Classifications

• • 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)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
  • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
  • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
  • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • 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
  • C12C11/00—Fermentation processes for beer
  • C12C11/003—Fermentation of beerwort
• • 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
  • 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
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • 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
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

• the invention relates to a novel enzyme composition and novel beer brewing process.
• Traditional beer production processes comprise a first hydrolysis stage in which starch is hydrolysed into saccharides and a second separate fermentation stage where these saccharides are subsequently fermented into alcohol.
• the fermentation is traditionally carried out with yeast cells.
• WO2013/167573 describes a method of preparing a wort with a high level of free amino acids, said method comprising the steps of a) mashing a composition comprising barley in the presence of exogenous enzymes comprising an alpha amylase, a beta glucanase, a pullulanase, a xylanase, and a lipase; and b) adding to said composition during mashing or after completion of mashing at least two different exogenous proteases, wherein one protease has endoprotease activity, and the other protease has exopeptidase activity.
• the endoprotease could be a metalloprotease, a proline-endoprotease or a glutamine endoprotease.
• Ondea Pro commercially available from Novozymes
• a metalloprotease and an exopeptidase were added in a mashing step.
• 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. After mashing when all the starch has been broken down, it was necessary to separate the liquid extract (the wort) from the residual solids (spent grains) and boil it. Hereby numerous substances including several proteins were denatured. After cooling and removal of precipitates, the wort was aerated and fermented to produce a beer. Due to the boiling, no alive enzymes could be present during the fermentation.
• some special alcoholic beverages such as sake may be produced in a process where saccharification and fermentation occur simultaneously within the same brewing tank.
• Haze is a well-known phenomenon in the beer industry. In a beer brewing process haze formation can occur at different stages. In “Enzymes in food processing" edited by T. Nagodawithana and G. Reed, 3 rd edition, Academic press Inc., San Diego, Chapter V, p.448-449, it has been proposed that the haze in beer is the result of interactions between beer proteins and polyphenolic procyanidins. [008] W02002046381 describes a method for the prevention or reduction of haze in a beverage wherein a prolyl-specific endoprotease (also referred to as “PEP”) is added to the beverage.
• PEP prolyl-specific endoprotease
• W02002046381 explains that the activity of prolyl-specific endoproteases is dependent on the pH. It describes for example that an endoprotease is added to a beverage having a maximum prolyl specific activity at a pH which corresponds to the pH of the beverage it is added to. Since haze formation often occurs in acidic beverages such as for example beer, wine and fruit juice, prolyl-specific endoproteases having a prolyl specific activity at a pH value below 7 are preferably used. W02002046381 subsequently provides several specific prolyl- specific endoproteases that have since then been very successful in reduction of haze in beverages.
• the “haze” in the beer may have a different composition and may be caused by a mixture of components.
• the haze may not only comprise the above mentioned interactions between beer proteins and polyphenolic procyanidins, but may also comprise fine particulate matter and/or polysaccharides resulting from the feedstock.
• the role of polysaccharides in haze formation is not well understood, but studies have shown effects of the polysaccharides on this haze formation.
• a novel enzyme composition, a novel process applying such novel enzyme and a novel method for producing such novel enzyme composition allowing one to speed up or otherwise improve a SSF process and/or to speed-up or otherwise improve the removal of fine particulate matter in the fermentation broth and/or fermentation tank and/or to improving the taste of the alcoholic beverage product, has now been found.
• the invention provides a composition (herein also referred to as “enzyme composition”), preferably for addition to a process for the production of an alcoholic beverage for human consumption, comprising or consisting of:
• a first enzymatic component comprising or consisting of a glutamine-specific protease and/or prolylspecific protease, preferably a glutamine-specific endoprotease and/or prolyl-specific endoprotease, most preferably a prolyl-specific endoprotease; and - a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.
• the second enzymatic component is derived from and/or produced by a Trichoderma reesei.
• the cellulolytic enzyme preferably cellulase
• the cellulolytic enzyme is derived and/or produced from Trichoderma reesei.
• a cellulolytic enzyme suitably cellulase, that is derived and/or produced from Trichoderma reesei produces the best results.
• the first enzymatic component and the second enzymatic component are both derived and/or produced from the same organism, preferably, Trichoderma reesei, which provides the benefit of an efficient production.
• the first aspect provides a composition comprising or consisting of:
• a first enzymatic component comprising or consisting of a prolyl-specific protease, preferably a prolylspecific endoprotease;
• a second enzymatic component comprising or consisting of a cellulolytic enzyme, preferably cellulase, wherein preferably the second enzymatic component is derived from and/or produced by a Trichoderma reesei.
• the invention provides a process for the preparation comprising the use of the above novel enzyme composition.
• the invention provides a process for the production of the above novel enzyme composition.
• the cellulase can be helpful to convert small residues of the feedstock in a SSF process and that the above invention can therefore help to improve to speed up or otherwise improve a home-brewing SSF process and/or speed-up or otherwise improve the removal of fine particulate matter in the fermentation broth and/or fermentation tank and/or improving the taste of a (home-made) beer would be an advancement in the art.
• the above novel enzyme composition can advantageously be useful in a process for the production of an alcoholic beverage for human consumption, preferably a beer. Therefore there is advantageously provided a novel process for the production of an alcoholic beverage for human consumption, preferably a novel beer brewing process, using the above novel enzyme composition.
• the endoprotease preferably proline-specific endoprotease
• the so-called active site(s) within an enzyme are those parts of the enzyme that are responsible for interaction with the substrate. This interaction may for example comprise the binding of and/or catalysing of the reaction in the substrate.
• the formation and presence of such so-called active site(s) can be dependent on the enzyme’s primary, secondary and/or tertiary structure. Examples of active sides include catalytic triad(s) and/or an oxyanion hole(s).
• the endoprotease preferably glutamine-specific and/or proline-specific endoprotease
• the endoprotease, preferably glutamine-specific and/or proline-specific endoprotease is an endoprotease, respectively glutamine-specific and/or proline-specific endoprotease, comprising a catalytic triad and an oxyanion hole.
• the catalytic triad comprises an amino acid sequence comprising or consisting of Serine, Histidine and Aspartic acid.
• a catalytic triad is herein also referred to as a Ser/Asp/His catalytic triad.
• the ND1 or Nd1 nitrogen atom of side chain of the histidine amino acid in the above catalytic triad is protonated, and the NE2 or Ne2 nitrogen atom (pK a of 6.0) of the side chain of the histidine amino acid is unprotonated.
• the histidine amino acid in the above catalytic triad is deprotonated.
• deprotonated histidine is herein preferably understood a histidine amino acid that has lost its proton (H + ion) on its side chain NE2 or Ne2 nitrogen atom.
• the oxyanion holes in hydrolytic enzymes are typically made up from hydrogen bond-donating groups of polar or basic amino acid side chains like Tyr, Trp, Arg, Lys, Asn, Gin, Ser, Thr or also often two backbone amides that hydrogen bond with the oxygen atoms of the negatively charged transition state.
• the enzyme component preferably comprises or consists of an endoprotease, preferably a prolyl-specific endoprotease, comprising an oxyanion hole comprising a protonated side chain of an acidic amino acid. More preferably this oxyanion hole comprises a glutamic acid amino acid, preferably in its protonated state (Glu-H).
• compositions as described herein are compositions, wherein the first enzymatic component is derived from and/or produced by a micro-organism, preferably a bacterium or fungus, most preferably a Trichoderma reesei.
• glutamine-specific and/or prolyl-specific endoproteases that are derived from an Aspergillus species but that are heterologously produced in a Trichoderma species, preferably Trichoderma reesei.
• cellulase enzyme or simply “cellulase” are used interchangeably herein.
• cellulase can hydrolyze beta- 1 ,4-D-glucosidic linkages in cellulose, thereby partially or completely degrading cellulose.
• the cellulase can be an “exocellulase”, “endocellulase” or “cellobiase”.
• the cellulase is an “endocellulase” or “cellobiase”, most preferably the cellulase is an “endocellulase”.
• the composition may comprise one, two, three, four, five, six or more (types of) cellulases.
• the composition may comprise any cellulase, for example, a lytic polysaccharide monooxygenase (e.g. GH61), a cellobiohydrolase, an endo-p-1 ,4-glucanase, a beta-glucosidase or a p-(1 ,3)(1 ,4)-glucanase.
• a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4-p-D-glucosidic linkages in cellulose or cellotetraose, releasing cellobiose from the ends of the chains.
• This enzyme may also be referred to as cellulase 1 ,4-p- cellobiosidase, 1 ,4-p-cellobiohydrolase, 1 ,4- -D-glucan cellobiohydrolase, avicelase, exo-1 ,4- -D- glucanase, exocellobiohydrolase or exoglucanase.
• the cellulase is a mixture of cellulase enzymes comprising one or more glucanase enzymes, more preferably one or more beta- glucanase enzymes.
• an endo-p-1 ,4-glucanase (EC 3.2.1.4) is preferred and is any polypeptide which is capable of catalyzing the endohydrolysis of 1 ,4-p-D-glucosidic linkages in cellulose, lichenin or cereal p-D-glucans. Such a polypeptide may also be capable of hydrolyzing 1 ,4-linkages in p-D- glucans also containing 1 ,3-linkages.
• a beta-glucosidase (EC 3.2.1.21) is also preferred and is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing p-D-glucose residues with release of p-D-glucose.
• a polypeptide may have a wide specificity for p-D-glucosides and may also hydrolyze one or more of the following: a p-D-galactoside, an a-L-arabinoside, a p-D-xyloside or a p-D-fucoside.
• This enzyme may also be referred to as amygdalase, p-D-glucoside glucohydrolase, cellobiase or gentobiase.
• a p-(1 ,3)(1 ,4)-glucanase (EC 3.2.1 .73) is also preferred and is any polypeptide which is capable of catalysing the hydrolysis of 1 ,4-p-D-glucosidic linkages in p-D-glucans containing
• Such a polypeptide may act on lichenin and cereal p-D-glucans, but not on p-D- glucans containing only 1 ,3- or 1 ,4-bonds.
• This enzyme may also be referred to as licheninase, 1 ,3-
• 1 .4-p-D-glucan 4-glucanohydrolase p-glucanase, endo-p-1 ,3-1 ,4 glucanase, lichenase or mixed linkage p-glucanase.
• An alternative for this type of enzyme is EC 3.2.1.6, which is described as endo- 1 ,3(4)-beta-glucanase.
• This type of enzyme hydrolyses 1 ,3- or 1 ,4-linkages in beta-D-glucanse when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3.
• Alternative names include endo-1 ,3-beta-glucanase, laminarinase, 1 ,3-(1 ,3;1 ,4)-beta-D-glucan 3 (4) glucanohydrolase.
• Substrates include laminarin, lichenin and cereal beta-D-glucans.
• the p- (1 ,3)(1 ,4)-glucanase (EC 3.2.1 .73) respectively endo-1 ,3(4)-beta-glucanase [EC 3.2.1 .6], are preferred as second enzymatic components.
• the second enzymatic component preferably comprises or consists of a glucanase, more preferably a beta-glucanase (also referred to as “p-glucanase”), most preferably a beta-glucanase derived from Trichoderma reesei.
• a glucanase more preferably a beta-glucanase (also referred to as “p-glucanase”), most preferably a beta-glucanase derived from Trichoderma reesei.
• Suitable cellulases can be derived from a plant, an animal, or a microorganism.
• the cellulase is derived from a microorganism, such as a fungus or a bacterium.
• Preferred cellulases are those derived from a bacterium or a fungus. Most preferred is a Trichoderma reesei derived cellulase.
• the second enzymatic component preferably cellulase, more preferably a beta-glucanase, is derived from and/or produced by a micro-organism, preferably a fungus, most preferably a Trichoderma reesei.
• the activity of the cellulase is dependent on the pH.
• the composition comprises a second enzymatic component, wherein this second enzymatic component preferably has its optimum activity at a pH in the range from equal to or more than pH 1 .0, more preferably from equal to or more than pH 1 .2 to equal to or less than pH 6.0, more preferably to equal to or less than pH 5.5, still more preferably to equal to or less than pH 5.0, even more preferably to equal to or less than pH 4.5 and most preferably to equal to or less than pH 4.0.
• the cellulase is a cellulase with an acidic pH optimum, i.e. a pH optimum below pH 7.0. More preferably the cellulase is a cellulase having its optimum activity at a pH in the range from equal to or more than pH 1 .0, more preferably from equal to or more than pH 1 .2 to equal to or less than pH 6.0, more preferably to equal to or less than pH 5.0, still more preferably to equal to or lower than pH 4.0, even more preferably to equal to or lower than pH 3.0 and most preferably to equal to or less than pH 2.5. Preferably the cellulase is a cellulase having its optimum activity at a pH in the range from pH 1 .0 to pH 5.0, even more preferably in the range from pH 1 .2 to pH 4.5.
• the cellulosic enzyme preferably cellulase
• the cellulosic enzyme is preferably produced in or derived from a microorganism, preferably a bacterium or a fungus.
• the cellulosic enzyme preferably cellulase, may be a natural occurring, recombinant or chemically modified cellulosic enzyme, respectively cellulase.
• cellulases examples include cellulases produced in or derived from the genera Bacillus, Pseudomonas, Streptomyces, Trichoderma, Humicola, Fusarium, Thielavia and Acremonium, and also cellulases produced in or derived from Humicola insolens, Myceliophthora thermophila or Fusarium oxysporum. Preferred are cellulases produced in or derived from Trichoderma.
• the cellulolytic enzyme may be produced recombinantly in a heterologous expression system, such as a microbial or fungal heterologous expression system.
• a heterologous expression system such as a microbial or fungal heterologous expression system.
• suitable heterologous expression systems include bacterial (e.g., E. coll, Bacillus sp.) and eukaryotic systems.
• Eukaryotic systems can employ yeast (e.g., Pichia sp., Saccharomyces sp.) or fungal (e.g., Trichoderma sp. such as T. reesei, Aspergillus species such as A. niger) expression systems.
• yeast e.g., Pichia sp., Saccharomyces sp.
• fungal e.g., Trichoderma sp. such as T. reesei, Aspergillus species such as A. niger
• Preferred cellulases are the cellulases disclosed in U.S. Pat. Nos. 4689297, 5814501 , 5324649, and International Patent Appl. Publ. Nos. WO92/06221 and WO92/06165, which are also herewith incorporated by reference.
• Exemplary commercial cellulases that could be used in this invention include CELLUZYME® and CAREZYME® (Novozymes A/S); CLAZINASE® and PURADAX® HA (DuPont Industrial Biosciences), LAMINEX® (IFF Bioscience) and KAC-500(B)®(Kao Corporation).
• the enzyme composition is a mixture of the enzyme composition.
• composition according to the invention is a composition comprising or consisting of:
• a first enzymatic component comprising or consisting of a prolyl-specific protease, preferably a prolylspecific endoprotease;
• a second enzymatic component comprising or consisting of a cellulolytic enzyme, preferably cellulase, wherein the molar ratio of moles cellulolytic enzyme, preferably cellulase, to moles prolyl-specific protease, preferably prolyl-specific endoprotease, is equal to or less than 1 :1 , more preferably equal to or less than 0.5:1 , even more preferably equal to or less than 0.1 :1 , still more preferably equal to or less than 0.05:1 and most preferably equal to or less than 0.01 :1 .
• composition according to the invention is a composition comprising or consisting of:
• a first enzymatic component comprising or consisting of a prolyl-specific protease, preferably a prolylspecific endoprotease;
• a second enzymatic component comprising or consisting of a cellulolytic enzyme, preferably cellulase, wherein the weight ratio of the weight of the first enzymatic component, preferably prolyl-specific protease, preferably prolyl-specific endoprotease, to the weight of the second enzymatic component, preferably cellulase, more preferably beta-glucanase, is:
• the weight ratio of the weight of the first enzymatic component, preferably prolyl-specific protease, preferably prolyl-specific endoprotease, to the weight of the second enzymatic component, preferably cellulase, more preferably beta-glucanase, is about 4:1.
• the cellulolytic enzyme preferably cellulase
• the cellulolytic enzyme is present in an amount of equal to or more than 0.001 % w/w, more preferably equal to or more than 0.005%w/w and still more preferably equal to or more than 0.01% w/w, and most preferably equal to or more than 0.05 % w/w, based on the total weight of the prolyl-specific protease, preferably a prolyl-specific endoprotease.
• the cellulolytic enzyme preferably cellulase
• the cellulolytic enzyme is present in an amount of equal to or less than 100% w/w, more preferably equal to or less than 50%w/w and still more preferably equal to or less than 10%w/w, and most preferably equal to or less than 5%w/w, based on the total weight of the prolyl-specific protease, preferably a prolyl-specific endoprotease.
• these ratios are advantageous in view of the differences in presence of polysaccharides and other components in the haze.
• composition according to the invention is preferably a composition, wherein the first enzymatic component and the second enzymatic component are derived from or produced by the same micro-organism, preferably the same bacterium or fungus, most preferably the same Trichoderma reesei.
• the enzyme composition is a liquid composition.
• the enzyme composition further comprises a solvent.
• the solvent comprises or consists of water.
• one or more cosolvents may or may not be present.
• the composition comprises water and optionally one or more co-solvents. If a co-solvent is present, the solvent can comprise water and one or more cosolvents.
• Preferred co-solvents include tripropylene glycol methyl ether, dipropylene glycol methyl ether, propylene glycol methyl ether, diethylene glycol butyl ether, dipropylene glycol, Methylene glycol, 1 ,2- propanediol, N-ethyl-2-pyrroldinone, isopropanol, ethanol, ethyl lactate, 1 ,3-propanediol, and/or any combinations thereof.
• the solvent may or may not comprise glycerol.
• the solvent comprises less than 50% v/v, more preferably less than 40% v/v, still more preferably less than 30 % v/v, even more preferably less than 20 % v/v, and most preferably less than 10 % v/v glycerol, based on the total volume of the solvent.
• the solvent does not comprise glycerol. More recently glycerol has been subject to a lot of price volatility and therefore is less preferred.
• the use of the solution stabilizer and/or osmolyte as described herein advantageously allows one to reduce the amount of glycerol in the solvent whilst still obtaining good stability and/or activity and/or avoiding precipitation.
• the composition has a pH value in the range from equal to or more than pH 1 .0, more preferably from equal to or more than pH 1 .2 to equal to or less than pH 6.0, more preferably to equal to or less than pH 5.5, still more preferably to equal to or less than pH 5.0, even more preferably to equal to or less than pH 4.5 and most preferably to equal to or less than pH 4.0.
• Y1 is a pH value in the range from equal to or more than pH 1 .0, more preferably from equal to or more than pH 1 .2 to equal to or less than pH 6.0, more preferably to equal to or less than pH 5.5, still more preferably to equal to or less than pH 5.0, even more preferably to equal to or less than pH 4.5 and most preferably to equal to or less than pH 4.0.
• the composition is a liquid composition having a pH in the range from equal to or more than pH 1 .0, more preferably from equal to or more than pH 1 .2 to equal to or less than pH 6.0, more preferably to equal to or less than pH 5.0, still more preferably to equal to or lower than pH 4.0, even more preferably to equal to or lower than pH 3.0 and most preferably to equal to or less than pH 2.5. More preferably the composition is a liquid composition having a pH in the range from pH 1 .0 to pH 5.0, even more preferably in the range from pH 1 .2 to pH 4.5.
• the enzyme composition can be provided as a frozen or freeze - dried form and be reconstituted before application by the addition of a solvent as described above during pre-processing.
• the enzyme composition may comprise one or more additional components, preferably chosen from the group consisting of:
• a salt preferably chosen from the group consisting of sodium chloride, potassium chloride and ammonium sulphate;
• carboxylic acid preferably having in the range from equal to or more than 1 to equal to or less than 10 carbon atoms, preferably in the range from equal to or more than 1 to equal to or less than 7 carbon atoms, and/or any ester and/or any salt thereof, more preferably formic acid, acetic acid, diacetic acid, ascorbic acid, lactic acid, citric acid, propionic acid, oxalic acid, malic acid and/or fumaric acid, and/or any ester thereof and/or any salt thereof;
• glyceryl monoacetate glyceryl diacetate, glyceryl triacetate, glyceryl monopropionate, glyceryl dipropionate, glyceryl tripropionate, glyceryl monobutanoate, glyceryl dibutanoate, glyceryl tributanoate, glyceryl monolactate, glyceryl dilactate, glyceryl trilactate;
• sorbitol mannitol, inositol, trehalose, sucrose, mannose and/or sucrolose, most preferably sorbitol;
• maltodextrin xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, amylopectin, and mixtures thereof.
• composition may or may not comprise glycerol.
• composition may or may not comprise benzoic acid or a derivative thereof.
• the composition further may or may not comprise one or more additional enzymes in addition to the endoprotease enzyme, such as for example acetolactate decarboxylate (ALDC), aminopeptidase or trehalase, glucoamylase, xylanase, maltogenic alpha-amylase, pullulanase, catalase or transglucosidase.
• the composition comprises one or more additional enzymes, preferably acetolactate decarboxylate (ALDC), glucoamylase and/or xylanase.
• the composition comprises or consists of the first enzymatic component, the second enzymatic component and xylanase.
• composition comprises or consists of the first enzymatic component, the second enzymatic component and acetolactate decarboxylate (ALDC).
• composition comprises or consists of the first enzymatic component, the second enzymatic component and glucoamylase.
• the invention further provides a bacterial or fungal strain, preferably a Trichoderma reesei strain, functionally expressing:
• a first nucleic acid sequence encoding a first enzymatic component comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease;
• a second nucleic acid sequence encoding a second enzymatic component comprising or consisting of a cellulolytic enzyme, preferably cellulase.
• the first nucleic acid sequence is:
• the invention also provides a bacterial or fungal strain, preferably Trichoderma reesei strain, functionally expressing:
• a first nucleic acid sequence encoding a first enzymatic component comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease;
• nucleic acid sequence encoding a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.
• first nucleic acid sequence is:
• the invention further provides a bacterial or fungal strain, preferably a Trichoderma reesei strain, functionally expressing:
• a first nucleic acid sequence encoding a recombinant first enzymatic component comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease;
• a second nucleic acid sequence encoding a natural occurring second enzymatic component comprising or consisting of a cellulolytic enzyme, preferably cellulase.
• the first nucleic acid sequence is:
• the above may advantageously cause the bacterial or fungal strain, preferably a Trichoderma reesei strain, to produce the first enzymatic component in a higher amount than the second enzymatic component.
• the first nucleic acid sequence is a heterologous nucleic acid sequence and the first enzymatic component is heterologous expressed.
• the second nucleic acid sequence is a endogenous nucleic acid sequence and the second enzymatic component is endogenous expressed.
• the invention also provides a process for the preparation of an alcoholic beverage, preferably a beer, comprising the use of the above novel enzyme composition.
• the invention further provides a process for the production of an alcoholic beverage for human consumption, comprising the addition of a composition according to the invention or the addition of a bacterial strain according to the invention.
• a process for the production of an alcoholic beverage for human consumption comprising the addition of a composition according to the invention or the addition of a bacterial strain according to the invention.
• a process for the production of an alcoholic beverage for human consumption comprising the addition of a composition according to the invention or the addition of a bacterial strain according to the invention.
• a process is a simultaneous saccharification and fermentation process (“SSF”)
• a process for the production of an alcoholic beverage for human consumption comprising the addition of a composition as described above or the addition of a bacterial or fungal strain, preferably a Trichoderma reesei strain, as described above.
• every beer brewing process typically comprises saccharification and fermentation.
• a feedstock such as starch
• saccharides can be converted into ethanol (alcohol).
• ethanol alcohol
• the second enzymatic component preferably cellulase
• the invention therefore also provides a beer brewing process comprising the addition of a composition according to the invention or the addition of a bacterial strain according to the invention, preferably during the saccharification or fermentation step.
• the process is a beer brewing process, wherein a composition according to the invention is added before or during fermentation, more preferably before fermentation, most preferably to the wort.
• such a beer brewing process is a simultaneous saccharification and fermentation process (“SSF”).
• SSF simultaneous saccharification and fermentation process
• the composition is added in such a process during the simultaneous saccharification and fermentation.
• composition according to the invention is thus preferably a composition that can be added to a process for the production of an alcoholic beverage for human consumption, more preferably a composition that can be added to a beer brewing process, which composition comprises or consists of:
• a first enzymatic component comprising or consisting of glutamine-specific protease and/or prolylspecific protease, preferably a glutamine-specific endoprotease and/or prolyl-specific endoprotease;
• the first enzymatic component is added to the processes as described herein in a dosage of equal to or more than 0.05 gram per hectoliter (gr/hl), more preferably equal to or more than 0.1 gr/hl, still more preferably equal to or more than 0.5 gr/hl and most preferably equal to or more than 1 .0 gr/hl and/or preferably equal to or less than 10Ogr/hl, more preferably equal to or less than 50 gr/hl, most preferably equal to or less than 10 gr/hl.
• the first enzymatic component is added before or during fermentation, preferably before fermentation, preferably to the wort in a beer brewing process.
• the second enzymatic component is added to the processes as described herein in a dosage of equal to or more than 0.001 gram per hectoliter (gr/hl), more preferably equal to or more than 0.01 gr/hl, still more preferably equal to or more than 0.05 gr/hl and most preferably equal to or more than 0.1 gr/hl and/or preferably equal to or less than 50gr/hl, more preferably equal to or less than 10 gr/hl, even more preferably equal to or less than 5 gr/hl and most preferably equal to or less than 1 gr/hl.
• gr/hl 0.001 gram per hectoliter
• the first enzymatic component and the second enzymatic component are added in a weight ratio, wherein the weight ratio of the weight of the first enzymatic component, preferably prolyl-specific protease, preferably prolyl-specific endoprotease, to the weight of the second enzymatic component, preferably cellulase, more preferably beta-glucanase, is:
• first enzymatic component and the second enzymatic component in such a weight ratio is especially advantageous in a process for the production of an alcoholic beverage for human consumption, more preferably a beer brewing process.
• first enzymatic component and the second enzymatic component are added in such a ratio before or during fermentation, for example to a wort.
• the addition of said enzyme composition in a process for the production of an alcoholic beverage for human consumption, especially a beer brewing process can positively affect the taste of the produced beverage, such as a beer, by increasing the amount of mono-, di-, and tri-saccharides which have a sweet taste and enhance fruity flavours. It is therefore preferable that the addition of the enzyme composition in a process for the production of an alcoholic beverage for human consumption, especially a beer brewing process, increases the amount of mono-, di-, and/or tri-saccharides in the produced alcoholic beverage, such as a beer.
• the addition of the enzymatic composition in particular a composition comprising both a prolylspecific endoprotease and a cellulase from Trichoderma reesei, in a process for the production of an alcoholic beverage for human consumption, especially a beer brewing process, can further improve the removal of fine particulate matter as is shown in the experimental section.
• the addition of a composition combining prolyl endoprotease and cellulase during beer fermentation can reduce the fouling of membranes during beer filtration and the filtration can advantageously be operated at an increased pressure and speed, gaining an economic advantage.
• the invention further provides an alcoholic beverage for human consumption obtained or obtainable by such a process.
• the invention provides an alcoholic beverage comprising, preferably at least partly inactivated:
• glutamine-specific and/or proline-specific protease preferably glutamine -specific and/or prolinespecific endoprotease, preferably as described herein above;
• NL-Fast is a product of DSM Food Specialties and contains a fungal cellulase, more specifically an endo-1 ,3 (4)- p-glucanase (a beta-glucanase), derived from a selected strain of Talaromyces emersonii.
• Laminex BG3 is a product of IFF and contains a fungal cellulase (beta-glucanase) from Trichoderma reesei.
• - Filtrase BR-X L is a product of DSM Food Specialties and contains a fungal cellulase, more specifically an endo-1 ,3 (4)- p-glucanase (a beta-glucanase), derived from a selected strain of Talaromyces emersonii and a hemicellulose, more specifically an endo-1 ,4- -xylanase (a xylanase), derived from a selected strain of Disporotrichum dimorphosporum.
• a fungal cellulase more specifically an endo-1 ,3 (4)- p-glucanase (a beta-glucanase), derived from a selected strain of Talaromyces emersonii and a hemicellulose, more specifically an endo-1 ,4- -xylanase (a xylanase), derived from a selected strain of Disporotrichum dimorphosporum.
• - Glucanex 100G is a product of Novozymes and contains a fungal cellulase, more specifically a beta-glucanase, from Trichoderma harzianum.
• Saflager S023 a bottom fermenting dried yeast (Saccharomyces pastorianus) from Lasaffre (Fermentis), which is suited for direct pitching.
• Heineken lager beer a 5% ABV pale lager produced by Heineken
• the filter system was assembled according to the manufacturer’s instructions by clamping the filter house to the bottom plate consisting of a perforated disc at the inner part and a connector (an 3/8- inch connector - ball valve - 3/8-inch connector and male luer lock (Z514691-1 EA from sigma), stainless steel and Teflon taped) affixed to a tripod at the outer part.
• a volume of 250 ml of cooled sample was poured into the pre-cooled filter house.
• the ball valve was opened to allow about 10 grams of sample to flow through by gravity after which the switch was closed ensuring complete fill of the connector exit without any air bubbles.
• the filter (Acrodisc PES 0.45pm 25mm, PN 4508; Pall Corporation) was rinsed by backflush with 5 ⁇ 1 ml degassed Heineken lager beer by attaching it to a disposable plastic syringe.
• the filter was slowly rinsed with the remainder of the beer during unscrew of the filter from the syringe to prevent air (bubbles) in the filter and the connection.
• the filter was attached to male luer lock at the filtration unit exit.
• the top plate of the filtration system was clamped to the filter house following the manufacturer’s instructions. The vent valve at the top plate was closed, and the filtration system was connected to a pressure valve after which a pressure of 1 bar was applied.
• a balance with an accuracy of 0.1g was put below the filtration system.
• a beaker was put on the balance.
• the automatic weight recording was started from weight 0. Thereafter, the valve is opened at an exact timepoint of the automated logging, such that the weight is recorded immediately. Every 10 seconds the weight is recorded for 6-7 minutes in total.
• ADF (Original extract - Apparent extract) / Original Extract *100
• Extract -460,234 + 662,649 x (SG) - 202,414 x (SG)2 wherein SG is the specific gravity of sample after fermentation and maturation (EBC method 9.4 Original, real and apparent extract and original gravity of beer - 2004).
• Haze was measured on fermentation samples after maturation with a Haffman’s VOS Rota 90/25 dual angle turbidity meter. This equipment measures the scattered light caused by particles. Particles smaller than 1 pm mainly cause scattered light and are measured under a 90° angle. Particles larger than 1 pm mainly cause forward-scattered light and are measured under a 25° angle. The measurement was performed in the provided cuvette by the manufacturer of the used equipment (EBC method 9.29 Haze in beer: calibration of haze meters-2015).
• glucanases The product obtained after the reaction with the cellulases (glucanases) is a glucan.
• the level of glucan in wort and fermentation samples after maturation was determined by Enzytec Color GlucaTest from R-Biopharm AG (EBC 4.16.3, MEBAK 3.1.4.9.2) according to the manufacturer’s instructions (protocol 06.04.2017). This method is accepted and recommended for inclusion in Analytical-EBC since 2005.
• Example 1 Use of the combination of a prolyl endoprotease and a cellulase during beer fermentation
• Table 2 Applied mashing profile for grist consisting of 60w% malt and 40w% barley
• the weight of the mash suspension was adjusted to 450 grams with hot tap water.
• the suspension was stirred and poured onto a folded filter (Macherey-Nagel; MN 614 % 0 320 mm REF 527032) in a glass funnel placed on a 500 ml Scott flask for collection of the filtrate.
• the filtration was stopped when the filter bed in the folded filter ran dry by gravity.
• the first 100 ml filtrate was collected and was fed back on top of the filter bed.
• the total collected filtrate, wort was cooled down to room temperature.
• the resulting wort solution had a gravity of 14.6 °P, a specific gravity of 1 .059582 g/cm3 and a fermentable sugar content of 95.8 g/L.
• the total wort generated via 16 individual mashes was divided into 300-gram portions in Scott flasks.
• the wort was cooled to 14°C after which the yeast (Saflager S23, Fermentis) was dosed at 100 g/hl and the enzyme(s) was (were) applied according to Table 3. Thereafter, the fermentation was performed for 8 days at 14°C and 100 rpm using an ANKOM gas fermentation system During fermentation, the cumulative pressure and the temperature were logged using the ANKOM RF Gas Production System (Ankom Technology).
• Table 3 Dosage of commercial enzyme to cold wort [142] At the end of the fermentation, beer maturation was performed by refrigeration at 0°C for 3 days. After maturation, the samples were centrifuged at 11 ,600g and 4°C for 10 min to remove the yeast. The supernatant was poured off and degassed by magnetic stirring at rT for 1 hour. This clarified beer was used for the compositional analysis and the physical analysis.
• the sugar composition of the final beer can have an influence on the taste perception.
• Mono-, di-, and tri-saccharides have a sweet taste, and are known to enhance fruity flavours.
• an increase in the presence of fermentable sugars in the final beer as can be detected in Experiments 1 B and 2B that are treated with a prolyl endoprotease and a cellulase, will favourably influence the flavour of the beer.
• glucan could be clearly detected in each of the control samples, “0”, “1 ” and “2”, in amounts ranging from 20 to 35 mg/L. Glucans can be problematic for filtration and may give haze/precipitates in the final beer.
• glucan was below detection limit in all experimental samples that contained cellulase activity. This indicates that all cellulases had been active during beer fermentation to further break down the glucan into the glucose. Glucose is sweeter than glucan and may also enhance fruity flavours, as explained also above. It can thus be concluded that during the fermentation in experiments 1A, 1 B, 1C, 1 D and 2A, 2B, 2C and 2D, in addition to the prolyl endoprotease activity, there has also been cellulase activity. The combination of these led to an improved break-down of glucan into the desired glucose, and an improved taste.
• Table 5 Haze formation measured at a 90° or 25° angle.
• Fouling rate was calculated from the data and is depicted in Table 6.
• Table 6 Shows that the fouling of membranes during clear beer filtration was reduced.
• filtration can be operated at an increased speed (cq pressure) gaining a process advantage compared to a regular clear beer filtration.
• cq pressure a speed to which different cellulases reduce filter fouling.
• Most effective in reducing fouling seems to be the cellulase of Trichoderma reesei. This is surprising since in the samples treated with the cellulase of Trichoderma reesei the haze formation before filtration was higher than in the controls with only prolyl endoprotease (see Table 5).

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Food Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Polymers & Plastics (AREA)
• Biotechnology (AREA)
• Nutrition Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biomedical Technology (AREA)
• Mycology (AREA)
• Medicinal Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Enzymes And Modification Thereof (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Applications Claiming Priority (8)

Publications (1)

Family

ID=92903444

Family Applications (1)

Country Status (4)

Family Cites Families (27)

• 2024
  • 2024-03-27 EP EP24713502.3A patent/EP4687490A1/de active Pending
  • 2024-03-27 WO PCT/EP2024/058288 patent/WO2024200534A1/en not_active Ceased
  • 2024-03-27 CN CN202480021721.2A patent/CN120957611A/zh active Pending
  • 2024-03-27 WO PCT/EP2024/058289 patent/WO2024200535A1/en not_active Ceased
• 2025
  • 2025-09-25 MX MX2025011394A patent/MX2025011394A/es unknown

Also Published As

Similar Documents

Legal Events