US20040219649A1 - Alcohol product processes - Google Patents

Alcohol product processes Download PDF

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US20040219649A1
US20040219649A1 US10/797,393 US79739304A US2004219649A1 US 20040219649 A1 US20040219649 A1 US 20040219649A1 US 79739304 A US79739304 A US 79739304A US 2004219649 A1 US2004219649 A1 US 2004219649A1
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Prior art keywords
amylase
alpha
gly
asp
ala
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Hans Olsen
Sven Pedersen
Rikke Festersen
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Novozymes AS
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Novozymes AS
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Priority to US10/797,393 priority Critical patent/US20040219649A1/en
Assigned to NOVOZYMES A/S reassignment NOVOZYMES A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FESTERSEN, RIKKE MONICA, PEDERSEN, SVEN, OLSEN, HANS SEJR
Publication of US20040219649A1 publication Critical patent/US20040219649A1/en
Priority to US12/138,681 priority patent/US8772001B2/en
Priority to US14/176,759 priority patent/US9670509B2/en
Priority to US15/495,027 priority patent/US20170226537A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C7/00—Preparation of wort
    • C12C7/04—Preparation or treatment of the mash
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2425—Beta-amylase (3.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to processes for production of an alcohol product from granular starch comprising a pre-treatment at an elevated temperature below the initial gelatinization temperature of the granular starch followed by simultaneous saccharification and fermentation.
  • Granular starch is found in grains, cereals or tubers of plants.
  • the granular starch is in the form of microscopic granules, which are insoluble in water at room temperature.
  • an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution.
  • this “gelatinization” process there is a dramatic increase in viscosity.
  • the starch has to be thinned or “liquefied” so that it can be handled. This reduction in viscosity is generally accomplished by enzymatic degradation in a process referred to as liquefaction.
  • liquefaction the long-chained starch is degraded into smaller branched and linear chains of glucose units (dextrins) by an alpha-amylase.
  • a conventional enzymatic liquefaction process may be carried out as a three-step hot slurry process.
  • the slurry is heated to between 80-85° and thermostable alpha-amylase added to initiate liquefaction.
  • the slurry is then jet-cooked at a temperature between 105-125° C. to complete gelatinization of the slurry, cooled to 60-95° C. and, generally, additional alpha-amylase is added to finalize hydrolysis.
  • the liquefaction process is generally carried out at pH between 5 and 6. Milled and liquefied whole grains are known as mash.
  • the dextrins from the liquefaction are further hydrolyzed to produce low molecular sugars DP1-3 that can be metabolized by yeast.
  • the hydrolysis is typically accomplished using glucoamylases, alternatively or in addition to glucoamylases, alpha-glucosidases and/or acid alpha-amylases can be used.
  • a full saccharification step typically last up to 72 hours, however, it is common only to do a pre-saccharification of, e.g., 40-90 minutes at a temperature above 50° C., followed by a complete saccharification during fermentation in a process known as simultaneous saccharification and fermentation (SSF).
  • SSF simultaneous saccharification and fermentation
  • Fermentation may be performed using a yeast, e.g., from Saccharomyces spp., which added to the mash.
  • yeast e.g., from Saccharomyces spp.
  • the fermentation is carried out, for typically 35-60 hours at a temperature of typically around 32° C.
  • the fermentation is carried out, for typically up to 8 days at a temperature of typically around 14° C.
  • the mash may be used, e.g. as a beer, or distilled to recover ethanol.
  • the ethanol may be used as, e.g., fuel ethanol, drinking ethanol, and/or industrial ethanol.
  • the present invention provides methods for producing an alcohol product from granular starch without prior gelatinization of said starch.
  • the invention provides a process for production of an alcohol product comprising the sequential steps of: (a) providing a slurry comprising water and granular starch, (b) holding said slurry in the presence of an acid alpha-amylase and a glucoamylase at a temperature of 0° C. to 20° C. below the initial gelatinization temperature of said granular starch for a period of 5 minutes to 12 hours, (c) holding said slurry in the presence of an acid alpha-amylase, and a glucoamylase, and a yeast at a temperature of between 10° C. and 35° C. for a period of 20 to 250 hours to produce ethanol and, (d) optionally recovering the ethanol.
  • step (b) is believed to result in swelling of starch granules enclosed in the plant cells resulting in the disruption of cell walls and release of the starch granules thereby rendering the starch granules more accessible to further hydration and the action of the enzymes.
  • step (b) the acid alpha-amylase degrades the starch granules to produce dextrins, which are degraded by the glucoamylase into glucose.
  • step (c) in which the glucose is continuously fermented to ethanol by the yeast, thereby maintaining the concentration of fermentable sugar at a relatively low concentration throughout the fermentation.
  • the present invention may be used to produce an alcohol product which has a reduced glycerol and/or methanol content compared to conventional processes.
  • the present invention provides a less energy consuming alternative to conventional processes which must employ significant amounts of energy to gelatinize the starch slurry.
  • Other advantages of the present invention include, without limitation, the ability to employ a low pH throughout the process, thus reducing the risk of unwanted microbial growth, and reducing or eliminating the need for expensive equipment to gelatinize the starch, such as, jetting installations and steam plant equipment.
  • the present invention relates to an enzyme composition
  • an enzyme composition comprising an acid alpha-amylase and a glucoamylase, wherein the ratio between the acid alpha-amylase activity and glucoamylase activity is from 0.30 to 5.00 AFAU/AGU wherein an additional enzyme activity is present; said enzyme activity is selected from the list consisting of cellulase, xylanase and phytase.
  • the present invention relates to a use of the enzyme composition of the second aspect in an alcohol product process or a starch hydrolysis process.
  • the present invention relates to a use of an enzyme composition comprising an acid alpha-amylase and a glucoamylase, wherein the ratio between the acid alpha-amylase activity and glucoamylase activity is from 0.30 to 5.00 AFAU/AGU, in an alcohol product process comprising hydrolysis of granular starch.
  • the present invention relates to a mashing process comprising application of an acid alpha-amylase.
  • alcohol product means a product comprising ethanol, e.g. fuel ethanol, potable and industrial ethanol.
  • the alcohol product may also be a beer, which beer may be any type of beer.
  • Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • granular starch means raw uncooked starch, i.e. starch in its natural form found in cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to 50° C. to 75° C. the swelling may be reversible. However, with higher temperatures an irreversible swelling called gelatinization begins.
  • initial gelatinization temperature means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50° C. and 75° C.; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C., Starch/Stärke, Vol. 44 (12) pp. 461-466 (1992).
  • the polypeptide “homology” means the degree of identity between two amino acid sequences.
  • the homology may suitably be determined by computer programs known in the art, such as, GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453.
  • GAP creation penalty 3.0
  • GAP extension penalty of 0.1.
  • acid alpha-amylase means an alpha-amylase (E.C. 3.2.1.1) which added in an effective amount has activity at a pH in the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0. Any suitable acid alpha-amylase may be used in the present invention.
  • the acid alpha-amylase is an acid fungal alpha-amylase or an acid bacterial alpha-amylase.
  • the acid fungal alpha-amylase is obtained from a strain of Aspergillus , preferably a strain of Aspergillus niger or a strain of a strain of Aspergillus oryzae .
  • the acid alpha-amylase is an acid alpha-amylase having at least 70% homology, such as at least 80% or even at least 90% homology to the acid fungal alpha-amylase having the amino acid sequence set forth in SEQ ID NO:1 or having at least at least at least 70% homology, such as at least 80% or even at least 90% homology to the acid fungal alpha-amylase having the amino acid in the sequence shown in SWISPROT No: P10529.
  • the acid alpha-amylase is an acid fungal alpha-amylase having the amino acid sequence set forth in SEQ ID NO:1 or variants thereof having one or more amino acid residues which have been deleted, substituted and/or inserted compared to the amino acid sequence of SEQ ID NO:1; which variants have alpha-amylase activity.
  • Preferred acid alpha-amylase for use in the present invention may be derived from a strain of B. licheniformis, B. amyloliquefaciens , and B. stearothermophilus . Also preferred are acid alpha-amylases having an amino acid sequence which has at least 50% homology, preferably at least 60%, 70%, 80%, 85% or at least 90%, e.g. at least 95%, 97%, 98%, or at least 99% homology to the sequences set forth in SEQ ID NO:2 or SEQ ID NO:3.
  • the acid alpha-amylase used for the process of the invention is one of the acid alpha-amylase variants and hybrids described in WO96/23874, WO97/41213, and WO99/19467, such as the Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variant having the following mutations delta(181-182)+N193F (also denoted 1181*+G182*+N193F) compared to the wild type amino acid sequence set forth in SEQ ID NO:2.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase
  • the acid bacterial alpha-amylase may also be a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase set forth in SEQ ID NO:3 and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens set forth in SEQ ID NO:4, which may further have the substitutions G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S using the numbering in SEQ ID NO:3.
  • alpha-amylase variants derived from Bacillus amyloliquefaciens and having at least 50% homology, such as at least 60%, at least 70%, at least 80%, or even 90% homology to the sequence set forth in SEQ ID NO:4.
  • variants having one or more of the mutations H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179.
  • Preferred commercial compositions comprising alpha-amylase include Mycolase from DSM (Gist Brochades), BANTM, TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM X and SANTM SUPER, SANTM EXTRA L (Novozymes A/S) and Clarase L-40,000, DEX-LOTM, Spezyme FRED, SPEZYMETM AA, and SPEZYMETM DELTA AA (Genencor Int.).
  • a glucoamylase (E.C.3.2.1.3) to be used in the processes of the invention may be derived from a microorganism or a plant.
  • glucoamylases of fungal origin such as Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102).
  • variants thereof such as disclosed in WO92/00381 and WO00/04136; the A. awamori glucoamylase (WO84/02921), A. oryzae (Agric. Biol. Chem.
  • glucoamylases include the glucoamylases derived from Aspergillus niger , such as a glucoamylase having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or even 90% homology to the amino acid sequence set forth in WO00/04136 and SEQ ID NO: 13. Also preferred are the glucoamylases derived from Aspergillus oryzae , such as a glucoamylase having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or even 90% homology to the amino acid sequence set forth in WO0/04136 SEQ ID NO:2.
  • glucoamylases include Talaromyces glucoamylases , in particular derived from Talaromyces emersonii (WO99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32, 153), Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215), Clostridium , in particular C. thermoamylolyticum (EP135,138), and C. thermohydrosulfuricum (WO86/01831).
  • compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SAN EXTRA L and AMGTM E (from Novozymes A/S); OPTIDEXTM 300 (from Genencor Int.); AMIGASETM and AMIGASTM PLUS (from DSM); G-ZYMETM G900, G-ZYMETM and G990 ZR (from Genencor Int.).
  • An additional enzyme that may be used in the processes of the invention includes xylanases, cellulases and phytases.
  • a xylanase used according to the invention may be derived from any suitable organism, including fungal and bacterial organisms, such as Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma.
  • Preferred commercially available preparations comprising xylanase include SHEARZYME®, BIOFEED WHEAT®, CELLUCLAST®, ULTRAFLO®, VISCOZYME® (from Novozymes A/S) and SPEZYME® CP (from Genencor Int.).
  • the cellulase activity may be a cellulase of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium ).
  • a filamentous fungus e.g., Aspergillus, Trichoderma, Humicola, Fusarium.
  • cellulase which may be used include CELLUCLAST®, CELLUZYME®, CEREFLO® and ULTRAFLO® (from Novozymes A/S), LAMINEXTM and SPEZYME® CP (from Genencor Int.) and ROHAMENT® 7069 W (from Röhm GmbH).
  • a phytase used according to the invention may be any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) or from any salt thereof (phytates).
  • Phytases can be classified according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
  • the phytase to be used in the invention may have any specificity, e.g., be a 3-phytase (E.C. 3.1.3.8), a 6-phytase (E.C. 3.1.3.26) or a 5-phytase (no E.C. number).
  • BIO-FEED PHYTASETM PHYTASE NOVOTM CT or L (Novozymes A/S), or NATUPHOSTM NG 5000 (DSM).
  • Another enzyme used in the process may be a debranching enzyme, such as an isoamylase (E.C. 3.2.1.68) or a pullulanases (E.C. 3.2.1.41).
  • Isoamylase hydrolyses alpha-1,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on alpha-limit dextrins.
  • Debranching enzyme may be added in effective amounts well known to the person skilled in the art.
  • the invention provides a process for production of ethanol, comprising the steps of: (a) providing a slurry comprising water and granular starch, (b) holding said slurry in the presence of an acid alpha-amylase and a glucoamylase at a temperature of 0° C. to 20° C. below the initial gelatinization temperature of said granular starch for a period of 5 minutes to 12 hours, (c) holding said slurry in the presence of an acid alpha-amylase, and a glucoamylase, and a yeast at a temperature of between 30° C. and 35° C.
  • steps (a), (b), (c) and (d) are performed sequentially; however, the process may comprise additional steps not specified in this description which are performed prior to, between or after any of steps (a), (b), (c) and (d).
  • the temperature under step (c) is between 28° C. and 36° C., preferably from 29° C. and 35° C., more preferably from 30° C. and 34° C., such as around 32° C.
  • step (c) preferably for a period of 25 to 190 hours, preferably from 30 to 180 hours, more preferably from 40 to 170 hours, even more preferably from 50 to 160 hours, yet more preferably from 60 to 150 hours, even yet more preferably from 70 to 140 hours, and most preferably from 80 to 130 hours, such as 85 to 110 hours.
  • the invention provides a process for production of a beer, comprising the steps of: (a) providing a slurry comprising water and granular starch, (b) holding said slurry in the presence of an acid alpha-amylase and a glucoamylase at a temperature of 0° C. to 20° C. below the initial gelatinization temperature of said granular starch for a period of 5 minutes to 12 hours, (c) holding said slurry in the presence of an acid alpha-amylase, and a glucoamylase, and a yeast at a temperature between 10° C. and 18° C. for a period of 20 to 200 hours to produce ethanol.
  • the steps (a), (b), and (c) are performed sequentially; however, the process may comprise additional steps not specified in this description which are performed prior to, between or after any of steps (a), (b), and (c).
  • the temperature under step (c) is between 10° C. and 18° C., preferably from 11° C. and 17° C., more preferably from 12° C. and 16° C., such as between 13° C. and 15° C., e.g. around 14° C. and the slurry is held in contact with the acid alpha-amylase, the glucoamylase and the yeast for a period of time sufficient to allow hydrolysis of the starch and fermentation of the released sugars during step (c), preferably for a period of 100 to 230 hours, preferably from 150 to 210 hours, more preferably from 170 to 200 hours.
  • the enzyme activities may preferably be dosed in form of the composition of the second aspect of the invention.
  • the acid alpha-amylase is preferably an acid bacterial alpha-amylase and/or an acid fungal alpha-amylase and/or a variant of an acid alpha-amylase derived from a bacterial and/or a fungal source.
  • the acid alpha-amylase is added in an effective amount, which is a concentration of acid alpha-amylase sufficient for its intended purpose of converting the granular starch in the starch slurry to dextrins.
  • the acid alpha-amylase is present in an amount of 10-10000 AFAU/kg of DS, in an amount of 500-2500 AFAU/kg of DS, or more preferably in an amount of 100-1000 AFAU/kg of DS, such as approximately 500 AFAU/kg DS.
  • the acid alpha-amylase activity is preferably present in an amount of 5-500000 AAU/kg of DS, in an amount of 500-50000 AAU/kg of DS, or more preferably in an amount of 100-10000 AAU/kg of DS, such as 500-1000 AAU/kg DS.
  • the glucoamylases is added in an effective amount, which is a concentration of glucoamylase amylase sufficient for its intended purpose of degrading the dextrins resulting from the acid alpha-amylase treatment of the starch slurry.
  • the glucoamylase activity is present in an amount of 20-200 AGU/kg of DS, preferably 100-1000 AGU/kg of DS, or more preferably in an amount of 200-400 AGU/kg of DS, such as 250 AGU/kg DS.
  • the glucoamylase activity is preferably present in an amount of 10-100000 AGI/kg of DS, 50-50000 AGI/kg of DS, preferably 100-10000 AGI/kg of DS, or more preferably in an amount of 200-5000 AGI/kg of DS.
  • the activities of acid alpha-amylase and glucoamylase are present in a ratio of between 0.3 and 5.0 AFAU/AGU. More preferably the ratio between acid alpha-amylase activity and glucoamylase activity is at least 0.35, at least 0.40, at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.85, or even at least 1.9 AFAU/AGU.
  • the ratio between acid alpha-amylase activity and glucoamylase activity should preferably be less than 4.5, less than 4.0, less than 3.5, less than 3.0, less than 2.5, or even less than 2.25 AFAU/AGU.
  • the activities of acid alpha-amylase and glucoamylase are preferably present in a ratio of between 0.4 and 6.5 AUU/AGI.
  • the ratio between acid alpha-amylase activity and glucoamylase activity is at least 0.45, at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or even at least 2.5 AUU/AGI.
  • the ratio between acid alpha-amylase activity and glucoamylase activity is preferably less than 6.0, less than 5.5, less than 4.5, less than 4.0, less than 3.5, or even less than 3.0 AUU/AGI.
  • step (b) and/or step (c) is performed in the presence of an additional enzyme activity selected from the list consisting of xylanase, cellulase and phytase.
  • the additional enzyme is preferably added together with the acid alpha-amylase and the glucoamylase.
  • Xylanases may be added in amounts of 1-50000 FXU/kg DS, preferably 5-5000 FXU/kg DS, or more preferably 10-500 FXU/kg DS.
  • Cellulases may be added in the amounts of 0.01-500000 EGU/kg DS, preferably from 0.1-10000 EGU/kg DS, preferably from 1-5000 EGU/kg DS, more preferably from 10-500 EGU/kg DS and most preferably from 100-250 EGU/kg DS.
  • the dosage of the phytase may be in the range 0.5-250000 FYT/kg DS, particularly 1-100000 FYT/kg DS, preferably in the range from 5-25000 FYT/kg DS, preferably 10-10000 FYT/kg, such as 100-1000 FYT/kg DS.
  • the starch slurry comprises water and 5-60% DS (dry solids) granular starch, preferably 10-50% DS granular starch, more preferably 15-40% DS, especially around 20-25% DS granular starch.
  • the granular starch to be processed in the processes of the invention may in particular be obtained from tubers, roots, stems, cobs, legumes, cereals or whole grain. More specifically the granular starch may be obtained from corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, bean, banana or potatoes.
  • the granular starch to be processed may preferably comprising milled whole grain or it may be a more refined starch quality, preferably more than 90%, 95%, 97% or 99.5% pure starch.
  • the raw material comprising the starch is preferably milled in order to open up the structure and allowing for further processing. Dry milling as well as wet milling may be used. When wet milling is applied it may be preceded by a soaking, or steeping step. Both dry and wet milling is well known in the art of alcohol manufacturing and is preferred for the processes of the invention.
  • the granular starch may preferably comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or even at least 90% granular starch derived from malted cereals, e.g. barley malt.
  • the pH during step (b) and/or (c) is preferably in the range of 3.0 to 7.0, more preferably from 3.5 to 6.0, or most preferably from 4.0-5.0, such as from 4.3 to 4.6.
  • step b The slurry is held in contact with the acid alpha-amylase and glucoamylase at an elevated temperature but below the initial gelatinization temperature for a period of time effective to render the starch granules susceptible for enzymatic degradation (step b), preferably for a period of 5 minutes to 12 hours, preferably from 10 minutes to 6 hours, more preferably from 15 minutes to 3 hours, even more preferably from 20 minutes to 11 ⁇ 2 hour, such as from 30 minutes to 11 ⁇ 4 hour, from 40 to 70 minutes, and even from 50 to 60 minutes.
  • the temperature during step (b) should always be adjusted to be below the initial gelatinization temperature of the particular granular starch to be processed, and will typically be between 45° C. and 75° C.
  • step (b) is conducted at a temperature from 0° C. to 20° C., preferably to from 0° C. 15° C., more preferably from 0° C. to 10° C., or even more preferably from 0° C. to 5° C. below the initial gelatinization temperature of the particular starch to be processed.
  • the actual temperature may be from 45° C. to 75° C., but is preferably from 55° C. to 65° C.
  • the temperature at which step (b) is conducted is at least 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C. or preferably at least 55° C., and preferably the temperature is no more than 74° C., 73° C., 72° C., 71° C., 70° C., 69° C., 68° C., 67° C., 66° C., 65° C., 64° C., 63° C. or preferably no more than 62° C.
  • the ethanol may optionally be recovered.
  • the ethanol recovery may be performed by any conventional manner such as e.g. distillation and may be used as fuel ethanol and/or potable ethanol and/or industrial ethanol.
  • the granular starch to be processed is derived from dry or wet milled cereal, such as wheat, barley, rye, and/or corn
  • the starch slurry has a DS of 20-40 percent
  • the temperature during step (b) is from 50° C. to 60° C., such as 55° C.
  • the duration of step (b) is from 30 minutes to 75 minutes, such as 60 minutes
  • step (c) is carried out for 60 to 90 hours.
  • the acid alpha amylase is dosed at 300 to 700 AFAU/kg DS, such as 500 AFAU/kg DS
  • the glucoamylase is dosed at 100 to 500 AGU/kg DS, such as 250 AGU/kg DS.
  • the ratio of acid alpha amylase to glucoamylase is from 1.0 to 3.0 AFAU/AGU or preferably from 1.5 to 2.5 AFAU/AGU, such as approximately 2.0 AFAU/AGU.
  • the ratio of acid alpha amylase to glucoamylase is from 1.3 to 4.0 AAU/AGI or preferably from 2.0 to 2.3 AAU/AGI, such as approximately 2.7 AAU/AGI.
  • the invention provides an enzyme composition which may be used in any process, including the process of the first aspect of the invention, said enzyme composition having a ratio between acid alpha-amylase activity and glucoamylase activity of at least 0.35, at least 0.40, at least 0.50, at least 0.60, at least 0.70, at least 0.80, at least 0.90, at least 1.00, at least 1.20, at least 1.30, at least 1.40, at least 1.50, at least 1.60, at least 1.70, at least 1.80, or even at least 1.85 AFAU/AGU.
  • said enzyme composition has a ratio between acid alpha-amylase activity and glucoamylase activity is less than 5.00, less than 4.50, less than 4.00, less than 3.00, less than 2.50, or even less than 2.25 AFAU/AGU.
  • the ratio of acid alpha amylase to glucoamylase in said enzyme composition is at least 0.45, at least 0.50, at least 0.60, at least 0.70, at least 0.80, at least 0.90, at least 1.00, at least 1.20, at least 1.30, at least 1.40, at least 1.50, at least 1.60, at least 1.70, at least 1.80, at least 1.90, at least 2.00, at least 2.10, at least 2.20, at least 2.30, at least 2.40 or even at least 2.50 AAU/AGI.
  • said enzyme composition has a ratio between acid alpha-amylase activity and glucoamylase activity is less than 6.50, less than 5.00, less than 4.50, less than 4.00, or even less than 3.50 AAU/AGI.
  • composition of the second aspect of the invention further comprises an additional enzyme activity is present; said enzyme activity is selected from the list consisting of cellulase, xylanase and phytase.
  • an acid alpha-amylase such as an acid alpha-amylase derived from a fungus, preferably of the genus Aspergillus , preferably from the species A. niger , and most preferably having at least 50%, at least 60%, at least 70%, at least 80% or even at least 90% homology to the sequence shown in SEQ ID No:1 is used in a brewing process.
  • Brewing processes are well-known in the art, and generally involve the steps of malting, mashing, and fermentation.
  • the malting serves the purpose of converting insoluble starch to soluble starch, reducing complex proteins, generating color and flavor compounds, generating nutrients for yeast development, and the development of enzymes.
  • the three main steps of the malting process are steeping, germination, and kilning.
  • Steeping includes mixing the barley kernels with water to raise the moisture level and activate the metabolic processes of the dormant kernel.
  • the wet barley is germinated by maintaining it at a suitable temperature and humidity level until adequate modification, i.e. such as degradation of starch and activation of enzymes, has been achieved.
  • the final step is to dry the green malt in the kiln.
  • Mashing is the process of converting starch from the milled barley malt and solid adjuncts into fermentable and unfermentable sugars to produce wort of the desired composition.
  • Traditional mashing involves mixing milled barley malt and adjuncts with water at a set temperature and volume to continue the biochemical changes initiated during the malting process. The mashing process is conducted over a period of time at various temperatures in order to activate the endogenous enzymes responsible for the degradation of proteins and carbohydrates.
  • the principal enzymes responsible for starch conversion in a traditional mashing process are alpha- and beta-amylases. Alpha-amylase very rapidly reduces insoluble and soluble starch by splitting starch molecules into many shorter chains that can be attacked by beta-amylase. The disaccharide produced is maltose.
  • the double-mash infusion system is the most widely used system for industrial production of beer, especially lager type beer.
  • This system prepares two separate mashes. It utilizes a cereal cooker for boiling adjuncts and a mash tun for well-modified, highly enzymatically active malts.
  • the traditionally mashing processes utilize the endogenous enzymes of the barley malt the temperature is maintained below 70° C. as inactivation of the enzymes would otherwise occur.
  • wort separation is important because the solids contain large amounts of protein, poorly modified starch, fatty material, silicates, and polyphenols (tannins).
  • the objectives of wort separation include the following:
  • the wort may be fermented with brewers yeast to produce a beer.
  • An acid alpha-amylase such as an acid alpha-amylase derived from a fungus, preferably of the genus Aspergillus , preferably from the species A. niger , and most preferably having at least 50%, at least 60%, at least 70%, at least 80% or even at least 90% homology to the sequence shown in SEQ ID No:1 be applied in any brewing process as a supplement to the enzymes comprised in the malted and/or unmalted grain or in a higher temperature mashing (HTM) process such as the one disclosed in WO 2004/011591.
  • HTM higher temperature mashing
  • the high temperature process preferably comprises, forming a mash comprising between 5% and 100% barley malt, adding prior to, during or after forming the mash a mashing enzyme composition, attaining within 15 minutes of forming the mash an initial incubation temperature of at least 70° C., followed by incubation of the mash at a temperature of at least 70° C. for a period of time, and separating the wort from the spent grains.
  • the period of time of mashing is sufficient to achieve an extract recovery of at least 80%.
  • the term “initial incubation temperature” is understood as the temperature regime during the initial part of the incubation in question.
  • enzyme activities needed for the mashing process to proceed are exogenously supplied and may be added to the mash ingredients, e.g. the water or the grist before forming the mash, or it may be added during or after forming the mash.
  • the enzymes are preferably supplied all at one time at the start of the process; however, one or more of the enzymes may be supplied at one or more times prior to, at the start, or during the process of the sixth aspect of the invention.
  • the enzyme activities added may comprise one or more of the following activities; a protease (E.C. 3.4.), cellulase (E.C. 3.2.1.4) and a maltose generating enzyme.
  • the maltose generating enzyme is preferably a beta-amylase (E.C. 3.2.1.2) or even more preferably a maltogenic alpha-amylase (E.C. 3.2.1.133).
  • a further enzyme is added, said enzyme being selected from the group consisting of laccase, lipase, glucoamylase, phospholipolase, phytase, phytin esterase, pullulanase, and xylanase.
  • a starch containing slurry, the mash is obtained by mixing a grist comprising at least 5%, or 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) barley malt with water.
  • a grist comprising at least 5%, or 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) barley malt with water.
  • at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 50%, at least 75% or even 100% of the barley malt is well modified barley malt.
  • the grist comprises other malted grain than barley malt, so that at least 10%, at least 25%, preferably at least 35%, more preferably at least 50%, even more preferably at least 75%, most preferably at least 90% (w/w) of the grist is other malted grain than barley malt.
  • the malted and/or unmalted grain Prior to forming the mash the malted and/or unmalted grain is preferably milled and most preferably dry milled.
  • the malted and/or unmalted grain used is a husk free (or hull free) variety or the husks are removed from the malted and/or unmalted grain before forming the mash. Removal of husks is preferably applied where the mashing programs comprising temperatures above 75° C., such as at temperatures above 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C. or even above 86° C.
  • the water may preferably, before being added to the grist, be preheated in order for the mash to attain the initial incubation temperature at the moment of mash forming. If the temperature of the formed mash is below the initial incubation temperature additional heat is preferably supplied in order to attain the initial incubation temperature. Preferably the initial incubation temperature is attained within 15 minutes, or more preferably within 10 minutes, such as within 9, 8, 7, 6, 5, 4, 2 minutes or even more preferably within 1 minute after the mash forming, or most preferably the initial incubation temperature is attained at the mash forming.
  • the initial incubation temperature is preferably at least 70° C., preferably at least 71° C., more preferably at least 72° C., even more preferably at least 73° C., or most preferably at least 74° C., such as at least 75° C., at least 76° C., at least 77° C., at least 78° C., at least 79° C., at least 80° C., at least 81° C., such as at least 82° C.
  • a preferred embodiment of the mashing process of the sixth aspect of the invention includes incubating the mash at the initial incubation temperature of at least 70° C.
  • At least 70° C. preferably at least 71° C., more preferably at least 72° C., even more preferably at least 73° C., or most preferably at least 74° C., such as at least 75° C., at least 76° C., at least 77° C., at least 78° C., at least 79° C., at least 80° C., at least 81° C., at least 82° C., at least 83° C., at least 84° C., or at least 85° C. i.e. a temperature that never falls below 70° C. for the duration of the incubation period.
  • the temperature is preferably held below 100° C., such as below 99° C., 98° C., 97° C., 96° C., 95° C., 94° C., 93° C., 92° C., 91° C., or even below 90° C.
  • the temperature may be held constant for the duration of the incubation, or, following a period of an essentially constant temperature (the initial incubation temperature) for the first part of the incubation the temperature may be raised, either as a slow continuously increase, or as one or more stepwise increment(s) during the incubation. Alternatively the temperature may be decreased during the incubation.
  • the initial incubation temperature is at least 70° C. and during the incubation the temperature is increased with at least 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C.
  • the initial incubation temperature is at least 75° C., or preferably at least 80° C., and the temperature is decreased during the incubation with at least 5° C., or preferably with at least 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C. or preferably with at least 10° C., or more preferably with at least 15° C.
  • the incubation comprises maintaining the mash at a temperature of at least 75° C., preferably at least 76° C., more preferably at least 77° C., even more preferably at least 78° C., such as at least 79° C., at least 80° C., at least 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C. or at least 90° C.
  • the incubation comprises maintaining the mash at a temperature of at least 75° C., preferably at least 76° C., more preferably at least 77° C., even more preferably at least 78° C., such as at least 79° C., at least 80° C., such as at least 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C. or at least 90° C.
  • the duration of the incubation is preferably at least 15 minutes, typically between 30 minutes and 21 ⁇ 2 hours, e.g. at least 45 minutes, at least 1 hour, at least 11 ⁇ 4 hour, at least 11 ⁇ 2 hour, at least 13 ⁇ 4 hour or at least 2 hours.
  • the grist may in addition to barley malt preferably comprise adjunct such as unmalted barley, or other malted or unmalted grain, such as wheat, rye, oat, corn, rice, milo, millet and/or sorghum, or raw and/or refined starch and/or sugar containing material derived from plants like wheat, rye, oat, corn, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane.
  • adjuncts may be obtained from tubers, roots, stems, leaves, legumes, cereals and/or whole grain.
  • the adjunct to be added to the mash of the sixth aspect of the invention has gelatinization temperatures at or below the process temperature. If adjuncts such as rice or corn, or other adjuncts with similar high gelatinization temperature, are to be used in the process of the sixth aspect of the invention, they may preferably be cooked separately to ensure gelatinization before being added to the mash of the sixth aspect of the invention, or the gelatinized adjunct starch may be mashed separately from the mash adding appropriate enzymes to ensure saccharification before being added to the mash. Methods for gelatinization and saccharification of brewing adjuncts are well known in the arts.
  • Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the barley malt mash before, during or after mashing process of the sixth aspect of the invention but is preferably added after the mashing process.
  • a part of the adjunct is treated with a protease and/or a beta-glucanase before being added to the mash of the sixth aspect of the invention.
  • starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins.
  • the mash is starch negative to iodine testing, before extracting the wort.
  • obtaining the wort from the mash typically includes straining the wort from the spent grains, i.e. the insoluble grain and husk material forming part of grist. Hot water may be run through the spent grains to rinse out, or sparge, any remaining extract from the grist.
  • the wort separation may comprise a centrifugation step.
  • the wort produced by the mashing process of the sixth aspect of the invention may be fermented to produce a beer. Fermentation of the wort may include pitching the wort with a yeast slurry comprising fresh yeast, i.e. yeast not previously used for the invention or the yeast may be recycled yeast.
  • the yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Saccharomyces spp. such as S. cerevisiae and S. uvarum , including natural or artificially produced variants of these organisms.
  • the methods for fermentation of wort for production of beer are well known to the person skilled in the arts.
  • Preferred beer types comprise ales, strong ales, stouts, porters, lagers, bitters, export beers, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • the enzymes to be applied in the sixth aspect of present invention should be selected for their ability to retain sufficient activity at elevated temperatures, such as at the process temperature of the processes, as well as for their ability to retain sufficient activity under the moderately acid pH regime in the mash and should be added in effective amounts.
  • the enzymes may be derived from any source, preferably from a plant or an algae, and more preferably from a microorganism, such as from a bacteria or a fungi.
  • Suitable proteases include microbial proteases, such as fungal and bacterial proteases.
  • Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.
  • Contemplated acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Torulopsis .
  • proteases derived from Aspergillus niger see, e.g., Koaze et al., (1964), Agr. Biol. Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida, (1954) J. Agr. Chem. Soc.
  • Contemplated are also neutral or alkaline proteases, such as a protease derived from a strain of Bacillus .
  • a particular protease contemplated for the invention is derived from Bacillus amyloliquefaciens and has the sequence obtainable at Swissprot as Accession No. P06832 (SEQ ID NO 5).
  • the proteases having at least 90% homology to amino acid sequence obtainable at Swissprot as Accession No. P06832 (SEQ ID NO 5) such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.
  • proteases having at least 90% homology to amino acid sequence disclosed as SEQ.ID.NO:1 in the Danish patent applications PA 2001 01821 and PA 2002 00005, such as at 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.
  • papain-like proteases such as proteases within E.C. 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).
  • cyste protease such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).
  • Proteases may be added in the amounts of 0.1-1000 AU/kg dm, preferably 1-100 AU/kg dm and most preferably 5-25 AU/kg dm.
  • the cellulase (E.C. 3.2.1.4) may be of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium ).
  • a filamentous fungus e.g., Aspergillus, Trichoderma, Humicola, Fusarium
  • Specific examples of cellulases include the endo-glucanase (endo-glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of FIG. 14 in WO 91/17244 and the 43 kD H. insolens endo-glucanase described in WO 91/17243.
  • a particular cellulase to be used in the processes of the sixth aspect of the invention may be an endo-glucanase, such as an endo-1,4-beta-glucanase.
  • Contemplated are beta-glucanases having at least 90% homology to amino acid sequence disclosed as SEQ.ID.NO:1 in Danish patent application PA2002 00130, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.
  • cellulase preparations which may be used include CELLUCLAST®, CELLUZYME®), CEREFLO® and ULTRAFLO® (available from Novozymes A/S), LAMINEXTM and SPEZYME® CP (available from Genencor Int.) and ROHAMENT® 7069 W (available from Röhm, Germany).
  • Beta-glucanases may be added in the amounts of 1.0-10000 BGU/kg dm, preferably from 10-5000 BGU/kg dm, preferably from 50-1000 BGU/kg dm and most preferably from 100-500 BGU/kg dm.
  • a particular alpha-amylase (EC 3.2.1.1) to be used in the processes of the sixth aspect of the invention may be any fungal alpha-amylase, preferably an acid alpha-amylase.
  • the acid alpha-amylase is derived from a fungus of the genus Aspergillus , preferably from the species A. niger , and most preferably having at least 50%, at least 60%, at least 70%, at least 80% or even at least 90% homology to the sequence shown in SEQ ID No:1 is used in a brewing process.
  • Fungal alpha-amylases may be added in an amount of 1-1000 AFAU/kg DM, preferably from 2-500 AFAU/kg DM, preferably 20-100 AFAU/kg DM.
  • Another acid alpha-amylase enzyme to be used in the processes of the sixth aspect of the invention may be a Bacillus alpha-amylase.
  • Bacillus alpha-amylases include alpha-amylase derived from a strain of B. licheniformis, B. amyloliquefaciens , and B. stearothermophilus .
  • Other Bacillus alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp.
  • a contemplated Bacillus alpha-amylase is an alpha-amylase as defined in WO99/19467 on page 3, line 18 to page 6, line 27.
  • a preferred alpha-amylase has an amino acid sequence having at least 90% homology to SEQ ID NO:4 in WO99/19467, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.
  • Most preferred variants of the maltogenic alpha-amylase comprise the variants disclosed in WO99/43794.
  • Contemplated variants and hybrids are described in WO96/23874, WO97/41213, and WO99/19467.
  • Bacillus alpha-amylases may be added in the amounts of 1.0-1000 NU/kg dm, preferably from 2.0-500 NU/kg dm, preferably 10-200 NU/kg dm.
  • a particular enzyme to be used in the processes of the sixth aspect of the invention is a maltogenic alpha-amylase (E.C. 3.2.1.133).
  • Maltogenic alpha-amylases glucan 1,4-alpha-maltohydrolase
  • hydrolyse amylose and amylopectin to maltose in the alpha-configuration are able to hydrolyse maltotriose as well as cyclodextrin.
  • maltogenic alpha-amylases may be derived from Bacillus sp., preferably from Bacillus stearothermophilus , most preferably from Bacillus stearothermophilus C599 such as the one described in EP 120.693.
  • This particular maltogenic alpha-amylase has the amino acid sequence shown as amino acids 1-686 of SEQ ID NO:1 in U.S. Pat. No. 6,162,628.
  • a preferred maltogenic alpha-amylase has an amino acid sequence having at least 90% homology to amino acids 1-686 of SEQ ID NO:1 in U.S. Pat. No.
  • 6,162,628 preferably at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.
  • Most preferred variants of the maltogenic alpha-amylase comprise the variants disclosed in WO99/43794.
  • Maltogenic alpha-amylases may be added in amounts of 0.1-1000 MANU/kg dm, preferably from 1-100 MANU/kg dm, preferably 5-25 MANU/kg dm.
  • Another particular enzyme to be used in the processes of the sixth aspect of the invention may be a beta-amylase (E.C 3.2.1.2).
  • Beta-amylases have been isolated from various plants and microorganisms (W. M. Fogarty and C. T. Kelly, Progress in Industrial Microbiology, vol. 15, pp. 112-115, 1979). These beta-amylases are characterized by having optimum temperatures in the range from 40° C. to 65° C. and optimum pH in the range from 4.5 to 7.0. Specifically contemplated beta-amylase include the beta-amylases SPEZYME® BBA 1500, SPEZYME® DBA and OPTIMALTTM ME, OPTIMALTTM BBA from Genencor Int. as well as the beta-amylases NOVOZYMTM WBA from Novozymes A/S. Beta-amylases may be added in effective amounts well known to the person skilled in the art.
  • a further particular enzyme to be used in the processes of the sixth aspect of the invention may be a glucoamylase (E.C.3.2.1.3) derived from a microorganism or a plant.
  • glucoamylases of fungal or bacterial origin selected from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as disclosed in WO92/00381 and WO00/04136; the A. awamori glucoamylase (WO84/02921), A. oryzae (Agric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.
  • Glucoamylases may be added in effective amounts well known to the person skilled in the art.
  • Another enzyme of the process of the sixth aspect of the present invention may be a debranching enzyme, such as an isoamylase (E.C. 3.2.1.68) or a pullulanases (E.C. 3.2.1.41).
  • Isoamylase hydrolyses alpha-1,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on alpha-limit dextrins.
  • Debranching enzyme may be added in effective amounts well known to the person skilled in the art.
  • the amylolytic activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.
  • KNU Kilo Novo alpha amylase Unit
  • the activity of any acid alpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units).
  • activity of acid alpha-amylase may be measured in AAU (Acid Alpha-amylase Units).
  • the acid alpha-amylase activity can be measured in AAU (Acid Alpha-amylase Units), which is an absolute method.
  • AAU Acid Amylase Unit
  • One Acid Amylase Unit (AAU) is the quantity of enzyme converting 1 g of starch (100% of dry matter) per hour under standardized conditions into a product having a transmission at 620 nm after reaction with an iodine solution of known strength equal to the one of a color reference.
  • Standard conditions/reaction conditions Substrate: Soluble starch. Concentration approx. 20 g DS/L. Buffer: Citrate, approx.
  • the starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as calorimetric indicator. Lintner starch is obtained by dilute hydrochloric acid treatment of native starch so that it retains the ability to color blue with iodine. Further details can be found in EP0140410B2, which disclosure is hereby included by reference.
  • Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 FAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.
  • Acid alpha-amylase an endo-alpha-amylase (1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzes alpha-1,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths.
  • the intensity of color formed with iodine is directly proportional to the concentration of starch.
  • Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions. Standard conditions/reaction conditions: Substrate: Soluble starch, approx. 0.17 g/L Buffer: Citrate, approx.
  • I2 0.03 M Iodine (I2): 0.03 g/L CaCl2: 1.85 mM pH: 2.50 ⁇ 0.05 Incubation temperature: 40° C. Reaction time: 23 seconds Wavelength: 590 nm Enzyme concentration: 0.025 AFAU/mL Enzyme working range: 0.01-0.04 AFAU/mL
  • Glucoamylase activity may be measured in AGI units or in AmyloGlucosidase Units (AGU)
  • Glucoamylase (equivalent to amyloglucosidase) converts starch into glucose.
  • the amount of glucose is determined here by the glucose oxidase method for the activity determination. The method described in the section 76-11 Starch-Glucoamylase Method with Subsequent Measurement of Glucose with Glucose Oxidase in “Approved methods of the American Association of Cereal Chemists”. Vol. 1-2 AACC, from American Association of Cereal Chemists, (2000); ISBN: 1-891127-12-8.
  • AGI glucoamylase unit
  • the starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as colorimetric indicator. Lintner starch is obtained by dilute hydrochloric acid treatment of native starch so that it retains the ability to color blue with iodine.
  • the Novo Glucoamylase Unit is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.
  • An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.
  • the xylanolytic activity can be expressed in FXU-units, determined at pH 6.0 with remazol-xylan (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka) as substrate.
  • a xylanase sample is incubated with the remazol-xylan substrate.
  • the background of non-degraded dyed substrate is precipitated by ethanol.
  • the remaining blue color in the supernatant is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions, i.e. at 50.0° C., pH 6.0, and 30 minutes reaction time.
  • the cellulytic activity may be measured in endo-glucanase units (EGU), determined at pH 6.0 with carboxymethyl cellulose (CMC) as substrate.
  • EGU endo-glucanase units
  • a substrate solution is prepared, containing 34.0 g/l CMC (Hercules 7 LFD) in 0.1 M phosphate buffer at pH 6.0.
  • the enzyme sample to be analyzed is dissolved in the same buffer. 5 ml substrate solution and 0.15 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 from Sofraser, France), thermostated at 40° C. for 30 minutes.
  • MIVI 3000 from Sofraser, France
  • One EGU is defined as the amount of enzyme that reduces the viscosity to one half under these conditions.
  • the amount of enzyme sample should be adjusted to provide 0.01-0.02 EGU/ml in the reaction mixture.
  • the arch standard is defined as 880 EGU/g.
  • the phytase activity is measured in FYT units, one FYT being the amount of enzyme that liberates 1 micromole inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37° C.; substrate: sodium phytate (C 6 H 6 O 24 P 6 Na 12 ) at a concentration of 0.0050 mole/l.
  • the proteolytic activity may be determined with denatured hemoglobin as substrate.
  • Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA).
  • TCA trichloroacetic acid
  • the amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.
  • One Anson Unit is defined as the amount of enzyme which under standard conditions (i.e. 25° C., pH 7.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.
  • NPC non-pressure cooking
  • a 20% D.S. slurry of the milled barley grain was made in room temperature (RT) tap water.
  • the NPC pre-treatment of the conventional ethanol process was performed in 6 ⁇ 1-litre tubs with stirring. Bacterial alpha-amylase was added and the tubs were placed in water bath at 65° C. When the temperature in the mash reached 55° C. the heating was increased to heat the mash to 90° C. over 60 minutes. The temperature was then adjusted to 32° C. and 3 ⁇ 250 g mash was portioned in 500 mL blue cap flasks with air locks. To all flasks 0.25 g dry bakers yeast was added (corresponding to 5-10 million vital cells/g mash). Enzyme activities were added according to the table below and each flask was weighed.
  • This example illustrates the use of an enzyme composition of the invention consisting of acid alpha-amylase, glucoamylase, cellulase and xylanase activity.
  • AFAU Acid alpha-amylase
  • AGU glucoamylase
  • EGU cellulase
  • FXU xylanase activity
  • Low Process of AFAU/AGU the invention AFAU/AGU 0.24 0.24 1.98 1.98 AFAU/kg DS 39 39 540 540 AGU/kg DS 163 163 273 273 FXU/kg DS 0 70 0 70 EGU/kg DS 0 175 0 175 Weight loss (g), 48 hours 9.9 11.2 12.8 14.3 Weight loss (g), 72 hours 12.1 13.8 15.5 16.5 Ethanol %, 48 Hrs 4.14 4.68 5.35 5.98 Ethanol %, 72 Hrs 5.06 5.77 6.48 6.90
  • This example illustrates the process of the invention using various raw materials.
  • a 20% D.S. slurry of the milled grain or corn meal was made in RT tab water.
  • 2 ⁇ 250 g was portioned in 500 mL blue cap flasks.
  • the pH was adjusted to 4.5
  • Enzymes were dosed according to table 3, 4 and 5, and a pre-treatment was carried out for one hour at 55° C. in a shaking water bath.
  • the flasks were cooled to 32° C. and 0.25 g dry bakers yeast added.
  • the flasks were placed in a water bath at 32° C. for 72 hours (90 hours for wheat).
  • TABLE 3 Rye the weight loss (g) at 48 hours and at 72 hours.
  • AFAU Acid alpha- amylase
  • AGU glucoamylase
  • This example illustrates a process of the invention using wheat.
  • a 20% D.S. slurry of milled wheat was made in RT tab water.
  • 2 ⁇ 250 g slurry was portioned in 500 mL blue cap flasks.
  • the pH was adjusted to 4.5 using 6 N HCl.
  • Enzyme activities were dosed according to table 6, and the flasks were incubated for one hour at 55° C. in a shaking water bath.
  • the flasks were cooled to 32° C. and 0.25 g dry bakers yeast added.
  • the flasks were placed in a water bath at 32° C. for 72 hours.
  • Weight loss data was recorded. At 50 and 72.5 hours the flasks were weighed and CO 2 weight loss measured for monitoring of the fermentation progress.
  • This example demonstrates the use of an acid alpha-amylase in a brewing process.
  • the enzymes used comprised an acid fungal alpha-amylase derived from Aspergillus niger having the sequence shown in SEQ ID NO:1.
  • a glucoamylase derived from Aspergillus niger A protease having the amino acid sequence shown as amino acids no. 1-177 of SEQ.ID.NO 2 in Danish patent applications WO 2003/048353.
  • a xylanase from Aspergillus aculeatus having the sequence amino acid disclosed as SEQ ID NO:2 in WO 9421785.
  • the acid alpha-amylase from Aspergillus niger SP288 was tested in a mashing set up using both Congress mashing and Higher Temperature Mashing (HTM) conditions. The effect was evaluated on formation of fermentable sugars in the wort, which is a key wort quality parameter. A bacterial heat stable alpha-amylase from Bacillus stearothermophilus was applied for comparison. All worts were added a xylanase 5 mg EP/kg DS, betaglucanase 5 mg EP/kg DS and protease 2.5 mg EP/kg DS.
  • HTM Higher Temperature Mashing
  • mashing trials were performed in 500 ml lidded vessels each containing a mash with 50 g grist and adjusted to a total weight of 450 ⁇ 0.2 g with water preheated to the initial incubation temperature+1° C. During mashing the vessels were incubated in water bath with stirring.
  • the second treatment comprised using HTM as disclosed in WO 2004/011591 with the following temperature profile: an initial incubation temperature of 70° C. for 65 minutes, increasing to 90° C., with 1.0° C./min for 20 minutes, followed by 90° C. for 15 minutes and finalized by cooling with 4.5° C./min to 20° C.
  • the recipe applied was: 50.0 g malt added 200 ml water from beginning, at the end of mashing the mash-cups are standardized to 300.0 g, which gives app. 13° P.
  • Fermentable sugars were analysed at 8.6° P using HPLC method equivalent to EBC: 8.7 TABLE 7 Extract E2, extract in dry malt, % (m/m) from Congress mashing (8.6° P) and HTM (13° P). To all treatments were added xylanase 5 mg EP/kg DS, betaglucanase 5 mg EP/kg DS and Protease 2.5 mg EP/kg DS. No additional 75 AFAU/kg DS + enzymes 61 AGU/kg DS 75 KNU/kg DS Congress mashing 80.90 81.79 81.97 HTM 81.42 81.23 81.33
  • Acid alpha-amylase showed very good effect on formation of fermentable sugars, predominantly the glucose concentration was increased in wort by the addition of acid alpha-amylase both compared to Termamyl SC and with out adding amylase. Overall the sum of fermentable sugars is increased from 94.91 g/L to 97.44 g/L from performance of acid alpha-amylase. This means that an increased part of the extract is now fermentable, which will yield higher alcohol amounts.

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WO2004080923A2 (en) 2004-09-23
ES2245621T1 (es) 2006-01-16
EP1604019B1 (de) 2010-01-06
ES2245621T3 (es) 2010-05-19
CN1788083B (zh) 2011-10-05
US20140154764A1 (en) 2014-06-05
US20090017511A1 (en) 2009-01-15
DE04718914T1 (de) 2006-02-23
DE602004024964D1 (de) 2010-02-25
US8772001B2 (en) 2014-07-08
CN102321705A (zh) 2012-01-18
CN102277341A (zh) 2011-12-14
CN1788083A (zh) 2006-06-14
US20170226537A1 (en) 2017-08-10
EP1604019A2 (de) 2005-12-14
US9670509B2 (en) 2017-06-06
CN102277341B (zh) 2015-07-22
EP2166091A3 (de) 2015-06-10
WO2004080923A3 (en) 2004-12-16
EP2166091A2 (de) 2010-03-24
ATE454446T1 (de) 2010-01-15

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