Enzyme composition and beer manufacturing process

Description

1 / 47 ENZYME COMPOSITION AND BEER MANUFACTURING PROCESS FIELD OF THE INVENTION

[0001] The invention relates to a new enzyme composition and a new brewing process. BACKGROUND OF THE INVENTION

[0002] Traditional beer production processes comprise a first hydrolysis step, in which starch is hydrolyzed into saccharides, followed by a second distinct fermentation step, in which these saccharides are subsequently fermented into alcohol. Fermentation is traditionally carried out with yeast cells.

[0003] For example, document WO2013 / 167573 describes a method for 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 this 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 may be a metalloprotease, a proline-endoprotease or a glutamine-endoprotease. In example 2, Ondea Pro (commercially available from Novozymes), a metalloprotease and an exopeptidase were added in a mashing step.According to document WO2013 / 167573, mashing is the process of converting the starch from milled barley malt and solid adjuncts into fermentable and non-fermentable sugars to produce wort with the desired composition. After mashing, when all the starch has been broken down, it is necessary to separate the liquid extract (the wort) from the residual solids (spent grains) and boil it. In this patent document, several substances, including various proteins, were denatured. After cooling and removal of the precipitates, the... Petition 870250086005, dated 09 / 23 / 2025, p. 38 / 92 2 / 47 of the wort was aerated and fermented to produce beer. Due to boiling, no live enzymes could be present during fermentation.

[0004] However, more recently, homebrewers have also started using a process called SSF (simultaneous saccharification and fermentation). In this process, the hydrolysis step (also known as the saccharification step) and the fermentation step are carried out simultaneously.

[0005] An example of such a simultaneous saccharification and fermentation process is described by Baltaci et al., in their article entitled The simultaneous saccharification and fermentation of malt dust and use in the acidification of mash, published online with DOI 10.1002 / jib.554 on February 26, 2019. They describe how an SSF process combines the saccharification of cellulosic materials using cellulolytic enzymes, which generate the sugars, and the fermentation that consumes them. The process is also preferable since it reduces the reaction volume and process time. Baltaci et al. discuss the issues of malt dust and wort acidification in the process.

[0006] In addition, some special alcoholic beverages, such as sake, can be produced in a process where saccharification and fermentation occur simultaneously in the same fermentation tank.

[0007] Turbidity is a well-known phenomenon in the brewing industry. Turbidity formation in a brewing process can occur at different stages. In the book Enzymes in Food Processing edited by T. Nagodawithana and G. Reed, 3rd edition, Academic Press Inc., San Diego, Chapter V, pp. 448-449, it was proposed that turbidity in beer is a result of interactions between beer proteins and polyphenolic procyanidins.

[0008] Document WO2002046381 describes a method for preventing or reducing turbidity in a beverage, in which a Petition 870250086005, dated 09 / 23 / 2025, page 39 / 92 3 / 47 A prolyl-specific endoprotease (also known as PEP) is added to the beverage. Document WO2002046381 explains that the activity of prolyl-specific endoproteases is pH-dependent. The text describes, for example, that an endoprotease is added to a beverage with maximum prolyl-specific activity at a pH that corresponds to the pH of the beverage to which it is added. Since turbidity formation frequently occurs in acidic beverages, such as beer, wine, and fruit juice, prolyl-specific endoproteases with prolyl-specific activity at a pH value below 7 are preferentially used. Document WO2002046381 subsequently presents several prolyl-specific endoproteases that have since been very successful in reducing turbidity in beverages.

[0009] The process as described in document WO2002046381 provides a good solution for turbidity in traditional brewing processes.

[0010] However, in the SSF processes described above, the turbidity in beer may have a different composition and may be caused by a mixture of components. Turbidity may not only comprise the interactions mentioned above between beer proteins and polyphenolic procyanidins, but may also include fine particulate matter and / or polysaccharides resulting from the raw material. The role of polysaccharides in turbidity formation is not well understood, but studies have shown effects of polysaccharides in this turbidity formation.

[0011] Providing an enzyme composition and / or a process that allows for the acceleration or otherwise improvement of an SSF process; and / or accelerating or otherwise improving the removal of fine particulate matter in the fermentation broth and / or fermentation tank; and / or improving the flavor of the alcoholic beverage product would be an advance in the art. Petition 870250086005, dated 09 / 23 / 2025, page 40 / 92 4 / 47 SUMMARY OF THE INVENTION

[0012] A new enzyme composition, a new process applying this new enzyme, and a new method for producing this new enzyme composition were then discovered, which allow for accelerating or otherwise improving an SSF process and / or accelerating or otherwise improving the removal of fine particulate matter in the fermentation broth and / or fermentation tank and / or improving the flavor of the alcoholic beverage product.

[0013] Consequently, in a first aspect, the invention provides a composition (herein also referred to as an 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 a prolyl-specific protease, preferably a glutamine-specific endoprotease and / or a prolyl-specific endoprotease, primarily a prolyl-specific endoprotease; and - a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.

[0014] Preferably, the second enzymatic component is derived from and / or produced by Trichoderma reesei. More preferably, the cellulolytic enzyme, preferably cellulase, is derived from and / or produced from Trichoderma reesei. As illustrated by the examples, a cellulolytic enzyme, suitably cellulase, that is derived from and / or produced from Trichoderma reesei, produces the best results. More preferably, the first enzymatic component and the second enzymatic component are both derived from and / or produced by the same organism, preferably Trichoderma reesei, which provides the benefit of efficient production. Petition 870250086005, dated 09 / 23 / 2025, page 41 / 92 5 / 47

[0015] Therefore, preferably, the first aspect provides a composition comprising or consisting of: - a first enzymatic component, comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease; and - 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 Trichoderma reesei.

[0016] In a second aspect, the invention provides a process for the preparation comprising the use of the novel enzyme composition described above.

[0017] In a third aspect, the invention provides a process for producing the novel enzyme composition described above.

[0018] Without adhering to any particular theory, it is believed that cellulase may be useful for converting small residues of raw material in an SSF process and that the said invention may therefore help to improve, accelerate or otherwise enhance an SSF process of home brewing and / or accelerate or otherwise enhance the removal of fine particulate matter in the fermentation broth and / or fermentation tank and / or enhance the flavor of a (homebrewed) beer would be an advance in the art.

[0019] The invention is illustrated by examples.

[0020] The new enzyme composition described above can be advantageously useful in a process for the production of an alcoholic beverage for human consumption, preferably a beer. Therefore, a new process for the production of an alcoholic beverage for human consumption, preferably a new beer-making process, using the new enzyme composition described above, is advantageously provided.

[0021] In a fourth aspect, the present invention provides Petition 870250086005, dated 09 / 23 / 2025, page 42 / 92 6 / 47 thus a process for the production of an alcoholic beverage for human consumption, preferably a brewing process, comprising the addition of a composition as described above or the addition of a bacterial or fungal strain, preferably a strain of Trichoderma reesei, as described below.

[0022] More preferably, the present invention provides a process for producing an alcoholic beverage for human consumption, preferably a brewing process, comprising the addition of a composition as described above, wherein the composition comprises or consists of: - a first enzymatic component, comprising or consisting of a glutamine-specific protease and / or a prolyl-specific protease, preferably a glutamine-specific endoprotease and / or a prolyl-specific endoprotease, primarily a prolyl-specific endoprotease; and - a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.

[0023] Furthermore, in a fifth aspect, the present invention provides an alcoholic beverage for human consumption obtained or that can be obtained by the above process. DESIGNS

[0024] The invention is illustrated with the following figures.

[0025] Figure 1, illustrating particle removal filtration performance in different beer samples. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0026] This application contains a Sequence Listing in a machine-readable format, which is incorporated herein by reference. An overview of the sequences is presented in the Table below. TABLE: Overview of sequence listings: Petition 870250086005, dated 09 / 23 / 2025, page 43 / 92 7 / 47 SEQ ID No: Enzyme / Gene Name Origin Type SEQ ID NO: 1 Endoprotease described in SEQ ID NO: 5 in document WO2002046381 Aspergillus niger Protein SEQ ID NO: 2 Endoprotease described in SEQ ID NO: 1 in document WO2022 / 266456 Aspergillus bertholletius Protein SEQ ID NO: 3 Endoprotease described in SEQ ID NO: 2 in document WO2022 / 266456 Aspergillus niger Protein SEQ ID NO: 4 Endoprotease described in SEQ ID NO: 3 in document WO2022 / 266456 Aspergillus transmontanensis Protein SEQ ID NO: 5 Endoprotease described in SEQ ID NO: 4 in document WO2022 / 266456 Aspergillus homomorphus Protein SEQ ID NO: 6 Endoprotease described in SEQ ID No. 16 in document WO2022 / 266456 Aspergillus niger Protein SEQ ID No. 7 endoprotease described in SEQ ID No. 4 in document WO2002046381 Aspergillus niger Protein SEQ ID No. 8 endoprotease described in SEQ ID No. 7 in document WO2002046381 Aspergillus niger Protein

[0027] Preferably, the endoprotease in the present invention is a polypeptide having an amino acid sequence of 40% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, much more preferably 95% or more, and preferentially equal Petition 870250086005, dated 09 / 23 / 2025, p. 44 / 92 8 / 47 or more than 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 above, giving priority to SEQ ID NO: 1. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0028] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this document have the same meaning as commonly understood by a person skilled in the art.

[0029] Throughout this descriptive report and the attached claims, the words "understand" and "include" and variations such as "understands," "comprising," "includes," and "including" should be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or whole numbers not specifically mentioned, where the context permits.

[0030] The articles a and an are used here to refer to one or more of (i.e., one or at least one) of the article's grammatical object. As an example, an element can mean one element or more than one element. When referring to a noun (e.g., a compound, an additive, etc.) in the singular, the plural must be included. When referring to a particular fraction, for example, a strain, this means at least one of that strain, e.g., at least one strain, unless otherwise specified.

[0031] When referring to a compound of which there are several isomers (for example, a D and an L enantiomer), the compound, in principle, includes all enantiomers, diastereomers and cis / trans isomers of that compound that can be used in the particular aspect of the invention; in particular, when referring to such a compound, it includes the natural isomer(s).

[0032] Unless explicitly stated otherwise, Petition 870250086005, dated 09 / 23 / 2025, p. 45 / 92 9 / 47 The various embodiments of the invention described herein can be cross-combined.

[0033] The terms peptide and oligopeptide are considered synonymous (as commonly recognized) and each term may be used interchangeably, as the context requires, to indicate a chain of at least two amino acids linked by peptide bonds. The word polypeptide is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences contained herein are written from left to right and in the amino-terminal to carboxy-terminal direction. References to amino acid names mentioned herein refer to amino acids as described in the textbook by Jeremy M. Berg, John L. Tymoczko, and Lubert Stryer, entitled Biochemistry, 6th edition, published in 2007 by W.H. Freeman and Company, New York, USA, Chapter 2. The one-letter code of the amino acids used herein is commonly known in the art and may be found, for example, in the textbook by Jeremy M. Berg, John L.Tymoczko and Lubert Stryer, entitled Biochemistry, 6th edition, published in 2007 by W.H. Freeman and Company, New York, USA, Chapter 2, Table 2.2.

[0034] Nucleic acid (i.e., polynucleotide) or protein (i.e., polypeptide) sequences can be native to or heterologous to the host cell genome.

[0035] With respect to a host cell, native, homologous, or endogenous means that the nucleic acid sequence occurs naturally in the host cell's genome or that the protein is naturally produced by that cell. The terms native, homologous, and endogenous are used interchangeably here.

[0036] As used herein, heterologous, in relation to the host cell, may refer to a polynucleotide that does not naturally occur in that form in the host cell's genome. Petition 870250086005, dated 09 / 23 / 2025, page 46 / 92 10 / 47 or that a polypeptide or protein is not naturally produced in that form by that cell. Heterologous protein expression involves the expression of a protein that is not naturally expressed in that form in the host cell. The term heterologous expression refers to the expression of heterologous nucleic acids in a host cell.

[0037] As used herein, a promoter is a DNA sequence that directs the transcription of a gene (structural) or another (part of) nucleic acid sequence. Properly, a promoter is located in the 5' region of a gene, close to the transcriptional start site of a gene (structural). Promoter sequences can be constitutive, inducing, or repressor. In some embodiments, an inducer (external) is not required. The first enzymatic component

[0038] The composition according to the invention suitably comprises a first enzymatic component, comprising or consisting of a glutamine-specific protease and / or a prolyl-specific protease, preferably a glutamine-specific endoprotease and / or a prolyl-specific endoprotease, primarily a prolyl-specific endoprotease.

[0039] The terms protease, protease enzyme, enzyme with protease activity, protein with protease activity and polypeptide with protease activity, peptidase, peptidase enzyme, enzyme with peptidase activity, protein with peptidase activity and polypeptide with peptidase activity are used interchangeably in this document.

[0040] In addition, the terms endoprotease, endoprotease enzyme, enzyme with endoprotease activity, protein with endoprotease activity and polypeptide with endoprotease activity, endopeptidase, endopeptidase enzyme, enzyme with endopeptidase activity, protein with activity of Petition 870250086005, dated 09 / 23 / 2025, page 47 / 92 11 / 47 endopeptidase and polypeptide with endopeptidase activity are used interchangeably in this document.

[0041] Furthermore, the terms prolyl-specific and proline-specific are used interchangeably in this document. Primarily, the first enzymatic component comprises or consists of a prolyl-specific endoprotease.

[0042] Glutamine-specific protease and / or proline-specific protease, respectively glutamine-specific endoprotease and / or proline-specific endoprotease, may be used in the invention in an isolated or purified form. An isolated or purified form is understood to mean a proline-specific protease removed from its native environment. For example, recombinantly produced proline-specific protease expressed in host cells is considered an isolate for the purposes of the invention, as are native or recombinant polypeptides that have been substantially purified by any suitable technique, such as, for example, the single-step purification method disclosed by Smith and Johnson, Gene 67:31-40 (1988).

[0043] A proline-specific protease suitable for use in the invention can be recovered from recombinant cell cultures by methods well known to those skilled in the art, including, for example, precipitation with ammonium sulfate or ethanol, acid extraction and chromatographic methods such as high-performance liquid chromatography (HPLC).

[0044] A proline-specific protease suitable for use in the invention may be a naturally purified product, a product of chemical synthesis, or a product produced by recombination technique from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, fungal, higher plant, insect, and mammalian cells.

[0045] Preferably, the first enzymatic component is derived from and / or produced by a microorganism, Petition 870250086005, dated 09 / 23 / 2025, page 48 / 92 12 / 47 preferably a bacterium or fungus, primarily Trichoderma reesei. That is, preferably the proline-specific protease, respectively the proline-specific endoprotease, is derived and / or produced by a microorganism, preferably a fungus, primarily Trichoderma reesei.

[0046] It should be understood, however, that the protease may be mixed with carriers or diluents that do not interfere with the intended purpose of the enzyme and still be considered isolated. A proline-specific protease suitable for use in the invention may also be in a substantially purified form. Consequently, the proline-specific protease may be present in a preparation in which more than 70%, for example, more than 80%, 90%, 95%, 98% or 99% of any protease present in the preparation is a proline-specific protease. Primarily, the proline-specific protease may be in a form free from or substantially free from any other protease.

[0047] A proline-specific protease suitable for use in the invention can be used in an immobilized form so that large quantities of protein-containing liquids can be treated. Ways of selecting appropriate support materials and suitable immobilization methods have been extensively described in the literature, for example, in Immobilization of Enzymes and Cells (ed. Gordon F. Bickerstaff; ISBN 0-89603-386-4).

[0048] In the context of the present invention, a prolyl-specific protease or endoprotease, respectively proline-specific, is defined as a protease, respectively endoprotease, that cuts proteins or peptides at sites where the protein or peptide contains a proline residue in its chain. The terms prolyl-specific and proline-specific are used interchangeably in this document. Petition 870250086005, dated 09 / 23 / 2025, page 49 / 92 13 / 47 Preferably, the prolyl-specific protease, or proline-specific protease, is an endoprotease that cleaves (hydrolyzes) proteins or peptides at sites where the protein or peptide contains a proline residue. In the method according to the invention, a proline-specific endoprotease that hydrolyzes the peptide bond at the carboxy-terminal end of the proline residues is preferably used. Examples of such enzymes are prolyl oligopeptidases (EC 3.4.21.26), as well as the prolyl endoprotease derived from Aspergillus niger reported in J. Agic Food Chem, Vol 53 (20), 7950-7957, 2005) and proline-specific dipeptidyl peptidases, such as DPP IV (EC 3.4.14.5). A proline-specific endoprotease that cleaves proline residues at their NH2-terminus is, for example, described in a publication in Nature of January 15, 1998, Vol. 391, pp. 301-304.

[0049] In the context of the present invention, a glutamine-specific protease is a protease that cuts proteins or peptides at sites where the protein or peptide contains a glutamine residue in its chain. Preferably, the glutamine-specific protease is a glutamine-specific endoprotease that cuts (hydrolyzes) proteins or peptides at sites where the protein or peptide contains a glutamine residue.

[0050] In a particularly preferred embodiment, the first enzymatic component comprises or consists of a protease, preferably an endoprotease, which is both glutamine-specific and proline-specific.

[0051] In a further aspect, the invention also provides a composition comprising or consisting of: - a first enzymatic component, comprising or consisting of a protease, preferably an endoprotease, and - a second enzymatic component, comprising or Petition 870250086005, dated 09 / 23 / 2025, pp. 50 / 92 14 / 47 consisting of a cellulolytic enzyme, preferably cellulase, in which the protease, respectively the endoprotease, is: - capable of hydrolyzing a substrate protein or peptide into one or more glutamine residues; and / or - capable of hydrolyzing a substrate protein or peptide into one or more proline residues.

[0052] Preferences for such composition and the first and second enzymatic components are further as described above and below.

[0053] Examples of an enzyme with both glutamine-specific and proline-specific action are the enzymes such as those described in document WO2022 / 266456. The protease, or endoprotease, of the first enzymatic component may also be a precursor, as described, that hydrolyzes a protein or peptide substrate into one or more glutamine residues.

[0054] In a first preferred embodiment, the first enzymatic component includes one or more enzymes having an amino acid sequence that exhibits at least 75, 80, 85, 90, 95, 98, 99, or 100% sequence identity with one or more of the SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and / or SEQ ID NO:16, as described in WO2022 / 266456, or an active endoprotease fragment thereof, such as a mature protein. The sequence listing of these enzymes, as presented in WO2022 / 266456, is incorporated herein by reference. These enzymes may be produced as described in WO2022 / 266456.

[0055] In a second preferred embodiment, the first enzymatic component includes one or more enzymes that have an amino acid sequence that exhibits at least 75, 80, 85, 90, 95, 98, 99, or 100% sequence identity with one or Petition 870250086005, dated 09 / 23 / 2025, pp. 51 / 92 15 / 47 plus SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, as described in document WO2020 / 025704 or an active endoprotease fragment thereof, as a mature protein. The sequence listing of these enzymes, as presented in document WO2020 / 025704, is incorporated herein by reference. These enzymes can be produced as described in document WO2020 / 025704.

[0056] Preferred examples also include the proline-specific proteases described in: - article entitled "Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger" by Miyazono et al., published in Biochemical and Biophysical Research Communications, Volume 591, February 5, 2022, pages 76-81; and - article entitled Extracellular Prolyl Endoprotease from Aspergillus niger and Its Use in the Debittering of Protein Hydrolysates by Edens et al, published in J. Agric. FoodChem. 2005, 53, 7950-7957; and - article entitled Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation by Montserrat et al., published in Biochemical and Biophysical Research Communications 591 (2022) pages 76-81, each incorporated herein by reference.

[0057] Preferably, the composition comprises a first enzymatic component, wherein this first enzymatic component preferably has its optimum activity at a pH in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.5, even more preferably equal to or less than pH 5.0, much more preferably equal to or less than pH 4.5 and preferentially equal to or less than pH 4.0.

[0058] Therefore, preferably, the activity of the endoprotease, preferably the glutamine-specific endoprotease and / or Petition 870250086005, dated 09 / 23 / 2025, pp. 52 / 92 16 / 47 proline-specific, is pH dependent. The endoprotease, preferably the glutamine-specific and / or proline-specific endoprotease, is an endoprotease, respectively glutamine-specific and / or proline-specific endoprotease, with an acidic optimum pH, that is, an optimum pH below pH 7.0. More preferably, the endoprotease, preferably the glutamine-specific and / or proline-specific endoprotease, is an endoprotease, respectively the proline-specific endoprotease, that exhibits its optimal activity at a pH in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.0, even more preferably equal to or less than pH 4.0, much more preferably equal to or less than pH 3.0 and preferentially equal to or less than pH 2.5.Preferably, the endoprotease, preferably the proline-specific endoprotease, is a proline-specific endoprotease that exhibits its optimal activity at a pH in the range of pH 1.0 to pH 5.0, even more preferably in the range of pH 1.2 to pH 4.5.

[0059] Primarily, the endoprotease, preferably glutamine-specific and / or proline-specific, is an endoprotease, more preferably a prolyle-specific endoprotease, primarily as described in EPA-1326957, WO2005027953, WO2002046381 or WO 2022 / 266456, which are incorporated herein by reference.

[0060] Preferably, the endoprotease, preferably a glutamine-specific and / or proline-specific endoprotease, more preferably a prolyl-specific endoprotease, is selected from the group consisting of: (a) a polypeptide having an amino acid sequence of 40% or more, more preferably 60% or more, even more preferably 70% or more, most preferably 80% or more Petition 870250086005, dated 09 / 23 / 2025, p. 53 / 92 17 / 47 preferably equal to or greater than 90%, even more preferably equal to or greater than 95%, and primarily equal to or greater than 99% overall amino acid sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or a fragment thereof, each being as described in document WO2002046381; (b) a polypeptide that is encoded by a polynucleotide that hybridizes with (i) the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or a fragment thereof that is at least 80% or 90% identical over 60, preferably over 100 nucleotides, more preferably at least 90% identical over 200 nucleotides, each being as described in WO2002046381, or (ii) a nucleic acid sequence complementary to the nucleic acid sequence of (i).

[0061] The so-called active site(s) within an enzyme is / are the part(s) of the enzyme that is / are responsible for interacting with the substrate. This interaction may, for example, involve binding and / or catalyzing the reaction on the substrate. The formation and presence of such so-called active site(s) may depend on the primary, secondary, and / or tertiary structure of the enzyme. Examples of active sites include catalytic triplet(s) and / or oxyanionic cavity(ies).

[0062] Preferably, the endoprotease, preferably a glutamine-specific and / or proline-specific endoprotease, is an endoprotease, respectively a glutamine-specific and / or proline-specific endoprotease, primarily a prolyl-specific endoprotease, comprising a catalytic triplet and / or an oxyanionic cavity. More preferably, the endoprotease, preferably a glutamine-specific and / or proline-specific endoprotease, is an endoprotease, respectively a glutamine-specific and / or proline-specific endoprotease, comprising a triplet Petition 870250086005, dated 09 / 23 / 2025, pp. 54 / 92 18 / 47 catalytic and an oxyanionic cavity.

[0063] Preferably, the catalytic triplet comprises an amino acid sequence comprising or consisting of Serine, Histidine, and Aspartic Acid. Such a catalytic triplet is also referred to as a Ser / Asp / His catalytic triplet. In a preferred embodiment of the invention, the nitrogen atom ND1 or Nd1 of the histidine amino acid side chain in the catalytic triplet above is protonated, and the nitrogen atom NE2 or Ne2 (pKa of 6.0) of the histidine amino acid side chain is not protonated. In another preferred embodiment of the invention, the histidine amino acid in the catalytic triplet above is deprotonated. Deprotonated histidine is preferably understood herein to be a histidine amino acid that has lost its proton (H+ ion) at the NE2 or Ne2 nitrogen atom of the side chain.

[0064] An oxyanionic cavity can be understood as a structural feature found in the active site of certain hydrolytic enzymes that stabilizes negatively charged transition states during catalysis, for example, in esterase, lipase, and peptidase enzymes. Appropriately, an oxyanionic cavity can stabilize a negative charge in the transition state on a deprotonated oxygen or alkoxide. Stabilization of this transition state can advantageously reduce the activation energy required for the reaction and thus promote catalysis. Oxyanionic cavities in hydrolytic enzymes are typically composed of hydrogen bond donor groups from polar or basic amino acid side chains, such as Tyr, Trp, Arg, Lys, Asn, Gln, Ser, Thr, or also frequently two amides of the main structure that hydrogen bond with the oxygen atoms of the negatively charged transition state.

[0065] However, in the composition according to the invention, the enzymatic component preferably comprises or consists of an endoprotease, preferably a prolyl endoprotease. Petition 870250086005, dated 09 / 23 / 2025, pp. 55 / 92 19 / 47 specific, comprising an oxyanionic cavity comprising a protonated side chain of an acidic amino acid. More preferably, this oxyanionic cavity comprises a glutamic acid amino acid, preferably in its protonated state (Glu-H).

[0066] The proline-specific protease is preferably produced in or derived from a microorganism, preferably a bacterium or a fungus. The proline-specific protease can be a natural, recombinant, or chemically modified proline-specific protease enzyme. Proline-specific proteases have been identified, for example, in species of Aspergillus (EP 0 522 428), Flavobacterium (EP 0 967 285), Aeromonas (J. Biochem. 113, 790-796), Xanthomonas, and Bacteroides. The proline-specific protease of the invention can be isolated from one of the microbial species mentioned above, particularly from a species of Aspergillus or Trichoderma. Preferably, the proline-specific endoprotease is isolated from a strain of Aspergillus niger or Trichoderma reesei. More preferably, the proline-specific endoprotease is isolated from an Aspergillus niger or Trichoderma reesei host genetically modified to overexpress a gene encoding a proline-specific endoprotease.

[0067] Other hosts, such as E. coli, may also be useful. For example, cloning and overproduction of proline-specific endoprotease derived from Flavobacterium, among others, E. coli, has made certain proline-specific endoproteases available in a pure form. An example of such an overproducing construct is presented in the World Journal of Microbiology & Biotechnology, Vol 11, pages 209-212.

[0068] Enzymes can be native to or heterologous to the host cell. In a preferred embodiment, the enzyme can be native to an Aspergillus species and be produced in that Aspergillus species, preferably Aspergillus niger. In Petition 870250086005, dated 09 / 23 / 2025, p. 56 / 92 20 / 47 another preferred embodiment, the enzyme can be derived from an Aspergillus species and the enzyme can be produced heterologously in another host cell, such as a Trichoderma reesei.

[0069] Preferred glutamine-specific and / or prolyl-specific endoproteases include glutamine-specific and / or prolyl-specific endoproteases derived from Aspergillus niger, Aspergillus transmontanensis, Aspergillus homomorphus, Aspergillus pseudocaelatus, Aspergillus neoauricomus, Aspergillus albertensis, Aspergillus albertensis, Aspergillus wentii, Aspergillus brasiliensis, Aspergillus sclerotioniger, Aspergillus bertholletius, Aspergillus awamori, Aspergillus tubegensis, Aspergillus accretis, Aspergillus foretidus, Aspergillus nidulans, Aspergillus japonica. Others include Oryzae and Aspergillus ficuum, and Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabrames, Myceliaceae Thermophilum.

[0070] Preferably, the compositions as described herein are compositions in which the first enzymatic component is derived from and / or produced by a microorganism, preferably a bacterium or fungus, primarily Trichoderma reesei.

[0071] In this context, glutamine-specific and / or prolyl-specific endoproteases are especially preferred, which are derived from an Aspergillus species, but are produced heterologously in a Trichoderma species, preferably Trichoderma reesei.

[0072] In another preferred embodiment, the host Aspergillus niger or the host Aspergillus niger or Trichoderma reesei is preferably used to produce a non-recombinant self-construct that uses A. niger promoters to target the expression of a gene encoding an A. niger-specific proline endoprotease. That is, Petition 870250086005, dated 09 / 23 / 2025, p. 57 / 92 21 / 47 preferentially the endoprotease, preferentially the proline-specific endoprotease, is a non-recombinant endoprotease, respectively a non-recombinant proline-specific endoprotease. The second enzymatic component

[0073] The composition further comprises a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.

[0074] The second enzymatic component is preferably derived from and / or produced by a microorganism, preferably a bacterium or fungus, primarily Trichoderma reesei.

[0075] As used herein, a cellulolytic enzyme is an enzyme capable of partially or completely degrading cellulose. This enzymatic process is also known as cellulolysis. Cellulose is a polysaccharide comprising a chain of beta-1,4-linked D-glucose monomeric units. Suitably, a cellulose polysaccharide may comprise in the range of 20 or more, more preferably 50 or more, even more preferably 100 or more, much more preferably 1000 or more, up to suitably 1,000,000 or less, preferably 100,000 or less monomeric units. The beta-1,4 linkages in cellulose are difficult to break, but can be broken by cellulolytic enzymes. A preferred cellulolytic enzyme is cellulase.

[0076] As used herein, a cellulase is any polypeptide with cellulase activity. A cellulase can appropriately be any enzyme capable of hydrolyzing beta-1,4-D-glycosidic linkages in cellulose. This hydrolysis advantageously allows for the partial or complete degradation of cellulose. A polypeptide capable of degrading cellulose is one that is capable of catalyzing the breakdown process. Petition 870250086005, dated 09 / 23 / 2025, pp. 58 / 92 22 / 47 of cellulose into smaller units, either partially, for example, into cellodextrins, or completely into glucose monomers. A cellulase according to the present invention can give rise to a mixed population of cellodextrins and glucose monomers. Such degradation will normally occur by means of a hydrolysis reaction.

[0077] The terms cellulase-active polypeptide, cellulase enzyme, or simply cellulase are used interchangeably herein. As indicated above, advantageously, such a cellulase can hydrolyze beta-1,4-D-glycosidic linkages in cellulose, partially or completely degrading the cellulose. The cellulase can be an exocellulase, endocellulase, or cellobiase. Preferably, the cellulase is an endocellulase or cellobiose, with priority given to the cellulase being an endocellulase.

[0078] The composition may comprise one, two, three, four, five, six or more (types of) cellulases. The composition may contain any cellulase, for example, a polysaccharide lytic monooxygenase (e.g., GH61), a cellobiohydrolase, an endo-β-1,4-glucanase, a beta-glucosidase or a β(1,3)(1,4)-glucanase.

[0079] As used herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide capable of catalyzing the hydrolysis of 1,4-εD-glycosidic linkages in cellulose or cellotetraose, releasing cellobiose from the chain ends. This enzyme may also be referred to as cellulase 1,4-β-cellobiosidase, 1,4-ε-cellobiohydrolase, 1,4-εD-glucan cellobiohydrolase, avicelase, exo-1,4-εD-glucanase, exocellobiohydrolase, or exoglucanase. Preferably, the cellulase is a mixture of cellulase enzymes comprising one or more glucanase enzymes, more preferably one or more beta-glucanase enzymes.

[0080] As used herein, an endo-ε-1,4-glucanase (EC Petition 870250086005, dated 09 / 23 / 2025, pp. 59 / 92 23 / 47 3.2.1.4) is preferred and is any polypeptide capable of catalyzing the endohydrolysis of 1,4-β-D-glycosidic linkages in cellulose, lichenin, or cereal β-D-glucans. Such a polypeptide may also be capable of hydrolyzing 1,4 linkages in β-D-glucans that also contain 1,3 linkages. This enzyme may also be referred to as cellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase, carboxymethylcellulase, celludextrinase, endo-1,4-β-D-glucanase, endo-1,4-β-D-glucan hydrolase, endo-1,4-β-glucanase, or endoglucanase.

[0081] As used herein, a beta-glucosidase (EC 3.2.1.21) is also preferred and is any polypeptide capable of catalyzing the hydrolysis of non-reducing terminal β-D-glucose residues, releasing β-D-glucose. Such a polypeptide may have broad specificity for β-D-glycosides and may also hydrolyze one or more of the following: a β-D-galactoside, an α-L-arabinoside, a β-D-xyloside, or a β-D-fucoside. This enzyme may also be referred to as amygdalase, β-D-glucoside glycohydrolase, cellobiase, or gentobiase.

[0082] As used herein, a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is also preferred and is any polypeptide capable of catalyzing the hydrolysis of 1,4-β-D-glycosidic linkages into β-D-glucans containing both 1,3- and 1,4- linkages. Such a polypeptide may act on lichenin and cereal β-D-glucans, but not on β-D-glucans containing only 1,3- or 1,4- linkages. This enzyme may also be referred to as licheninase, 1,3-1,4-β-D-glucan 4-glucan hydrolase, βglucanase, endo-β-1,3-1,4-glucanase, lichenase, or mixed-linkage β-glucanase. An alternative to this type of enzyme is EC 3.2.1.6, which is described as endo-1,3(4)-beta-glucanase. This type of enzyme hydrolyzes 1,3- or 1,4- linkages in beta-D-glucans, when the glucose residue, whose reducing group is present, is affected. Petition 870250086005, dated 09 / 23 / 2025, pp. 60 / 92 24 / 47 involved in the linkage to be hydrolyzed 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)-glucan hydrolase. Substrates include laminarin, lichenin, and cereal beta-D-glucans. β-(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 secondary enzymatic components.

[0083] In view of the foregoing, the second enzymatic component comprises or consists preferably of a glucanase, more preferably a beta-glucanase (also known as βglucanase), primarily a beta-glucanase derived from Trichoderma reesei.

[0084] Suitable cellulases may be derived from a plant, an animal, or a microorganism. Preferably, the cellulase is derived from a microorganism, such as a fungus or a bacterium. The preferred cellulases are those derived from a bacterium or a fungus. The most preferred is a cellulase derived from Trichoderma reesei.

[0085] That is, preferably the second enzymatic component, preferably the cellulase, more preferably a beta-glucanase, is derived from and / or produced by a microorganism, preferably a fungus, and primarily a Trichoderma reesei.

[0086] Preferably, cellulase activity is pH dependent.

[0087] Preferably, the composition comprises a second enzymatic component, wherein this second enzymatic component preferably has its optimum activity at a pH in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.5, even more preferably equal to or less than pH 5.0, much more preferably equal to or less than pH 4.5 and preferentially equal to or less than pH 4.0. Petition 870250086005, dated 09 / 23 / 2025, pp. 61 / 92 25 / 47

[0088] Therefore, preferably, the cellulase is a cellulase with an acidic optimum pH, that is, an optimum pH below 7.0. More preferably, the cellulase is a cellulase that exhibits its optimum activity at a pH in the range of pH 1.0 or higher, more preferably pH 1.2 or higher, and pH 6.0 or lower, more preferably pH 5.0 or lower, even more preferably pH 4.0 or lower, much more preferably pH 3.0 or lower, and preferentially pH 2.5 or lower. Preferably, the cellulase is a cellulase that exhibits its optimum activity at a pH in the range of pH 1.0 to pH 5.0, even more preferably in the range of pH 1.2 to pH 4.5.

[0089] The cellulosic enzyme, preferably cellulase, is preferably produced in or derived from a microorganism, preferably a bacterium or a fungus. The cellulosic enzyme, preferably cellulase, may be a natural, recombinant, or chemically modified cellulosic enzyme, respectively cellulase. Examples of suitable cellulases 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 cellulases are produced in or derived from Trichoderma.

[0090] The cellulolytic enzyme, namely cellulase, can be produced recombinantly in a heterologous expression system, such as a microbial or fungal heterologous expression system. Examples of suitable heterologous expression systems include bacterial (e.g., E. coli, Bacillus sp.) and eukaryotic systems. Eukaryotic systems may employ yeasts (e.g., Pichia sp., Saccharomyces sp.) or fungal expression systems (e.g., Trichoderma sp. such as T. reesei, Aspergillus species such as A. niger). Petition 870250086005, dated 09 / 23 / 2025, pp. 62 / 92 26 / 47 Among these fungal expression systems, Trichoderma reesei is particularly preferred. For example, a cellulase can be conveniently produced as described in U.S. Patent Nos. 4435307, 5776757, and 7604974, which are incorporated herein by reference.

[0091] Suitable examples of cellulases are described in U.S. Pat. Nos. 4435307, 5648263, 5691178, 5776757, 6562612 and 7604974 and are incorporated herein by reference.

[0092] The preferred cellulases are the cellulases disclosed in U.S. Pat. Nos. 4689297, 5814501, 5324649 and International Patent Application Publications Nos. WO92 / 06221 and WO92 / 06165, which are also incorporated herein by reference.

[0093] Exemplary embodiments of 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 composition of enzymes

[0094] Preferably, the 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 prolyl-specific endoprotease; and - a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase; wherein the molar ratio of moles of cellulolytic enzyme, preferably cellulase, to moles of 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, much more preferably equal to or less than 0.1:1, even more preferably equal to or less than 0.05:1, and preferentially equal to or less than 0.01:1. Petition 870250086005, dated 09 / 23 / 2025, pp. 63 / 92 27 / 47

[0095] More preferably, the 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 prolyl-specific endoprotease; and - a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase; where 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: - equal to or greater than 0.1:1, more preferably equal to or greater than 0.5:1, even more preferably equal to or greater than 1:1, much more preferably equal to or greater than 2:1, and even more preferably equal to or greater than 3:1; and / or - equal to or less than 1000:1, more preferably equal to or less than 100:1, even more preferably equal to or less than 50:1, and much more preferably equal to or less than 20:1.

[0096] In a particularly preferred embodiment, 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.

[0097] Preferably, the cellulolytic enzyme, preferably cellulase, is present in an amount equal to or greater than 0.001% w / w, more preferably equal to or greater than 0.005% w / w, even more preferably equal to or greater than 0.01% w / w, and preferentially equal to or greater than 0.05% w / w, based on the total weight of the prolyl-specific protease, preferably a prolyl-specific endoprotease. Petition 870250086005, dated 09 / 23 / 2025, pp. 64 / 92 28 / 47

[0098] Preferably, the cellulolytic enzyme, preferably cellulase, is present in an amount equal to or less than 100% w / w, more preferably equal to or less than 50% w / w, even more preferably equal to or less than 10% w / w, and preferentially equal to or less than 5% w / w, based on the total weight of the prolyl-specific protease, preferably a prolyl-specific endoprotease. These proportions are advantageous in view of the differences in the presence of polysaccharides and other components in the turbidity.

[0099] The composition, according to the present invention, is preferably a composition in which the first enzymatic component and the second enzymatic component are derived from or produced by the same microorganism, preferably by the same bacterium or fungus, primarily by the same Trichoderma reesei.

[0100] Preferably, the enzyme composition is a liquid composition. Preferably, the enzyme composition further comprises a solvent.

[0101] Preferably, the solvent comprises or consists of water. In addition to water, one or more cosolvents may or may not be present. Preferably, the composition comprises water and, optionally, one or more cosolvents. If a cosolvent is present, the solvent may comprise water and one or more cosolvents. Preferred cosolvents 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.

[0102] The solvent may or may not comprise glycerol. Preferably, the solvent comprises less than 50% v / v, more preferably less than 40% v / v, even more preferably Petition 870250086005, dated 09 / 23 / 2025, pages 65 / 92 29 / 47 less than 30% v / v, much more preferably less than 20% v / v, primarily less than 10% v / v of glycerol, based on the total volume of the solvent. Preferably, the solvent does not comprise glycerol. More recently, glycerol has been subject to high price volatility and is therefore less preferred. The use of a solution stabilizer and / or osmolyte, as described herein, advantageously allows for a reduction in the amount of glycerol in the solvent, while still maintaining good stability and / or activity and / or preventing precipitation.

[0103] Preferably, the composition has a pH value in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.5, even more preferably equal to or less than pH 5.0, much more preferably equal to or less than pH 4.5 and preferentially equal to or less than pH 4.0.

[0104] More preferably, the composition is a liquid composition having a pH in the range of pH 1.0 or higher, more preferably pH 1.2 or higher to pH 6.0 or lower, more preferably pH 5.0 or lower, even more preferably pH 4.0 or lower, much more preferably pH 3.0 or lower, and most preferably pH 2.5 or lower. More preferably, the composition is a liquid composition having a pH in the range of pH 1.0 to pH 5.0, even more preferably in the range of pH 1.2 to pH 4.5.

[0105] In an alternative embodiment, the enzyme composition may be provided in a frozen or lyophilized form and reconstituted prior to application by the addition of a solvent, as described above, during preprocessing.

[0106] In addition, the enzyme composition may comprise one or more additional components, preferably chosen Petition 870250086005, dated 09 / 23 / 2025, pp. 66 / 92 30 / 47 of the group consisting of: - a salt, preferably chosen from the group consisting of sodium chloride, potassium chloride and ammonium sulfate; - a carboxylic acid, preferably having in the range of 1 or more to 10 or fewer carbon atoms, preferably in the range of 1 or more to 7 or fewer 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; - a glyceryl monoacetate, glyceryl diacetate, glyceryl triacetate, glyceryl monopropionate, glyceryl dipropionate, glyceryl tripropionate, glyceryl monobutanoate, glyceryl dibutanoate, glyceryl tributanoate, glyceryl monolactate, glyceryl dilactate, glyceryl trilactate glyceryl; - a monosaccharide, a disaccharide and / or an alcohol derivative or chloride derivative of such monosaccharide or disaccharide; - a sorbitol, mannitol, inositol, trehalose, sucrose, mannose and / or sucralose, primarily sorbitol; - maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, amylopectin and mixtures thereof.

[0107] As indicated above, the composition may or may not include glycerol.

[0108] In addition, the composition may or may not include benzoic acid or a derivative thereof. [010 9] The composition may or may not include one or more additional enzymes besides the endoprotease enzyme, such as, for example, acetolactate decarboxylase (ALDC), aminopeptidase or trehalase, glucoamylase, xylanase, maltogenic alpha-amylase, Petition 870250086005, dated 09 / 23 / 2025, pp. 67 / 92 31 / 47 pullulanase, catalase, or transglycosidase. In a preferred embodiment, the composition comprises one or more additional enzymes, preferably acetolactate decarboxylase (ALDC), glucoamylase, and / or xylanase. In a more preferred embodiment, the composition comprises or consists of the first enzymatic component, the second enzymatic component, and xylanase. In a second more preferred embodiment, the composition comprises or consists of the first enzymatic component, the second enzymatic component, and acetolactate decarboxylase (ALDC). In a third more preferred embodiment, the composition comprises or consists of the first enzymatic component, the second enzymatic component, and glucoamylase. Other realizations

[0110] The invention further provides a bacterial or fungal strain, preferably a strain of Trichoderma reesei, which functionally expresses: - a first nucleic acid sequence encoding a first enzymatic component, comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease; and - a second nucleic acid sequence encoding a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.

[0111] In such a bacterial or fungal strain, preferably a strain of Trichoderma reesei, preferably the first nucleic acid sequence is: - operationally linked to a promoter; or - present in 2, 3, 4, 5, 6 or more copies.

[0112] Therefore, the invention also provides a bacterial or fungal strain, preferably the Trichoderma reesei strain, which functionally expresses: - a first nucleic acid sequence that codes for a Petition 870250086005, dated 09 / 23 / 2025, pp. 68 / 92 32 / 47 first enzymatic component, comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease; and - a second nucleic acid sequence encoding a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase. where the first nucleic acid sequence is: - operationally linked to a promoter; or - present in 2, 3, 4, 5, 6 or more copies.

[0113] The invention further provides a bacterial or fungal strain, preferably a strain of Trichoderma reesei, which functionally expresses: - a first nucleic acid sequence encoding a first recombinant enzymatic component, comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease; and - a second nucleic acid sequence encoding a second naturally occurring enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.

[0114] In such a bacterial or fungal strain, preferably a strain of Trichoderma reesei, preferably the first nucleic acid sequence is: - operationally linked to a promoter; or - present in 2, 3, 4, 5, 6 or more copies.

[0115] The above may cause the bacterial or fungal strain, preferably a strain of Trichoderma reesei, to advantageously produce the first enzymatic component in a greater quantity than the second enzymatic component.

[0116] Preferably, the first nucleic acid sequence is a heterologous nucleic acid sequence and the first enzymatic component is expressed heterologously. Preferably, the second nucleic acid sequence is a Petition 870250086005, dated 09 / 23 / 2025, pp. 69 / 92 33 / 47 endogenous nucleic acid sequence and the second enzymatic component is expressed endogenously. Process

[0117] The invention also provides a process for preparing an alcoholic beverage, preferably a beer, comprising the use of the novel enzyme composition described above.

[0118] 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. Preferably, such a process is a simultaneous saccharification and fermentation (SSF) process.

[0119] More preferably, a process is provided 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 strain of Trichoderma reesei, as described above.

[0120] A wide variety of beer production processes are used worldwide. However, every beer-making process generally comprises saccharification and fermentation. During saccharification, a raw material, such as starch, can be converted into saccharides. During fermentation, such saccharides can be converted into ethanol (alcohol). Depending on the efficiency of saccharification, it may be beneficial to have the second enzymatic component, preferably cellulase, present during fermentation, according to the invention. The invention, therefore, also provides a beer-making 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.

[0121] Primarily, the process is a beer-making process, in which a composition according to the Petition 870250086005, dated 09 / 23 / 2025, pp. 70 / 92 34 / 47 The invention is added before or during fermentation, more preferably before fermentation, primarily to the wort.

[0122] In a particularly preferred embodiment, such a brewing process is a simultaneous saccharification and fermentation (SSF) process. Primarily, the composition is added in such a process during the simultaneous saccharification and fermentation process.

[0123] The composition according to the invention is therefore 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 brewing process, the composition of which comprises or consists of: - a first enzymatic component, comprising or consisting of a glutamine-specific protease and / or a prolyl-specific protease, preferably a glutamine-specific endoprotease and / or a prolyl-specific endoprotease; and - a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase.

[0124] Preferably, the first enzymatic component is added to the processes, as described herein, at a dosage equal to or greater than 0.05 grams per hectoliter (g / hL), more preferably equal to or greater than 0.1 g / hL, even more preferably equal to or greater than 0.5 g / hL and preferably equal to or greater than 1.0 g / hL and / or preferably equal to or less than 100 g / hL, more preferably equal to or less than 50 g / hL, preferably equal to or less than 10 g / hL. Preferably, the first enzymatic component is added before or during fermentation, preferably before fermentation, preferably to the wort in a manufacturing process of Petition 870250086005, dated 09 / 23 / 2025, pp. 71 / 92 35 / 47 beer.

[0125] Preferably, the second enzymatic component is added to the processes, as described herein, at a dosage equal to or greater than 0.001 grams per hectoliter (g / hL), more preferably equal to or greater than 0.01 g / hL, even more preferably equal to or greater than 0.05 g / hL and preferentially equal to or greater than 0.1 g / hL and / or preferably equal to or less than 50 g / hL, more preferably equal to or less than 10 g / hL, much more preferably equal to or less than 5 g / hL and preferentially equal to or less than 1 g / hL.

[0126] Primarily, 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: - equal to or greater than 0.1:1, more preferably equal to or greater than 0.5:1, even more preferably equal to or greater than 1:1, much more preferably equal to or greater than 2:1, and even more preferably equal to or greater than 3:1; and / or - equal to or less than 1000:1, more preferably equal to or less than 100:1, even more preferably equal to or less than 50:1, and much more preferably equal to or less than 20:1.

[0127] The addition of the 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 production process. Primarily, the first enzymatic component and the second enzymatic component are added in such a ratio before or during fermentation, for example, to a wort. Petition 870250086005, dated 09 / 23 / 2025, pp. 72-92 36 / 47

[0128] The addition of the aforementioned enzyme composition in a process for the production of an alcoholic beverage for human consumption, especially a brewing process, can positively affect the flavor of the beverage produced, such as beer, by increasing the amount of monosaccharides, disaccharides, and trisaccharides that have a sweet taste and enhance fruity flavors. Therefore, it is preferable that the addition of the enzyme composition in a process for the production of an alcoholic beverage for human consumption, especially in a brewing process, increases the amount of monosaccharides, disaccharides, and trisaccharides in the alcoholic beverage produced, such as beer.

[0129] The addition of an enzymatic composition, in particular a composition comprising both a prolyl-specific 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 enhance the removal of fine particulate matter, as demonstrated in the experimental section. The addition of a composition combining prolyl-endoprotease and cellulase during beer fermentation can reduce membrane fouling during beer filtration, and filtration can advantageously be operated at higher pressure and speed, providing an economic advantage. Alcoholic beverage

[0130] The invention also provides an alcoholic beverage for human consumption obtained or that can be obtained by such a process.

[0131] More preferably, the invention provides an alcoholic beverage comprising, preferably, at least partially inactivated: - glutamine-specific and / or proline-specific protease, preferably glutamine-specific and / or proline-specific endoprotease, preferably as described above; Petition 870250086005, dated 09 / 23 / 2025, pp. 73 / 92 37 / 47 and - cellulase, preferably as described above. EXAMPLES Materials and methods Materials

[0132] The following materials were used in the examples: Brewers Clarex is a product of DSM Food Specialties and contains a prolyl-specific endoprotease derived from a selected autocloned strain of Aspergillus niger. Brewers Clarex XF is a product of DSM Food Specialties and contains a prolyl-specific endoprotease derived from a selected autocloned strain of Aspergillus niger. - Filtrase NL-Fast is a product of DSM Food Specialties and contains a fungal cellulase, more specifically an endo1,3(4)-e-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)-ε-glucanase (a beta-glucanase), derived from a selected strain of Talaromyces emersonii and a hemicellulose, more specifically an endo-1,4-ε-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 dry, bottom-fermenting yeast (Saccharomyces pastorianus) from Lasaffre (Fermentis), suitable for direct inoculation. - Heineken Lager Beer: a light beer with 5% ABV produced by Heineken. - Malt: Standard EBC 21st EBC malt (European Brewery) Petition 870250086005, dated 09 / 23 / 2025, pp. 74 / 92 38 / 47 Convention). - Barley: IFBM (Institut Français des Boissons de la Brasserie et de la Malterie) 2 RS Barley - Planet variety, 2020 harvest. Analytical Methods Filtration

[0133] After maturation, the samples were centrifuged at 11,600g (Sorvall RC6 Plus, rotor F10S-6*500Y) and 4°C for 10 min. The supernatant was removed and degassed by magnetic stirring at room temperature for 1 hour. The recovered beer was diluted five times by weight by the addition of similarly degassed Heineken lager beer. Before filtration, the diluted sample and the stainless steel filter housing of the Seitz EDF-14-2 filtration system were cooled in ice water.

[0134] The filtration system was assembled according to the manufacturer's instructions, attaching the filter housing to the lower plate, which consists of a perforated disc on the inside and a connector (a 3 / 8-inch connector - ball valve - 3 / 8-inch connector and a male luer lock (Z514691-1EA from Sigma), in stainless steel and Teflon tape) fixed to a tripod on the outside. A volume of 250 mL of chilled sample was poured into the pre-chilled filter housing. The ball valve was opened to allow approximately 10 grams of the sample to flow by gravity, and then the valve was closed, ensuring complete filling of the connector outlet without air bubbles. The filter (Acrodisc PES 0.45 pm 25 mm, PN 4508; Pall Corporation) was rinsed by backwashing with 5 ± 1 mL of degassed Heineken lager beer by connecting it to a disposable plastic syringe.The filter was slowly rinsed with the remaining beer while unscrewing the filter from the syringe to prevent air (bubbles) from entering the filter and the connection. The filter was connected to the male luer lock at the outlet of the filtration unit. The top plate of the filtration system was then secured. Petition 870250086005, dated 09 / 23 / 2025, pp. 75 / 92 39 / 47 to the filter housing following the manufacturer's instructions. The vent valve on the top plate was closed, and the filtration system was connected to a pressure valve, and then a pressure of 1 bar (100000 Pa) was applied. A scale with an accuracy of 0.1 g was placed below the filtration system. A beaker was placed on the scale. The automatic weight recording was started from weight 0. Then, the valve is opened at an exact moment of the automated recording, so that the weight is recorded immediately. Every 10 seconds, the weight is recorded for a total of 6-7 minutes.

[0135] The weight of the filtrate, transferred in arbitrary units (U), over time was used to determine fouling. The fouling of the 0.45 pm filter, expressed in hour-1* bar-1 (1 bar = 100000 Pa), is the inverse of the slope obtained from the linear relationship applied (complete blockage applied as a filtration model) between the flow, expressed in M(million)U / (m2*h*bar) (1 bar = 100000Pa), in relation to the filter load in MU / m2. Determination of Specific Gravity, Plato, ABV and ADF

[0136] The specific gravity (SG) and alcohol content (Alcohol by Volume) of the must and fermentation samples after maturation were determined using a digital hydrometer (Anton Paar; type DMA58) following the supplier's instructions. The specific gravity was measured at 20°C.

[0137] The Apparent Degree of Fermentation (ADF) was calculated according to ADF = (Original Extract - Apparent Extract) / Original Extract * 100

[0138] The original extract expressed in degrees Plato (°P) was calculated using the formula: Extract = - 460.234 + 662.649 x (SG) 202.414 x (SG)2, where SG is the specific gravity of the wort (EBC 8.3 method, Extract of wort - 2004).

[0139] The apparent extract expressed in degrees Plato (°P) was calculated using the formula: Extract = - 460.234 + 662.649 x (SG) Petition 870250086005, dated 09 / 23 / 2025, pp. 76 / 92 40 / 47 202.414 x (SG)2, where SG is the specific gravity of the sample after fermentation and maturation (EBC 9.4 method, Original, real and apparent extract and original gravity of beer - 2004). Analysis of fermentable sugars

[0140] The analysis of the fermentation samples after maturation was performed on a Thermo Fisher Scientific High Performance Ion Exchange Chromatography (Dionex) system coupled with pulsed amperometric detection (HPAEC-PAD) on a CarboPac PA20 column, with an Amino Trap shielding column and a Borate Trap pre-column. The mobile phase was a gradient composed of 3 solutions. The gradient configurations for analyzing fermentable sugars with this system are shown below in Table 1. TABLE 1: Gradient settings for sugar analysis: Time [min] % A % C % D Curve - 15 80 20 0 5 0 80 20 0 5 30 64 16 20 5 31 0 0 100 5 36 0 0 100 5 37 80 20 0 5 Mobile phase A: Milli Q purified water Mobile phase C: 500 mM NaOH Mobile phase D: 100 mM NaOH / 1 M NaOAc

[0141] The peak area of ​​fermentable sugars (glucose, The amount of maltose and maltotriose present in the samples was quantified using calibration curves of glucose, maltose, and maltotriose references. Analysis of beer haze

[0142] Turbidity was measured in fermentation samples after maturation using a Haffman's VOS Rota 90 / 25 double-angle turbidimeter. This equipment measures scattered light caused by particles. Particles smaller than 1 µm mainly cause Petition 870250086005, dated 09 / 23 / 2025, pp. 77 / 92 41 / 47 the light scattering and are measured at a 90° angle. Particles larger than 1 pm cause mainly forward light scattering and are measured at a 25° angle. The measurement was performed in the cuvette provided by the manufacturer of the equipment used (method EBC 9.29, Haze in beer: calibration of haze meters - 2015). Glucan analysis

[0143] The product obtained after the reaction with cellulases (glucanases) is a glucan. The glucan level in the must and fermentation samples after maturation was determined by the 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 has been accepted and recommended for inclusion in the Analytical-EBC since 2005. Example 1: The use of a combination of prolyl endopeptidase and cellulase during beer fermentation.

[0144] For this example, the brewing was carried out with 80 grams of milled grain (grist), composed of 60% by weight of disc-milled malt (Standard Malt EBC 21st EBC) and 40% by weight of hammer-milled barley (IFBM 2 RS Barley - Planet variety, 2020 harvest), to which 200 grams of tap water were added and mixed.

[0145] The suspension was introduced into a conversion vessel preheated to 48°C in a Lochner LB Electronic. A volume of 1 mL of calcium chloride (0.61 M liquid stock) was added and the pH was adjusted to 5.4 using 80% lactic acid while mixing at 100 rpm. Subsequently, the mash profile was applied in LB Electronic, as shown in Table 2. Meanwhile, the completion of saccharification was verified by performing an iodine test (Lugol's solution; Sigma 32922, according to Lugol). At the end of the mash, no starch was detected by the iodine test. TABLE 2: Brewing profile applied to milled grains Petition 870250086005, dated 09 / 23 / 2025, pages 78 / 92, composed of 60% malt by weight and 40% barley by weight. Brewing profile for brewing °C min 1. Rest 48 35 2. Rest 65 50 3. Rest 72 30 4. Rest 78 5 Ramp [°C per min] 1.0 Total rest time [min] 120 Total heating time [min] 30 Total [min] 150

[0146] After completion of the mashing scheme, the weight of the mashing suspension was adjusted to 450 grams with hot tap water. The suspension was stirred and poured over a folded strainer (Macherey-Nagel; MN 614 ¼ 0 320 mm REF 527032) into a glass funnel placed in a 500 mL Scott flask for filtrate collection. Filtration was stopped when the filter bed in the folded strainer dried by gravity. The first 100 mL of filtrate were collected and reintroduced to the top of the filter bed. The total filtrate collected, the wort, was cooled to room temperature.

[0147] The resulting must solution had a gravity of 14.6°P, a specific density of 1.059582 g / cm3 and a fermentable sugar content of 95.8 g / L.

[0148] The total wort generated through 16 individual brews was divided into 300-gram portions in Scott flasks. The wort was cooled to 14°C, and then the yeast (Saflager S23, Fermentis) was dosed at 100 g / hL and the enzyme(s) was / were applied according to Table 3. Subsequently, fermentation was carried out for 8 days at 14°C and 100 rpm using an ANKOM gas fermentation system. During fermentation, the cumulative pressure and temperature were recorded using the ANKOM RF Gas Production System (Ankom Technology). Petition 870250086005, dated 09 / 23 / 2025, pp. 79 / 92 43 / 47 TABLE 3: Dosage of commercial enzyme for cold wort Experiment Number Commercial enzyme dosage (g / hL) Filtrase NL Fast Laminex BG3 Filtrase BRXL Glucanex 100G Brewers Clarex Brewers Clarex ^^^B 2 ^^^B 1D ^^^B ^^^B ^^^B 0.5 2 ^^^B Control 1 ^^^B ^^^B ^^^B ^^^B 2 ^^^B 2A 0.5 ^^^B ^^^B ^^^B ^^^B 2 2B ^^^B 0.5 ^^^B ^^^B ^^^B 2 2C ^^^B ^^^B 0.5 ^^^B ^^^B 2 2D ^^^B ^^^B ^^^B 0.5 ^^^B 2 Control 2 ^^^B ^^^B ^^^B ^^^B ^^^B 2 Control 0 ^^^B ^^^B ^^^B ^^^B ^^^B ^^^B

[0149] At the end of fermentation, the beer was matured 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 removed and degassed by magnetic stirring at room temperature for 1 hour. This clarified beer was used for compositional and physical analysis. RESULTS Improved flavor

[0150] To illustrate the improvement in flavor, a compositional analysis was performed.

[0151] The Apparent Degree of Fermentation (ADF) of all samples ranged between 78 and 82%, and the alcohol content between 6.20 and 6.45%. The presence of fermentable sugars in the final beer is shown in Table 4. TABLE 4: Presence of glucose, maltose, and maltotriose in the samples. The total fermentable sugars are the sum of these. Glucose Maltose Maltotriose Fermentable sugars [g / L] [g / L] [g / L] [g / L] Petition 870250086005, dated 09 / 23 / 2025, pp. 80-92 44 / 47 Glucose Maltose Maltotriose Fermentable sugars Experiment 1A 0.16 4.43 1.84 6.42 Experiment 1B 0.24 6.18 2.51 8.92 Experiment 1C 0.23 5.98 2.32 8.53 Experiment 1D 0.16 4.70 1.95 6.81 Control 1 0.15 4.32 1.85 6.32 Experiment 2A 0.17 4.87 2.00 7.04 Experiment 2B 0.22 5.29 2.09 7.60 Experiment 2C 0.20 4.00 1.59 5.79 Experiment 2D 0.15 3.96 1.58 5.69 Control 2 0.14 3.75 1.62 5.51 Control 0 0.15 4.27 1.68 6.10

[0152] The sugar composition of the final beer can influence flavor perception. Monosaccharides, disaccharides, and trisaccharides have a sweet taste and are known to enhance fruity flavors. Therefore, an increase in the presence of fermentable sugars in the final beer, as can be detected in Experiments 1B and 2B which are treated with a prolyl-endoprotease and a cellulase, is expected to favorably influence the beer's flavor.

[0153] Furthermore, 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 can result in turbidity / precipitates in the final beer.

[0154] However, glucan was below the detection limit in all experimental samples that contained cellulase activity. This indicates that all cellulases were active during beer fermentation to further break down glucan into glucose. Glucose is sweeter than glucan and can also enhance fruity flavors, as explained above. Therefore, it can be concluded that, during fermentation in experiments 1A, 1B, 1C, 1D and 2A, 2B, 2C and 2D, in addition to prolyl-endoprotease activity, there was also cellulase activity. The combination of these led to a better breakdown of glucan into glucose. Petition 870250086005, dated 09 / 23 / 2025, pp. 81 / 92 45 / 47 desired and with an enhanced flavor. Improved removal of fine particulate matter.

[0155] To illustrate the improvement in the removal of fine particulate matter, a physical analysis was carried out.

[0156] To test the effect of enzymatic treatment on the possible presence of fine particulate matter in fermentation and to monitor the rate of removal of this matter, the turbidity at the end of fermentation after maturation and the filterability of the clarified beer were tested. a) Test for the presence of fine particulate matter

[0157] The turbidity measured at the end of fermentation after maturation is presented in Table 5 TABLE 5: Turbidity formation measured at an angle of 90° or 25°. H90 H25 Experiment 1A 4.69 5.48 Experiment 1B 8.18 13.34 Experiment 1C 6.15 9.97 Experiment 1D 4.30 5.31 Control 1 5.29 6.81 Experiment 2A 3.97 5.42 Experiment 2B 5.46 7.94 Experiment 2C 3.25 4.27 Experiment 2D 3.16 2.86 Control 2 4.52 4.86 Control 0 out of range out of range

[0158] This experiment shows that the addition of a prolyl endoprotease has a significant effect on reducing turbidity in beer at the end of fermentation. In control 0, without the addition of prolyl endoprotease, turbidity formation was very high and outside the range of the measuring instrument. The turbidity in all samples with the addition of prolyl endoprotease, however, could be measured within the range. The combination of prolyl Petition 870250086005, dated 09 / 23 / 2025, pages 82 / 92 46 / 47 endoprotease with cellulases in some cases showed either a slight increase or a slight additional decrease in turbidity, depending on the origin of the cellulase. However, any turbidity formation at the end of fermentation may not be significant for turbidity formation in the final bottled beer. An important step here is the removal of fine particulate matter during the filtration stage.

[0159] In other words, the results above show that, at the end of fermentation after maturation, there may be the presence of fine particulate matter and the removal of this matter is desirable. b) Filtration

[0160] In regular beer production, fine particulate matter is removed by a filtration step after fermentation and maturation. To test the effect of enzymatic treatment on the capacity and flow of beer filtration, samples were initially diluted 5x with commercial degassed Heineken Lager beer. This dilution allows for a more accurate assessment, as the time until filter blockage is increased. Before filtration, the diluted sample and the stainless steel filter housing of the Seitz EDF-14-2 filtration system were chilled in ice water.

[0161] The amount of filtrate was monitored over time and the results are presented in Figure 1. All samples containing cellulase in combination with prolylendoprotease showed an improvement in filtrate yield before filter blockage. The filtration rate, compared to the 3 control samples that were not treated with cellulase, was also improved.

[0162] The fouling rate was calculated from the data and is shown in Table 6. Clearly, with all samples in which prolyl-endoprotease was combined with cellulase during beer fermentation, membrane fouling during filtration of light beer was reduced. Therefore, filtration Petition 870250086005, dated 09 / 23 / 2025, pages 83 / 92 47 / 47 filtration can be performed at a higher speed (CQ pressure), gaining an advantage in the process compared to regular light beer filtration. Surprisingly, there is a clear difference in the extent to which different cellulases reduce filter fouling. Trichoderma reesei cellulase appears to be the most effective in reducing fouling. This is surprising, since in samples treated with Trichoderma reesei cellulase, turbidity formation before filtration was greater than in controls treated with prolyl-endoprotease alone (see Table 5). TABLE 6: Fouling rate of the filters used to clarify the different beer samples. Fouling (hour*bar)-1 Experiment 1A 96.5 Experiment 1B 36.6 Experiment 1C 100.3 Experiment 1D 139.9 Control 1 179.4 Experiment 2A 91.1 Experiment 2B 37.4 Experiment 2C 81.9 Experiment 2D 137.5 Control 2 187.4 Control 0 215.5 (1 bar = 100,000 Pa) Petition 870250086005, dated 09 / 23 / 2025, pages 84 / 92

Claims

1 / 5 CLAIMS 1. Composition characterized in that it comprises or consists of: - a first enzymatic component, comprising or consisting of a glutamine-specific protease and / or a prolyl-specific protease, preferably a glutamine-specific endoprotease and / or a prolyl-specific endoprotease, primarily a prolyl-specific endoprotease; and - 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 Trichoderma reesei.

2. Composition according to claim 1, characterized in that the first enzymatic component is derived from and / or produced by a microorganism, preferably a bacterium or fungus, primarily Trichoderma reesei.

3. Composition, according to claim 1 or 2, characterized in that the first enzymatic component comprises or consists of a prolyl-specific endoprotease and that the cellulolytic enzyme, preferably cellulase, is present in an amount equal to or less than 50% w / w, based on the total weight of the prolyl-specific endoprotease.

4. Composition, according to any of the preceding claims, characterized in that - the first enzymatic component consists of a prolyl-specific endoprotease; and - the second enzymatic component consists of a cellulolytic enzyme, preferably cellulase, wherein the second enzymatic component is derived from and / or produced by Trichoderma reesei, wherein the composition does not comprise one or more additional enzymes. Petition 870250086005, dated 09 / 23 / 2025, page 85 / 92 2 / 5 5. A composition, according to any of the preceding claims, characterized in that the composition is for addition during the saccharification and / or fermentation stage of a brewing process.

6. Composition, according to any of the preceding claims, characterized in that the composition is for improving the removal of fine particulate matter in a fermentation broth and / or fermentation tank and / or for improving the flavor of the alcoholic beverage product.

7. Composition, according to any of the preceding claims, characterized in that the first enzymatic component and the second enzymatic component are derived from or produced by the same microorganism, preferably by the same bacterium or fungus, primarily by the same Trichoderma reesei.

8. Composition, according to any of the preceding claims, characterized in that the first enzymatic component has its optimum activity at a pH in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.5, even more preferably equal to or less than pH 5.0, much more preferably equal to or less than pH 4.5 and preferentially equal to or less than pH 4.

0.

9. Composition, according to any of the preceding claims, characterized in that the second enzymatic component has its optimum activity at a pH in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.5, even more preferably equal to or less than pH 5.0, much more preferably equal to or less than pH 4.5 and preferentially equal to or less than pH 4.

0.

10. Composition, according to any of the preceding claims, characterized in that the composition comprises water and, optionally, one or more cosolvents.

11. Composition, according to any of the preceding claims, characterized in that the composition has a pH value in the range equal to or greater than pH 1.0, more preferably equal to or greater than pH 1.2 to equal to or less than pH 6.0, more preferably equal to or less than pH 5.5, even more preferably equal to or less than pH 5.0, much more preferably equal to or less than pH 4.5 and preferentially equal to or less than pH 4.

0.

12. Composition, according to any of the preceding claims, characterized in that the composition comprises 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 sulfate; - a carboxylic acid, preferably having in the range of 1 or more to 10 or less carbon atoms, preferably in the range of 1 or more to 7 or less 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, Petition 870250086005, dated 09 / 23 / 2025, page 87 / 92 4 / 5 glyceryl tributanoate, glyceryl monolactate, glyceryl dilactate, glyceryl trilactate; - a monosaccharide, a disaccharide and / or an alcohol derivative or chloride derivative of such monosaccharide or disaccharide; - sorbitol, mannitol, inositol, trehalose, sucrose, mannose and / or sucralose; - maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, amylopectin and mixtures thereof.

13. Composition, according to any of the preceding claims, characterized in that the composition either includes or does not include glycerol.

14. Composition, according to any of the preceding claims, characterized in that the composition either comprises or does not comprise benzoic acid or a derivative thereof.

15. A bacterial or fungal strain, preferably a strain of Trichoderma reesei, characterized in that it functionally expresses: - a first nucleic acid sequence encoding a first enzymatic component, comprising or consisting of a prolyl-specific protease, preferably a prolyl-specific endoprotease; and - a second nucleic acid sequence encoding a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably cellulase, wherein the second enzymatic component is preferably derived from Trichoderma reesei.

16. A bacterial or fungal strain, preferably the Trichoderma reesei strain, according to claim 15, characterized in that it functionally expresses: - a first nucleic acid sequence encoding a first enzymatic component, comprising or consisting of a glutamine-specific protease and / or a prolyl-specific protease, preferably a glutamine-specific endoprotease and / or a prolyl-specific endoprotease; and - a second nucleic acid sequence encoding a second enzymatic component, comprising or consisting of a cellulolytic enzyme, preferably a cellulase; wherein the first nucleic acid sequence is: - operationally linked to a promoter; or - present in 2, 3, 4, 5, 6 or more copies.

17. Bacterial or fungal strain, preferably the Trichoderma reesei strain, according to claim 15 or 16, characterized in that the first enzymatic component is produced in a greater quantity than the second enzymatic component.

18. Process for the production of an alcoholic beverage for human consumption, characterized in that it comprises the addition of a composition as defined in any one of claims 1 to 14 or the addition of a bacterial or fungal strain, preferably a strain of Trichoderma reesei, as defined in claim 15 or 16.

19. Process according to claim 18, characterized in that the process is a brewing process, and that a composition, as defined in any one of claims 1 to 14, is added before or during fermentation, more preferably before fermentation, primarily to the wort.

20. Alcoholic beverage for human consumption characterized by the fact that it is obtained or can be obtained by the process as defined in claim 18 or 19. Petition 870250086005, dated 09 / 23 / 2025, pp. 89 / 92

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