40 ml glucose/sophorose mix (Danisco, Jamsa, Finland) was added to the LD-medium and made up to 1 L. 6 L fermentors containing 4 L of medium were inoculated with the pre-culture, and grown at pH 3.5 for approximately 16 hours at 34°C, until CER/OUR (Carbondioxide Excretion Rate/Oxygen Uptake Rate) started falling. Then temperature was lowered to 28°C, pH was raised to 5.5 and the fermentation was continued for approximately 80 hours. The cell culture is harvested and media clarified by centrifugation (4000 rpm at 25 min.) and filtration (VacuCap 90, 0.2 μm). Following, the ferment was concentrated and stored at -20°C. Example 2 - Protein Determination Methods

Protein Determination by Stain Free Imager Criterion

[0001] Protein was quantified by SDS-PAGE gel and densitometry using Gel Doc™ EZ imaging system. Reagents used in the assay: Concentrated (2x) Laemmli Sample Buffer (Bio- Rad, Catalogue #161-0737); 26-well XT 4-12% Bis-Tris Gel ( Bio-Rad, Catalogue #345-0125); protein markers "Precision Plus Protein Standards" (Bio-Rad, Catalogue #161- 0363); protein standard BSA (Thermo Scientific, Catalogue #23208) and SimplyBlue Safestain (Invitrogen, Catalogue #LC 6060. The assay was carried out as follow: In a 96well-PCR plate 50μΙ. diluted enzyme sample were mixed with 50 μL, sample buffer containing 2.7 mg DTT. The plate was sealed by Microseal 'B' Film from Bio-Rad and was placed into PCR machine to be heated to 70°C for 10 minutes. After that the chamber was filled by running buffer, gel cassette was set. Then 10 μL, of each sample and standard (0.125-1.00 mg/mL BSA) was loaded on the gel and 5 μL, of the markers were loaded. After that the electrophoresis was run at 200 V for 45 min.

Following electrophoresis the gel was rinsed 3 times 5 min in water, then stained in Safestain overnight and finally destained in water. Then the gel was transferred to Imager. Image Lab software was used for calculation of intensity of each band. By knowing the protein amount of the standard sample, the calibration curve can be made. The amount of sample can be determined by the band intensity and calibration curve. The protein quantification method was employed to prepare samples of glucoamylase enzyme used for assays shown in subsequent Examples.

Example 3 - Sugar analysis by HPLC

All standards: Glucose, Maltose, Maltotriose and Maltotetraose were prepared in double distilled water (ddH20) and filtered through 0.45 μm syringe filters. A set of each standard was prepared ranging in concentration from 10 to 100,000 ppm.

All wort samples containing active enzymes were inactivated by heating the sample to 95°C for 10 min. Subsequently wort samples were prepared in 96 well MTP plates (Corning, NY, USA) and diluted minimum 4 times in ddH20 and filtered through 0.20 μm 96 well plate filters before analysis (Corning filter plate, PVDF hydrophile membrane, NY, USA). All samples were analyzed in duplicates. Instrumentation

Quantification of sugars: DPI, DP2, DP3, DP4 and DP5+ were performed by HPLC. Analysis of samples was carried out on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a DGP-3600SD Dual-Gradient analytical pump, WPS-3000TSL thermostated autosampler, TCC-3000SD thermostated column oven, and a RI-101 refractive index detector (Shodex, JM Science). Chromeleon datasystem software (Version 6.80, DU10A Build 2826, 171948) was used for data acquisition and analysis. Chromatographic conditions

The samples were analyzed using a RSO oligosaccharide column, Ag ⁺ 4% crosslinked (Phenomenex, The Netherlands) equipped with an analytical guard column (Carbo-Ag ⁺ neutral, AJO-4491, Phenomenex, The Netherlands) operated at 70°C. The column was eluted with double distilled water (filtered through a regenerated cellulose membrane of 0.45 μm and purged with helium gas) at a flow rate of 0.3 ml/min. Isocratic flow of 0.3 ml/min was maintained throughout analysis with a total run time of 45 min and injection volume was set to 10 μL. Samples were held at 20°C in the thermostated autosampler compartment. The eluent was monitored by means of a refractive index detector (RI-101, Shodex, JM Science) and quantification was made by the peak area relative to the peak area of the given standard (DPI : glucose; DP2: maltose; DP3 : maltotriose and peaks with a degree of four or higher maltotetraose was used as standard).

Example 4 - Pairwise application of glucoamylases for increased wort saccharification

Mashing operation for wort production

Glucoamylases was tested in mashing operation with 55% Pilsner malt (Pilsner malt; Fuglsang Denmark, Batch 13.01.2016) and 45% Corn grits (Nordgetreide GmBH Liibec, Germany, Batch: 02.05.2016.), using a water to grist ratio of 3.8: 1. Pilsner malt was milled at a Buhler Miag mill (0.5 mm setting). The corn adjunct was liquefied in the follow way: corn grits (31.5g), Malt (milled pilsner malt, 5.5g) and tap water (141g) was mixed in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.4 with 2.5M sulphuric acid. The adjunct was mashed with the program; heated to 43°C and kept for 1 minute for mashing in; heated to 72°C for 14.5 minutes by increasing temperature with 2°C/minute; kept at 72°C for 5 minutes; heated to 74°C for 1 minute by increasing temperature with 2°C/minute; kept at 74°C for 2 minutes; heated to 76°C for 1 minute by increasing temperature with 2°C/minute; kept at 76°C for 2 minutes; heated to 78°C for 1 minute by increasing temperature with 2°C/minute; kept at 78°C for 2 minutes, heated to 80°C for 1 minute by increasing temperature with 2°C/minute; kept at 80°C for 5 minutes; heated to 95°C for 7.5 minutes by increasing temperature with 2°C/minute; kept at 95°C for 15 minutes and mashing off. Hereafter the adjunct was cooled to 64°C and combined with the main mash. In the main mash, malt (milled pilsner malt, 122g) and tap water (122g) was mixed in a mashing bath (Lochner, LG-electronics) cup and pH adjusted to pH 5.4 with 2.5M sulphuric acid. The main mash was heated to 43 °C by increasing temperature with 2°C/minute; kept at 43 °C for 10 minutes and heated to 64°C for 21 minutes by increase temperature with l°C/minute. The adjunct and main mash was combined and substrate was aliquoted out in 15.0 g samples (in small wheaton glass containers with cap) to continue the mashing with enzyme addition.

After enzyme addition in the 15g sample mashing was continued in dry bath at 64°C with magnetic stirring for 240 minutes, followed by heating to 79°C (held for 15 minutes) and sugar analysis was carried out by HPLC as described in example 3.

Five different glucoamylases were tested in different pairwise combinations: TrGA

(Trichoderma reesie, glucoamylase), TrGA variant 1 (protein engineered variant of

Trichoderma reesie, glucoamylase including the mutations L417V, T430A, Q511H, A539R and N563I), TrGA variant 2 (protein engineered variant of Trichoderma reesie, glucoamylase including the mutations D44R and A539R), AfuGA (Aspergillus fumigatus, glucoamylase) and FvGA (Fusarium verticillioides, glucoamylase).

The following glucoamylase dose was used to obtain a conversion of starch into approximate 93% DPI (e.g. glucose and fructose). Table 2 Concentration in ppm of GA in mash application to obtain 93% DPI conversion

The five glucoamylases were mixed in all 10 unique pairwise combinations: TrGA variant 2:TrGA_variant 1, TrGA variant 2:TrGA, TrGA variant 2:AfuGA, TrGA variant 2:FvGA, TrGA variant l :TrGA, TrGA variant 1 : AfuGA, TrGA variant l :FvGA, TrGA: AfuGA, TrGA:FvGA and AfuGA:FvGA. Furthermore, varying pair ratios were applied ranging: [10:0], [8:2], [6:4], [5:5], [4:6], [2:8] and [0: 10]. Thus, A mix of AfuGA:FvGA [6:4] would equal 0,6 x 60.4 ppm AfuGA + 0.4 x 120.9ppm FvGA in application. The following relative % of DPI released after mashing by applying all 10 unique pairwise combinations with the 7 different ratios is shown in table 3.

Table 3 Relative concentration in % of DPI after mashing 240 minutes at 64C. Values are normalized to the relative DPI concentration obtained by the single glucoamylases, e.g. [10:0] and [0:10] in each mix, which is set to 93%. Normalization of value obtained by two glucoamylases are weighted by the relative ratio in the mix.

The relative concentration of DPI after mashing 240 minutes at 64°C is normalized to the concentration obtained by the single glucoamylases alone (93%) for better evaluation of potential pairwise synergies by glucoamylase pairs. As seen from the relative part of DPI released in table 3, the use of a single glucoamylase resulted in 93% DPI, whereas the vast majority of glucoamylase pairs resulted in a higher DPI release when combined. This may be explained as a positive synergy between two different GA, increasing DPI . Only 7 specific combinations out the 50 tested lowered DPI produced, whereas 1 was unchanged and the remaining increased DPI . Thus, several pairs of the different glucoamylases showed synergy, with an increased DPI released (as high as 94.63% obtained by TrGA var 2:FvGA [8:2]). Notably, higher DPI release was obtained in all pairs including either AfuGA or FvGA, whereas a lower increase was obtained combining TrGA with either TrGA variant 1 or 2 in any of the tested combinations. The latter showed for some pairs a decreased DPI release (< 93%). Thus it's obvious that more effective release of DPI during mashing may be accomplished by applying the right combination of two glucoamylases compared to applying one glucoamylase.

Example 5 -AfuGA and FvGA for increased wort saccharification

The AfuGA {Aspergillus fumigatus, glucoamylase) and FvGA (Fusarium verticillioides, glucoamylase) were tested in the setup described in example 4 for increased wort

saccharification. Dose-response experiments of the two glucoamylases were performed individually together with a dose-response of a 1 : 1 molar mix of AfuGA and FvGA (termed 1 : 1 AfuGA:FvGA). Glucoamylases were dosed based on the total amount of GA in the mashing. The following concentrations were applied: AfuGA [Oppm, 26ppm, 52ppm, 65ppm, 78ppm, 93ppm], FvGA [Oppm, 29ppm, 58ppm, 73ppm, 88ppm] and 1 : 1 AfuGA:FvGA [Oppm, 20ppm, 30ppm, 40ppm, 50ppm, 60ppm, 70ppm, 80ppm]. The relative concentration of DPI (of all sugars) after mashing 240 minutes at 64°C is shown in figure 1 for the three GA preparations. Figure 1 shows that it's possible to obtain a relative higher concentration of DPI sugar in the mash by the addition of both FvGA and AfuGA together than what can be achieved by the two glucoamylase individually. In addition, the combination of the two glucoamylases enables a certain DPI concentration by a lower total glucoamylase dosage compared to the DPI concentration achieved by two glucoamylases individually.

Example 6 -Application of single and pairwise glucoamylases in brew analysis with determination of real degree of fermentation (RDF) Glucoamylases was tested in mashing operation with 55% Pilsner malt (Pilsner malt; Fuglsang Denmark, Batch 13.01.2016) and 45% Corn grits (Nordgetreide GmBH Liibec, Germany, Batch: 02.05.2016.), using a water to grist ratio of 3.8: 1. Pilsner malt was milled at a Buhler Miag mill (0.5 mm setting).

The corn adjunct was liquefied in the follow way: Corn grits (31.5g), Malt (milled pilsner malt, 5.5g) and tap water (141g) was mixed in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.4 with 2.5M sulphuric acid. The adjunct was mashed with the program; heated to 43°C and kept for 1 minute for mashing in; heated to 72°C for 14.5 minutes by increasing temperature with 2°C/minute; kept at 72°C for 5 minutes; heated to 74°C for 1 minute by increasing temperature with 2°C/minute; kept at 74°C for 2 minutes; heated to 76°C for 1 minute by increasing temperature with 2°C/minute; kept at 76°C for 2 minutes; heated to 78°C for 1 minute by increasing temperature with 2°C/minute; kept at 78°C for 2 minutes, heated to 80°C for 1 minute by increasing temperature with 2°C/minute; kept at 80°C for 5 minutes; heated to 95°C for 7.5 minutes by increasing temperature with 2°C/minute; kept at 95°C for 15 minutes and mashing off. Hereafter the adjunct was cooled to 64°C and combined with the main mash. In the main mash, malt (milled pilsner malt, 122g) and tap water (122g) was mixed in a mashing bath (Lochner, LG-electronics) cup and pH adjusted to pH 5.4 with 2.5M sulphuric acid.

The main mash was heated to 43 °C by increasing temperature with 2°C/minute; kept at 43 °C for 10 minutes and heated to 64°C for 21 minutes by increase temperature with l°C/minute. Thus, to each cup with 178 g adjunct (liquefied corn, malt and water) enzymes where added when temperature reached 64°C. Following the mash where held at 64°C for 240 minutes followed by a ramp to 79°C by l°C/minute. The mash was finally held at 79°C for 15 min. Iodine negative was tested when temperature had reached 72°C. The time in minutes that was required to get iodine negative was noted.

At the end of mashing, the mashes were made up to 350 g and filtered. Filtrate volumes were measured after 30 minutes. The pH was adjusted to pH 5.2 with 2.5 M sulphuric acid and one pellet of bitter hops from Hopfenveredlung, St. Johann: Alpha content of 16,0 % (EBC 7.7 0 specific HPLC analysis, 01.10.2013), was added to each flask (210 g). The wort samples were boiled for 60 minutes in a boiling bath and wort were cooled down to 17°C and filtered. 100 g of each wort was weighted out into a 500 ml conical flask for fermentation adding 0.5 % W34/70 (Weihenstephan) freshly produced yeast (0.50 g) to the wort having 17°C. The remaining of the filtered wort was used for analysis, see below. The wort samples were fermented at 18°C and 150 rpm after yeast addition. Analysis was performed when fermentation had finished.

The following combinations of glucoamylase enzymes were tested: 50ppm AfuGA, 50ppm FvGA, 50ppm TrGA_varl, 50ppm TrGA, 25ppm AfuGA + 25ppm FvGA, 25ppm AfuGA + 25ppm TrGA_varl, 25ppm FvGA + 25ppm TrGA_varl and 25ppm TrGA_varl + 25ppm TrGA.

In this experiment, the dosage of glucoamylase was constant at 50ppm independent on addition of one or the blend of two glucoamylases.

Wort analysis: Original Extract (OE), Extract in the wort samples after mashing was measured using Anton Paar (Lovis) following Dupont Standard Instruction Brewing, 23.8580- B28Fermentable sugars (% total + g/100 ml) by HPLC were DPI, DP2, DP3 and DP4+ was determined after mashing following Dupont Standard Instruction Brewing, 23.8580-B20. Beer analysis: RDF was measured using an Anton Paar (DMA 5000) following Standard

Instruction Brewing, 23.8580-B28 and alcohol by Dupont Standard Instruction Brewing, 23.8580-B28.

Real degree of fermentation (RDF) value may be calculated according to the equation below:

Where: is the specific gravity of

the standardised worts before fermentation and is the specific gravity of the fermented worts expressed in degree Plato.

In the present context, Real degree of fermentation (RDF) was determined from the specific gravity and alcohol concentration.

Specific gravity and alcohol concentration was determined on the fermented samples using a Beer Alcolyzer Plus and a DMA 5000 Density meter (both from Anton Paar, Gratz, Austria). Based on these measurements, the real degree of fermentation (RDF) value was calculated according to the equation below:

Where: E(r) is the real extract in degree Plato (°P) and OE is the original extract in °P.

Original Extract (OE) Extract in the beer samples after mashing was measured using an Anton Paar (DMA 5000) following Dupont Standard Instruction Brewing, 23.8580-B28..

The result of UPLC and extract analysis of the wort after saccharification by different glucoamylases are shown in table 4. The relative sugar composition in the wort is shown in table 5.

Table 4 HPLC and original extract analysis of wort composition

Table 5 Relative distribution of sugars from HPLC analysis of wort

It is seen that a more effective conversion of starch into DPI is achieved with combinations of glucoamylases in the mashing. Especially the blends: 25ppm AfuGA + 25ppm FvGA, 25ppm AfuGA + 25ppm TrGA varl and 25ppm FvGA + 25ppm TrGA varl lead to an increased DPI concentration compared to using the glucoamylases individually.

After wort fermentation % RDF was determined. The results are shown in table 6.

Table 6 RDF after fermentation of wort produced with different glucoamylases.

The obtained RDF values corresponds with the analysis of sugar composition in the applied wort, thus a relative higher content of DPI sugar in the wort lead to a higher % RDF value of the fermentation. Thus, the blends: 25ppm AfuGA + 25ppm FvGA, 25ppm AfuGA + 25ppm TrGA varl and 25ppm FvGA + 25ppm TrGA varl showed increased RDF values compared to the individually glucoamylases and the blend TrGA varl (25 ppm) + TrGA (25 ppm). Thus, certain combinations of glucoamylase may increase the fermentability by a more effective saccharification of starch into DPI (being primarily glucose).

Example 7 - Sequence relationship of glucoamylases

A multiple alignment of SEQ ID NO: 1-5 is shown below generated by the Genious software package (Geneious® Pro 5.6.7) using CustalW alignment algorithm with a BLOSUM cost matrix and a gap opening cost of 10 and extension cost of 0.1.

The calculated pairwise sequence identity is shown in table 7. The sequence identity is calculated as (# Identities)/(# Identities + # Differences) * 100 %, using the following defition: # Identities, displays the absolute number of positions where two sequence have an identical residue and # Differences, displays the absolute number of positions where two sequence have non-identical residues on the alignment.

It's clear that TrGA, TrGA varl and TrGA_var2 are closely related with more than 99.2% sequence identity whereas both AfuGA and FvGA are only distantly related to any of the other present GA's with maximum of 57.2% and 50.7% pair-wise identity respectively.

Table 7 Pairwise sequence identity in percent (%) between the different glucoamylases with seq ID no. 1-5.

Example 8 - The glucoamylase synergy on DPI generation and sequence relationship

The synergy of applying two glucoamylase was calculated as the additional DPI generated by applying two glucoamylase compared to one. Thus, the additional DPI generated by 2 GA's in example 4 was used to calculate the maximum concentration of DPI obtained by a combination of two GA's subtracted the DPI concentration of 93% obtained by one glucoamylase. The additional DPI content (synergy) is shown in table 8 (calculated from results in example 4) together with the pairwise sequence identity in percent as shown in example 7.

Table 8 The pairwise glucoamylase synergies given as additional DPI (above 93%) and the pairwise sequence identity in percent (%).

The result clearly shows that the combination of more distantly related sequences, e.g.

TrGA:FvGA gave rise to higher synergy (1.16 %) compared to the combination of highly related sequences such as, e.g. TrGA:TrGA_varl (0.16 %). Considering these findings, its indeed relevant that more distantly related sequences may have more diverse substrate specificities and thus give rise to higher synergies in e.g. the mashing operation within brewing.

Example 9 - Application of three glucoamylases in mashing analysis

Glucoamylases was tested in mashing operation with 55% Pilsner malt (Pilsner malt; Fuglsang Denmark, Batch 13.01.2016) and 45% Corn grits (Nordgetreide GmBH Liibec, Germany, Batch: 02.05.2016.), using a water to grist ratio of 3.8: 1. Pilsner malt was milled at a Buhler Miag mill with a 0.5 mm setting. The corn adjunct was liquefied in the follow way: Corn grits (31.5g), Malt (milled pilsner malt, 5.5g) and tap water ( 141 g) was mixed in mashing bath (Lockner, LG- electronics) cups and pH adjusted to pH 5.4 with 2.5M sulphuric acid. The adjunct was mashed with the program; heated to 43°C and kept for 1 minute for mashing in; heated to 72°C for 14.5 minutes by increasing temperature with 2°C/minute; kept at 72°C for 5 minutes; heated to 74°C for 1 minute by increasing temperature with 2°C/minute; kept at 74°C for 2 minutes; heated to 76°C for 1 minute by increasing temperature with 2°C/minute; kept at 76°C for 2 minutes;

heated to 78°C for 1 minute by increasing temperature with 2°C/minute; kept at 78°C for 2 minutes, heated to 80°C for 1 minute by increasing temperature with 2°C/minute; kept at 80°C for 5 minutes; heated to 95°C for 7.5 minutes by increasing temperature with 2°C/minute; kept at 95°C for 15 minutes and mashing off. Hereafter the adjunct was cooled to 64°C and combined with the main mash. In the main mash, malt (milled pilsner malt, 122g) and tap water (122g) was mixed in a mashing bath (Lochner, LG-electronics) cup and pH adjusted to pH 5.4 with 2.5M sulphuric acid. The main mash was heated to 43°C by increasing temperature with 2°C/minute; kept at 43 °C for 10 minutes and heated to 64°C for 21 minutes by increase temperature with l°C/minute. Thus, to each cup with 178 g adjunct (liquefied corn, malt and water) enzymes where added according to table 9 below when temperature reached 64°C. Table 9 Combinations of the individuual glucoamylases added mashing either pairwise or as triplet and the total glucoamylase concentration in ppm.

Following the mash where held at 64°C for 240 minutes followed by a ramp to 79°C by l°C/minute. The mash was finally held at 79°C for 15 min. At the end of mashing, the mashes were made up to 350 g and filtered. Filtrate volumes were measured after 30 minutes. The pH was adjusted to pH 5.2 with 2.5 M sulphuric acid and one pellet of bitter hops from

Hopfenveredlung. St. Johann: Alpha content of 16.0 % (EBC 7.7 0 specific HPLC analysis. 01.10.2013). was added to each flask (210 g). The wort samples were boiled for 60 minutes in a boiling bath and wort were cooled down to 17°C and analysed by HPLC and Original Extract (OE) Extract in the beer samples after mashing was measured using an Anton Paar (DMA 5000) following Dupont Standard Instruction Brewing, 23.8580-B28..

The result of HPLC and extract analysis of the wort after saccharification by different glucoamylses are shown in table 10 with the relative sugar composition in the wort. Table 10 Relative distribution of sugars from HPLC analysis of wort and OE.

It is seen that a more effective conversion of starch into DPI is achieved with a triplet combination of AfuGA + FvGA + TrGA_varl compared to AfuGA + FvGA also used in example 6. For example, the use of AfuGA (18.75 ppm), FvGA (19.65 ppm) and TrGA_varl (5.80 ppm) gave 93.2% DPI whereas AfuGA (25 ppm) and FvGA (25 ppm) in the current mash produced 92.8% DPI .