Method for obtaining plant-based food ingredients

Abstract

The present invention relates to a method for producing a plant-based food ingredient comprising treating a slurry of plant material with an alpha amylase, a xylanase and optionally at least one additional enzyme. The invention further includes plant-based food ingredients produced by the method. The plant-based food ingredients produced by the method can be used to produce dairy alternative food products.

Description

[0001] References to sequence lists This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0002] This invention relates to the use of enzymes in an improved method for extracting carbohydrates and proteins from plant materials to produce plant-based food ingredients. Background Technology

[0003] In recent years, there has been an increase in the number of people pursuing vegan, vegetarian, or non-dairy diets for health reasons. Furthermore, food products made from animal milk (such as cow's milk) are increasingly attracting attention due to their high environmental costs. These factors are driving growing demand for dairy alternatives to foods traditionally derived from milk, including milk, non-dairy creamer, cheese, yogurt, and ice cream.

[0004] Dairy alternative food products are typically derived from high-starch plant materials, such as cereal grains, nuts, or legume pods. Generally, in order to convert high-starch plant materials into dairy alternative food products or food ingredients included in dairy alternative food products, starch must be hydrolyzed. Starch conversion typically includes a gelatinization step, in which starch granules are dissolved to form a viscous suspension; a liquefaction step, in which the starch is partially hydrolyzed with a loss of viscosity; and optionally, a subsequent saccharification step, which involves producing glucose and maltose through further hydrolysis.

[0005] Examples of dairy alternative food products that have received considerable attention in recent years are those based on oats. Oats are considered healthy for many reasons: they are a good source of vitamins, minerals, fiber, antioxidants, and essential amino acids, and the health benefits associated with consuming oats include lowering blood cholesterol levels and reducing the risk of heart disease.

[0006] The use of enzymes to improve the conversion of high-starch plant materials into non-alcoholic plant-based food ingredients and plant-based dairy alternatives remains a topic of interest in the industry. The object of this invention is to identify improved methods for producing non-alcoholic plant-based food ingredients and plant-based dairy alternatives, particularly methods for increasing the raw material utilization and / or nutritional properties of the food ingredients and products, while maintaining or improving the sensory properties of the resulting plant-based dairy alternatives. Summary of the Invention

[0007] This invention relates to an improved method for producing non-alcoholic plant-based food ingredients, which offers increased production capacity, lower production costs, and / or better raw material utilization. This improved method includes selective enzymes that enhance the solubilization of carbohydrates, proteins, and / or fiber in the starting plant material.

[0008] This invention provides a method for obtaining alcohol-free plant-based food ingredients, the method comprising: a) Obtaining a pulp of plant material in water; b) Provides α-amylase, xylanase, and optionally at least one other enzyme; and c) Treat the slurry with the enzyme to produce hydrolyzed plant material, wherein the hydrolyzed plant material is a plant-based food ingredient.

[0009] The present invention also provides a method for obtaining oat-based food ingredients, the method comprising: a) Obtain a slurry of oat material in water; b) Provides an enzyme composition comprising amylase, xylanase, optionally β-glucanase, and optionally protein deamidase; and c) Treat the slurry with the enzyme composition to produce hydrolyzed plant material, wherein the hydrolyzed plant material is an oat-based food ingredient.

[0010] This invention provides plant-based and oat-based food ingredients obtained by these methods, which can have an improved nutritional profile compared to similar plant-based ingredients prepared using similar methods known in the art. These ingredients can be further processed to produce dairy alternative food products that can also have an improved nutritional profile.

[0011] The present invention further provides the use of α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase in the production of plant-based food ingredients to improve the extraction of β-glucan and / or protein from plant materials.

[0012] definition Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context clearly indicates otherwise. Unless otherwise defined or clearly indicated by the context, all percentages are weight percentages (percentage w / w or “% (w / w)”).

[0013] Unless otherwise defined or explicitly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0014] α-Amylase (1,4-α-D-glucanase, EC 3.2.1.1) is a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides.

[0015] β-glucanase: The term “β-glucanase” encompasses polypeptides having β-1,6-glucanase activity and / or exonuclease and / or endo-β-1,3-glucanase activity.

[0016] Expression: The term “expression” refers to any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0017] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., promoter) of the polypeptide or nucleic acid is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence comes from a gene other than the gene encoding the mature polypeptide.

[0018] Host strain or host cell: A "host strain" or "host cell" is an organism in which an expression vector, bacteriophage, virus, or other DNA construct (including a polynucleotide encoding the polypeptide of the present invention) has been introduced. An exemplary host strain is a microbial cell (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the target polypeptide and / or fermentable sugar. The term "host cell" includes protoplasts produced by cells.

[0019] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other designated material or component that has been separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Therefore, the isolated polypeptide, nucleic acid, cell, or other material exists in a form not found in nature. Isolated polypeptides include, but are not limited to, culture media containing secreted polypeptides expressed in host cells.

[0020] Mature peptide: The term “mature peptide” refers to a peptide that has been processed at its N-terminus and / or C-terminus (e.g., removal of the signal peptide) to be in its mature form.

[0021] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0022] Purified: The term "purified" means nucleic acids, peptides, or cells that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified peptides or nucleic acids form discrete bands). Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., weight percentage or molar percentage). In a relevant sense, a composition is enriched with the molecule when the concentration of the molecule increases significantly after the application of purification or enrichment techniques. The term “enrichment” refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specified materials or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0023] In one respect, the term "purified," as used herein, means that the polypeptide or cell is substantially free of components (especially insoluble components) from the producing organism. In another respect, the term "purified" means that the polypeptide is substantially free of insoluble components (especially insoluble components) from the natural organism from which it was obtained. In one respect, the polypeptide is separated from some soluble components of the organism from which it was recovered and the culture medium. The polypeptide can be purified (i.e., separated) by one or more of the following methods: unit operation filtration, precipitation, or chromatography.

[0024] Accordingly, peptides can be purified so that only small amounts of other proteins, particularly other peptides, are present. As used herein, the term "purified" can refer to the removal of other components present in the cells from which the peptide originates, particularly other proteins and most particularly other enzymes. A peptide can be "substantially pure," meaning it is free of other components from the organism that produced it (e.g., the host organism used to recombinantly produce the peptide). In one aspect, the peptide is at least 40% pure by weight of the total peptide material present in the formulation. In another aspect, the peptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total peptide material present in the formulation. As used herein, "substantially pure peptide" can mean a peptide formulation containing, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of the peptide and other peptide material associated with it, either naturally or recombinantly.

[0025] Therefore, it is preferred that the substantially pure polypeptide, based on the weight of the total polypeptide material present in the formulation, is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure. The polypeptides of the present invention are preferably in a substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated with it, either naturally or recombinantly). This can be achieved, for example, by preparing the polypeptide using well-known recombinant methods or classical purification methods.

[0026] Recombination: The term "recombination," used in its conventional sense, refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group different from that found in nature. The term recombination refers to cells, nucleic acids, polypeptides, or vectors that have been modified from their natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) forms, or express natural genes at different levels or under different conditions compared to those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0027] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) is used. J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) determines the sequence identity between two amino acid sequences as the output of "longest identity," an algorithm such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, 48: 443-453). Trends Genet. The method is implemented in the Niedel procedure of [Trends in Genetics] 16: 276-277 (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel procedure to report the longest identity, the non-brief (-nobrief) option must be specified on the command line. The Niedel-marked "Longest Identity" output is calculated as follows: (Identical residues x 100) / (Alignment length - Total number of vacancies in the alignment) For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity," as implemented in the Niedel program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4). For the Niedel program to report the longest identity, a non-simplified option must be specified on the command line. The Niedel-marked "longest identity" output is calculated as follows: (Identical deoxyribonucleotides x 100) / (Alignment length – Total number of vacancies in the alignment) Signal peptide: A signal peptide is an amino acid sequence that attaches to the N-terminal portion of a protein and promotes its secretion outside the cell. The mature form of extracellular proteins lacks a signal peptide, which is cleaved during the secretion process.

[0028] Variants: The term "variant" refers to a polypeptide that has enzymatic activity and contains artificial mutations (i.e., substitutions, insertions (including extensions), and / or deletions (e.g., truncations)) at one or more positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0029] Wild-type: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is natural or naturally occurring. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., protein, amino acid, or nucleic acid sequences). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

[0030] Xylanase: The term "xylanase" refers to glucuronide arabinoxylan endo-1,4-β-xylanase (EC3.2.1.136), which catalyzes the endo-hydrolysis of 1,4-β-D-xylosyl bonds in some glucuronide arabinoxylans. Detailed Implementation

[0031] This invention relates to a method for producing non-alcoholic plant-based food ingredients, the method comprising treating a slurry of plant material with α-amylase, xylanase, and optionally at least one other enzyme. The invention further relates to a method for producing non-alcoholic oat-based food ingredients, the method comprising treating a slurry of oat material with α-amylase, xylanase, optionally β-glucanase, and optionally protein deamidase. The invention further includes non-alcoholic plant-based food ingredients produced by this method. Compared to methods that do not treat oat material with α-amylase, xylanase, optionally β-glucanase, and optionally protein deamidase, the non-alcoholic oat-based food ingredients produced by this method can have increased amounts of β-glucan and / or protein, thereby providing increased nutritional value and optionally increased viscosity. The oat-based food ingredients produced by the method of the present invention can be improved oat bases superior to methods in the art, making it possible to require less oat base to produce the desired plant-based food product. The plant-based food ingredients produced by this method can be used to produce dairy alternative food products.

[0032] "Plant-based food ingredients" refers to plant-based compositions that can be combined with other food ingredients to produce food products. Plant-based food ingredients and plant-based food products can be solids or liquids, such as beverages. In some embodiments, plant-based food ingredients are alcohol-free. The term "alcohol-free" means that the plant-based food ingredient contains no alcohol or contains no significant amount of alcohol. In some embodiments, alcohol-free plant-based food ingredients contain less than 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or less than 0.01% alcohol by volume.

[0033] In some embodiments, plant-based food ingredients may be combined with other food ingredients to produce dairy alternative food products. The other food ingredients may be any food ingredients that a person skilled in the art would find useful. The other food ingredients may be solid or liquid. The other food ingredients may or may not be plant-based. In some embodiments, the other food ingredient is water.

[0034] "Dairy alternative food products" refer to food products that can be used as substitutes for dairy food products. Dairy alternative food products are plant-based and do not contain dairy-derived food ingredients. Dairy alternative food products include plant-based beverages, non-dairy creamers, cheese, ice cream, and yogurt. In some embodiments, dairy alternative food products are plant-based beverages. In some embodiments, dairy alternative food products are oat-based beverages.

[0035] In some embodiments, plant-based food ingredients can be used as substrates for fermentation to produce dairy alternative beverages (such as buttermilk) or dairy alternative yogurt.

[0036] In some embodiments, plant-based food ingredients may be further processed. Further processing may include water removal. In some embodiments, water removal concentrates the hydrolysis products, namely the released carbohydrates, proteins, and fiber. In some embodiments, water removal increases the viscosity of the plant-based food ingredients.

[0037] In some embodiments, plant-based food ingredients may be further processed and combined with other food ingredients to produce dairy alternative ice cream. In some embodiments, plant-based food ingredients may be further processed and combined with other food ingredients to produce dairy alternative cheese.

[0038] In some embodiments, plant-based food ingredients may be directly combined with other food ingredients to produce ready-to-drink dairy alternative beverages. Examples of dairy alternative beverages include oatmeal beverages, cashew beverages, fava bean beverages, lentil beverages, soy beverages, rice beverages, barley beverages, quinoa beverages, flaxseed beverages, potato beverages, pea beverages, almond beverages, tiger nut beverages, macadamia nut beverages, coconut beverages, or beverages containing any combination thereof.

[0039] Dairy alternative food products can be fortified with plant-based dairy alternative powders (such as soy milk powder) or with concentrated or isolated proteins (such as soy / pea protein isolates or soy / pea protein concentrates). In this example, the dairy alternative food product is fortified, such as an oat-based beverage fortified with pea protein.

[0040] In some embodiments, the plant-based food ingredient has a protein content of at least about 0.5% (w / w), 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or at least about 4.0% (w / w). In some embodiments, the dairy alternative food product has a protein content of up to 4% (w / w). In some embodiments, the plant-based food ingredient has a protein content of about 1% (w / w).

[0041] Other food ingredients that can be combined with plant-based food ingredients include, but are not limited to, lipids such as oils, especially vegetable oils; sugars such as sucrose; proteins, various forms of synthetic amino acids; dietary fiber; salts; minerals; flavorings; vitamins; and any combination thereof.

[0042] In this embodiment, the lipid may be a vegetable oil or a mixture of vegetable oils. The lipid may be selected from rapeseed oil, flaxseed oil, safflower oil, soybean oil, olive oil, sunflower oil, palm oil, and combinations thereof. In one embodiment, the lipid is rapeseed oil, sunflower oil, or a combination thereof. The appropriate choice of lipid depends on the type of plant-based dairy alternative food product desired.

[0043] Lipids may be added in an amount between about 1% and about 5% (w / w) (e.g., about 3% (w / w)) relative to the weight of the final product (such as a dairy alternative food product). In some embodiments, the dairy alternative food product has a lipid content of at least about 0.5% (w / w), 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or at least about 5.0% (w / w). In some embodiments, the dairy alternative food product has a lipid content of up to 5.0% (w / w).

[0044] In some embodiments, salt is combined with plant-based food ingredients. The salt can be sodium chloride, dicalcium carbonate, calcium hydrogen phosphate, tricalcium phosphate, calcium carbonate, or any combination thereof.

[0045] In some embodiments, vitamins and / or minerals are combined with plant-based food ingredients. Vitamins may be vitamin A, vitamin C, vitamin D, vitamin E, vitamin B12, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, vitamin K, folic acid (vitamin B9), and mixtures thereof. Minerals may be calcium, phosphorus, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper, and mixtures thereof.

[0046] It can standardize and / or homogenize dairy alternative food products. It can pasteurize or otherwise heat-treat dairy alternative food products.

[0047] The plant-based food ingredients of this invention are derived from plant materials, which are or are derived from the edible parts of plants. In some embodiments, the plant materials are derived from the edible parts of plants that also have a high starch content. In some embodiments, the edible parts of the plant may be tubers, roots, stems, spikes, legume pods, fruits, nuts, or seeds. In some embodiments, the plant is a cereal and the plant material is or is derived from cereal grains (also called whole grains). In other embodiments, cereal grains may be derived from corn, rice, barley, wheat, flax, buckwheat, millet, sorghum, quinoa, oats, or rye. In some embodiments, the plant material is or is derived from tubers or roots (including rhizomes), such as potatoes, sweet potatoes, cassava, tiger nuts, canna lilies, or tapioca. In some embodiments, the plant material is or is derived from fruits or nuts, such as cashews, macadamia nuts, almonds, coconuts, bananas, jackfruit, or breadfruit. In some embodiments, the plant material is or is derived from sago, peas, or legumes, such as soybeans, broad beans, or lentils.

[0048] In some embodiments, the plant material is heat-treated. In some embodiments, the plant material is dehydrated. In some embodiments, the plant material is dehulled, ground, wet-milled, and / or dry-milled. In some embodiments, the plant material is corn flour, rice flour, barley flour, wheat flour, buckwheat flour, millet flour, quinoa flour, oat flour, rye flour, potato flour, sweet potato flour, cassava flour, tiger nut flour, tapioca flour, nut flour, pea flour, legume flour, hulled oats, hulled barley, hulled wheat, hulled peas, hulled legumes, or any combination thereof. In some embodiments, the plant material is pulverized or ground to produce a paste.

[0049] In some embodiments, the plant material is oat material. In other embodiments, the oat material is oat flour, oat flakes, oat bran, hulled oats, hulled oat groat, or a combination thereof. In still further embodiments, the oat material may be oat flour (such as heat-treated oat flour), or the oat material may be milled oat kernels (such as wet-milled hulled and heat-treated oat kernels), or the oat material may be any other oat material known in the art. In some embodiments, the oat material is heat-treated oat flour, oat flakes, or oat bran. The non-alcoholic oat-based food ingredients produced by the method of the present invention may also be referred to as "oat-based ingredients".

[0050] In the method of the present invention, plant material is suspended in water to produce a slurry, wherein the ratio of plant material to water is 1:1 to 1:16 (w / w). In some embodiments, the ratio of plant material to water is 1:1 to 1:4 (w / w). In some embodiments, the ratio of plant material to water is 1:3 to 1:8 (w / w). In some embodiments, the plant material is oat material, which is suspended in water to produce a slurry, wherein the ratio of oat material to water in the slurry is 1:3 to 1:8 (w / w). In another embodiment, the ratio of oat material to water is 1:4 to 1:6 (w / w).

[0051] In the method of the present invention, the plant material has a protein content of at least 5% (w / w), at least 10%, at least 15%, or at least 20%. In some embodiments, the plant material has a protein content of up to 20% (w / w). In some embodiments, the plant material has a protein content of about 15% (w / w). In some embodiments, the plant material is oat material having a protein content of at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (w / w).

[0052] enzymes In the method of the present invention, α-amylase, xylanase, and optionally at least one additional enzyme are added to a slurry of plant material in water. This allows the enzymes to act on the plant material substrate, and the resulting hydrolysate is a non-alcoholic plant-based food ingredient. The α-amylase can be any α-amylase suitable for the method according to the present invention. In some embodiments, the α-amylase is a bacterial endo-α-amylase, which is preferably obtained from Bacillus spp. (…). Bacillus (Preferably Bacillus amyloliquefaciens) Bacillus amyloliquefaciens Endoamylases obtained from bacteria or variants thereof. An example of a bacterial endoamylase is BAN®, available from Novozymes A / S. In some embodiments, the α-amylase is a fungal endoamylase, preferably obtained from *Aspergillus* ( ). Aspergillus (Preferably Aspergillus oryzae) Aspergillus oryzae The endo-α-amylase obtained or a variant thereof. An example of a fungal endo-α-amylase is Fungamyl®, available from Novozymes. Another example of a suitable α-amylase is Termomyl®, available from Novozymes.

[0053] In some embodiments of the present invention, the α-amylase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4. In some embodiments, the α-amylase comprises the amino acid sequence of SEQ ID NO: 4.

[0054] In some embodiments, α-amylase is a raw starch hydrolytic α-amylase. As used herein, raw starch hydrolytic α-amylase (also known as raw starch degrading α-amylase) refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch. Sources of raw starch degrading enzymes include those obtained from the genus *Aspergillus* (such as species of *Aspergillus oryzae*, *Aspergillus niger*, etc.). Aspergillus niger ) and Aspergillus ( Aspergillus kawachii Examples of such starch-degrading enzymes include those described in WO2005 / 003311, WO 2006 / 0692, WO 2006 / 060289 and WO 2004 / 080923.

[0055] In some embodiments, the starch-degrading α-amylase is an acidic α-amylase. "Acidic α-amylase" is an α-amylase (4-α-D-glucan hydrolase, EC 3.2.1.1) that, when added in an effective amount, has activity in the pH range of 3.0 to 7.0, preferably 3.5 to 6.0, or more preferably 4.0 to 5.0. The source of the starch-degrading acidic α-amylase is the acidic α-amylase of *Aspergillus niger* disclosed in the Swiss-prot / TREMBL database under accession number P56271 as "AMYA_ASPNG" and described in more detail in WO1989 / 01969 (Example 3). The *Aspergillus niger* acidic α-amylase is also shown in WO 2004 / 080923 (Novozymes) (which is hereby incorporated by reference) as SEQ ID NO: 1. A suitable commercially available acidic fungal α-amylase derived from *Aspergillus niger* is product SP288 (SEQ ID NO: 1, U.S. Patent No. 7,244,597; commercially available from Novozymes). Other sources of acidic α-amylase include those derived from the genus *Rhizopus* (…). Rhizomucor ) and Polyporaceae ( Meripilus strains, such as *Rhizopus microphylla* ( Rhizomucor pusillus (WO 2004 / 055178) or macroporous fungi ( Meripilus giganteus The strain was identified as *Aspergillus oryzae*. In another embodiment, the acidic α-amylase was derived from *Aspergillus oryzae* and was developed by Kaneko et al. J. Ferment. Bioeng. [Journal of Fermentation and Biotechnology] 81:292-298(1996) “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii [Molecular cloning and determination of the nucleotide sequence of the gene encoding an acid-stable α-amylase from Aspergillus whiteus]” disclosed; and further disclosed as EMBL: #AB008370. In some embodiments, the raw starch-degrading α-amylase has a carbohydrate-binding module (CBM) that binds to starch. In some embodiments, the CBM preferentially binds to starch, especially to untreated granular starch. Such a CBM may also be referred to as a starch-binding domain (SBD). SBDs are known to exist in 15 CBM families, namely CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82, and 83.

[0056] In some embodiments of the present invention, the α-amylase is a raw starch-degrading α-amylase comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5. In some embodiments, the α-amylase is a raw starch-degrading α-amylase comprising the amino acid sequence of SEQ ID NO: 5.

[0057] In the method of this invention, xylanase is provided to a slurry of plant material and water. "Xylanase" refers to glucuronide arabinoxylan endoglucanase (EC3.2.1.136), which catalyzes the endo-hydrolysis of 1,4-β-D-xylosyl bonds in some glucuronide arabinoxylan. Xylanase activity can be determined at 37°C, in 0.01% TRITON® X-100 and 200 mM sodium phosphate (pH 6) using 0.2% AZCL-glucuronide xylan as a substrate. One unit of xylanase activity is defined as the production of 1.0 μmol of azurin per minute from 0.2% AZCL-glucuronide xylan as a substrate at 37°C, pH 6, and 200 mM sodium phosphate (pH 6). Examples of enzyme preparations with xylanase activity include MULTIFECT® xylanase (Genencor) and HSP 6000 xylanase (DSM).

[0058] The xylanase used in the method of the present invention can be GH3 xylanase, GH5 xylanase, GH8 xylanase, GH10 xylanase, GH11 xylanase, GH30 xylanase, GH43 xylanase, and GH98 xylanase. In some embodiments, the xylanase is GH10, GH30, or GH5 xylanase. In some embodiments, the xylanase is GH5_21 or GH30 xylanase, which are known xylanases that act on complex xylans. In some embodiments, the xylanase can be Aspergillus GH10 xylanase, Bassilago farfara (… Talaromyces GH10 xylanase, Chlorella vulgaris ( ChryseobacteriumGH5_21 xylanase or Bacillus GH30 xylanase. In some embodiments, the xylanase may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11. In some embodiments, the xylanase may comprise an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11.

[0059] Xylooligosaccharides (XOS) are polymers of xylose with a degree of polymerization (DP) typically ranging from 2 to 10. XOS can act as a prebiotic, selectively stimulating the gut microbiota of humans or mammals (such as Bifidobacteria). Bifidobacteria ) or Lactobacillus ( Lactobacilli The growth of XOS is beneficial to gut health. In some embodiments, the method of the present invention can produce plant-based food ingredients that contain XOS and can act as prebiotics when consumed.

[0060] In the method of this invention, a protein-modifying enzyme (such as a deamidase, transglutaminase, or peptidase) can be used to treat the slurry of plant material in water. In another embodiment, a protein deamidase can be used to treat the slurry of plant material. In this invention, "protein deamidase" or "deamidase" refers to an enzyme that acts directly on the amide groups of the amino acid side chains constituting a protein to cause deamidation and release ammonia without cleaving the protein and / or cross-linking the peptide bonds of the protein.

[0061] The term "deamidase" refers to the activity of protein-glutamine glutaminase (also known as glutamylpeptide glutaminase), as described in EC 3.5.1.44, which catalyzes the hydrolysis of γ-amides of glutamines substituted at the carboxyl position or both the α-amino and carboxyl positions (e.g., L-glutamylglycine and L-phenylalanyl-L-glutamylglycine). Therefore, deamidases can deamidate glutamine residues in proteins to glutamate residues, and deamidases are also referred to as protein glutamine deamidases. Deamidases contain a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al., “Crystal structures of protein glutaminase and its proforms converted into enzyme-substrate complex”, Journal of Biological Chemistry, Vol. 286, No. 44, pp. 38691–38702) and belong to InterPro entry IPR041325.

[0062] Another example of a protein deamidase is a protein asparaginase, which acts directly on the amide group of the side chain of an asparagine residue in a protein, thereby releasing ammonia and thus converting the asparagine residue into an aspartic acid residue. In this invention, either a protein glutaminase or a protein asparaginase, or a combination of both, can be used as the protein deamidase. In some embodiments, the protein deamidase used in this invention is a protein glutaminase.

[0063] Deamidase activity can be measured using an assay consisting of two separate components: (1) an enzymatic step in which ammonia is formed by the catalytic action of a protein deamidase; and (2) a non-enzymatic assay step in which the ammonia formed in step (1) is derivatized into an indophenol blue compound with maximum absorption at 630 nm. One unit (expressed in indophenol assay units: IPA(U)) is defined as the amount of enzyme that produces 1 μmol of ammonia per minute at 37 °C. Activity can be determined relative to known intensity standards.

[0064] The protein deamidase used in the method of the present invention can be obtained from any genus of microorganism. For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0065] There are no particular limitations on the type or source of the protein deamidase used in this invention. Examples of protein deamidases include those derived from the genera *Chlorella* and *Flavobacterium*. Flavobacterium genus), Stenotrophomonas ( Empedobacter genus), Sphingosine Bacillus ( Sphingobacterium genus), Chlorella ( Aureobacterium genus) or genus of mushrooms ( Myroides Protein deamidases (genus). These protein deamidases can be obtained from the culture medium of the aforementioned microorganisms and are used in this invention. In some embodiments, the protein deamidases may be derived from the genus *Chlorobacterium*, such as species of the genus *Chlorobacterium*. 62563 Beer God Golden Bacterium ( C. gambrini Culex pipiens ( ), C. culicis ), wastewater vulcanii ( C. defluvii ) or pyruvum valerate ( C. proteolyticum In some embodiments, the deamidase in the method of the present invention is derived from or obtained from species of the genus *Chlorella*. -62563 EP1839491 discloses the presence of Corynebacterium glutamicum (…). Corynebacterium glutamicum This is a clone of a protein glutaminase from *Chlorella vulgaris* expressed in [the sample / sample]. The protein glutaminase from *Chlorella vulgaris* is commercially available, for example, as "Amano" 500 (manufactured by Amano Enzyme Inc.).

[0066] In some embodiments of the invention, α-amylase, xylanase, protein deamidase, and optionally additional enzymes are added to a slurry of plant material in water and allowed to act on the plant material substrate to produce a non-alcoholic plant-based food ingredient. In some embodiments, the protein deamidase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In other embodiments, the protein deamidase comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0067] In some embodiments of the invention, α-amylase, xylanase, and protein deamidase are added to a slurry of plant material in water and allowed to act on the plant material substrate to produce plant-based food ingredients. In other embodiments, the α-amylase is a starch-degrading α-amylase. In some embodiments, the xylanase is GH10 xylanase. In some embodiments, the xylanase is GH5-21 xylanase.

[0068] In another embodiment, α-amylase, xylanase, and protein deamidase are added to an oat material slurry in water and kept at 50°C-65°C for 30-90 minutes to produce a non-alcoholic plant-based food ingredient, wherein: -α-amylase is a starch-degrading α-amylase containing an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5. - Xylanase is GH10 xylanase or GH5_21 xylanase, comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 11; and - Protein deamidases contain an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0069] In some embodiments, the enzyme and slurry are kept at 60°C or lower for 60 minutes or less.

[0070] In some embodiments, the resulting oat-based food ingredients contain more soluble proteins compared to food ingredients produced by similar methods that do not include protein deamidases. In some embodiments, the resulting oat-based food ingredients contain more soluble β-glucan compared to food ingredients produced by similar methods that do not include GH10 or GH5_21 xylanases.

[0071] In some methods of the present invention, oat-based food ingredients are separated into solid streams and liquid streams, and the liquid stream is harvested as oat-based food ingredients (i.e., oat substrate). In some embodiments, the resulting oat substrate contains more soluble proteins compared to liquid streams produced by similar methods that do not include protein deamidases. In some embodiments, the resulting oat substrate contains more soluble β-glucan compared to food ingredients produced by similar methods that do not include GH10 or GH5_21 xylanases.

[0072] In some embodiments of the invention, one or more additional enzymes are added to the slurry containing plant material. These additional enzymes may be α-amylase, glucosylamylase, maltodextrin-producing amylase, β-amylase, isoamylase, cyclodextrin-glucantransferase, endopeptidase, protein deamidase, protease, hemicellulase, cellulase, pectinase, glucosidase, glucanase, xylanase, arabinofuranase, pullulanase, and / or lipase, or any combination thereof. The one or more additional enzymes may be of any source, including mammalian, plant, and microbial (bacterial, yeast, or fungal) origin.

[0073] In some embodiments of the invention, α-amylase, xylanase, β-glucanase, and optionally additional enzymes are added to a slurry of plant material in water and allowed to act on the plant material substrate to produce a non-alcoholic plant-based food ingredient. The β-glucanase may have β-1,6-glucanase activity and / or exo- and / or endo-β-1,3-glucanase activity, and may also have other enzymatic activities. These enzymes having β-1,6-glucanase activity and / or exo- and / or endo-β-1,3-glucanase activity are members of the glycoside hydrolase family selected from GH16, GH64, and GH5. In some embodiments, the β-glucanase is derived from GH5.

[0074] In some embodiments, the enzyme having β-glucanase activity may be a formulation of an endo-α-amylase with β-glucanase secondary activity obtained from Bacillus species (such as, for example, Bacillus amylolyticus). In some embodiments, the enzyme having β-glucanase activity may be in a cellulase formulation. In another embodiment, the cellulase formulation may be obtained from Trichoderma reesei (… Trichoderma reesei Examples of enzyme preparations with β-glucanase activity include BAN®, Celluclast®, or Ultraflo® Prime, each available from Novozymes. These enzyme preparations are believed to contain β-glucanase. Ultraflo® Prime contains both β-glucanase and GH10 xylanase.

[0075] In some embodiments of the invention, α-amylase, xylanase, β-glucanase, and optionally additional enzymes are added to a slurry of plant material in water and allowed to act on the plant material substrate to produce a non-alcoholic plant-based food ingredient. In some embodiments, the β-glucanase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12. In other embodiments, the β-glucanase comprises the amino acid sequence of SEQ ID NO: 12.

[0076] In some embodiments of the invention, α-amylase, xylanase, β-glucanase, and protein deamidase are added to an oat material slurry in water and allowed to act on a plant material substrate to produce a plant-based food ingredient. In other embodiments, the xylanase is GH10 xylanase or GH5_21 xylanase, comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 11. In some embodiments, the protein deamidase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the β-glucanase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12. In some embodiments, the α-amylase is a raw starch-degrading α-amylase, and these enzymes act on the plant material substrate at a temperature of 60°C or lower for 60 minutes or less. In some embodiments, the α-amylase is not a raw starch-degrading α-amylase.

[0077] In some embodiments, the additional enzyme is glucosylamylase (also known as amylase). Glucoamylases, such as exoglucosylamylase, hydrolyze maltose into glucose. A suitable glucosylamylase may be Amylase® AG, available from Novozymes.

[0078] In some embodiments, the additional enzyme is maltose-producing amylase. Maltose-producing α-amylase (EC 3.2.1.133) can be derived from Bacillus. It can also be derived from Bacillus thermophilus (…). B. stearothermophilusThe maltose-producing α-amylase of strain NCIB 11837 is commercially available from Novozymes under the trademark Novamyl®. Maltose-producing α-amylases can also be variants of maltose-producing α-amylases from *Bacillus stearothermophilus*, such as those disclosed in, for example, WO1999 / 043794, WO 2006 / 032281, or WO 2008 / 148845, such as Novamyl® 3D. Another suitable maltose-producing amylase can be Maltogenase®, also available from Novozymes.

[0079] In some embodiments, the additional enzyme is a hemicellulase or hemicellulase. The term "hemicellulase" or "hemicellulase" refers to one or more (e.g., several) enzymes that hydrolyze hemicellulosic materials. See, for example, Shallom and Shoham, 2003, Microbial hemicellulases. Current Opinion in Microbiology [Current Views in Microbiology] 6(3): 219-228. Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to: acetylmannan esterase, acetylxylan esterase, arabinonanase, arabinofuranylase, coumarin esterase, ferulic esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. The substrates of these enzymes (hemicellulose) are heterogeneous groups of branched and linear polysaccharides that are linked to cellulose microfibrils in the plant cell wall via hydrogen bonds, thereby crosslinking them into a stable network. Hemicellulose is also covalently attached to lignin, thus forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the synergistic action of many enzymes to achieve its complete degradation. The catalytic modules of hemicellulases are either glycosidases (GH) that hydrolyze glycosidic bonds, or carbohydrate esterases (CE) that hydrolyze ester bonds on the side groups of acetic acid or ferulic acid. These catalytic modules are assigned to the GH and CE families by numerical labeling based on their primary sequence homology. Some families with generally similar folds can be further grouped into alphabetically labeled clans (e.g., GH-A). Information and updated classifications of these and other carbohydrate-active enzymes are available in the Carbohydrate-Activated Enzymes (CAZy) database. Hemicellulase activities can be determined according to Ghose and Bisaria, 1987. Pure & Appl. Chem. [Pure and Applied Chemistry] 59: 1739-1752, measurements were taken at suitable temperatures (e.g., 50°C, 55°C, or 60°C).

[0080] In one embodiment, the hemicellulase comprises a commercial hemicellulase formulation. Examples of commercial hemicellulase formulations suitable for use in this invention include, for example, SHEARZYME™ (Novozymes), CELLIC® HTec (Novozymes), CELLIC® HTec2 (Novozymes), VISCOZYME® (Novozymes), ULTRAFLO® (Novozymes), PULPZYME® HC (Novozymes), ACCELLERASE® XY (Genetronics), ACCELLERASE® XC (Genetronics), ECOPULP® TX-200A (AB Enzymes), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK).

[0081] In some embodiments, the additional enzyme is a protease. The protease may be derived from the genus *Bacillus*, such as *Bacillus amyloliquefaciens*. Suitable proteases may be Neutrase® or Formea®, each available from Novozymes.

[0082] In some embodiments, the additional enzyme is a cyclodextrin dextrantransferase (CGT enzyme). Suitable CGT enzymes may be cyclodextrin dextrantransferase "Amano" (manufactured by Amano Enzyme Products Co., Ltd.) or Toruzyme® (Novozymes).

[0083] Variant naming conventions: In the context of this invention, the term "variant" means a polypeptide having enzymatic activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids (e.g., 1-5 amino acids) adjacent to and immediately following an amino acid occupying a position.

[0084] Amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine fragments, antigenic epitopes, or binding domains.

[0085] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific reactivity are known in the art and, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*. Academic Press [Academic Publishers], New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0086] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, these amino acid alterations can affect the peptide's thermal stability, change its substrate specificity, and alter its optimal pH.

[0087] Procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989), can be used. Science [Science] 244: 1081-1085) is used to identify essential amino acids in polypeptides. In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the enzymatic activity of the resulting mutant molecule is tested to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996. , J. Biol. Chem [Journal of Biochemistry] 271: 4699-4708. The active site of the enzyme or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations in the amino acids at the hypothetical contact site. See, for example, de Vos et al., 1992. Science [Science] 255: 306-312; Smith et al., 1992, J. Mol. Biol. [Journal of Molecular Biology] 224:899-904; Wlodaver et al., 1992, FEBS Lett.[Circular of the Federation of European Biochemical Societies] 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related polypeptides.

[0088] Using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested. These screening procedures include those developed by Reidhaar-Olson and Sauer, 1988. Science [Science] 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 86: 2152-2156; WO 95 / 17413; or those disclosed in WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry [Biochemistry] 30: 10832-10837; US Patent No. 5,223,409; WO 92 / 06204) and regional directed mutagenesis (Derbyshire et al., 1986, Gene [Gene] 46: 145; Ner et al., 1988, DNA 7: 127).

[0089] Mutagenesis / recombination methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenic peptides expressed by host cells (Ness et al., 1999). Nature Biotechnology [Nature Biotechnology] 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the peptide.

[0090] Enzyme treatment The enzymes used in the methods of this invention can be added to a slurry containing plant material in any suitable form, such as a liquid (especially a stabilizing liquid), or they can be added as a substantially dry powder or granules. For example, granules can be produced as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0091] Enzymes can be added to slurries containing plant material in any suitable manner, such as adding them as individual components (adding enzymes separately or sequentially), or adding them together in a step or a composition, or any combination thereof.

[0092] Adding an enzyme to the slurry and maintaining the mixture at 20°C–65°C allows for the hydrolysis of the plant material. The step of incubating the slurry with the enzyme and maintaining the mixture at the temperature that allows for the hydrolysis of the plant material for the duration permitted by the enzyme can also be called enzyme treatment, or enzyme-treated slurry.

[0093] In some embodiments, the temperature of the slurry is maintained between 25°C-40°C, 30°C-45°C, 35°C-50°C, 40°C-55°C, 50°C-60°C, or 50°C-65°C. In other embodiments, the temperature of the slurry is maintained between approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, approximately 60°C, or approximately 65°C. In some embodiments, the temperature of the slurry is maintained between 50°C-55°C, 55°C-60°C, 58°C-62°C, or 60°C-65°C.

[0094] In some embodiments, the slurry containing the added enzyme is maintained at 25°C–65°C for at least 10 minutes to allow enzymatic hydrolysis of the plant material. In some embodiments, the slurry is maintained for about 10, about 15, about 20, about 25, about 30, about 60, about 120, about 180, about 240, or at least about 240 minutes to allow enzymatic hydrolysis of the plant material. In some embodiments, the slurry is maintained for at least about 10, 30, or 60 minutes. In some embodiments, the slurry is maintained for about 60 minutes.

[0095] Starch-degrading enzymes can directly degrade raw starch granules below the starch gelatinization temperature. The gelatinization temperature range for starch can be from 51°C to 78°C, as the gelatinization initiation temperature can vary between approximately 51°C and 68°C. When oat flour is used, starch-degrading α-amylases can directly degrade raw starch at a gelatinization temperature of approximately 55°C to 62°C. In some methods of the present invention, starch-degrading amylases and additional enzymes are provided to a slurry of plant material, and the enzymatic treatment of the slurry can be sustained at 50°C-55°C, 55°C-62°C, 58°C-62°C, approximately 50°C, approximately 55°C, or approximately 60°C for at least 20, 30, 40, or 60 minutes.

[0096] In some embodiments, the slurry is maintained at different temperatures depending on the enzymes added to it. In some embodiments, enzyme treatment is performed in two steps at different temperatures (hereinafter referred to as a two-step method). In the context of this invention, the two-step method includes maintaining the slurry at different temperatures for a certain period of time, wherein the temperature in a given step is set to match the optimal temperature for the enzyme activity of one or more enzymes added to the slurry. For example, the slurry may be heated to a temperature in the range of 70°C to 90°C in one step, one or more first enzymes may be added, and the slurry may be maintained at that temperature range for a period of time (e.g., like 30 minutes). The slurry may then be cooled to a temperature in the range of 20°C to 60°C (e.g., like 50°C to 60°C), followed by the addition of one or more second enzymes different from the first enzyme, and the slurry may be maintained at that temperature range for, for example, 30 minutes. Alternatively, in the context of this invention, a method comprising the following steps (hereinafter referred to as the one-step method) may be performed: providing a slurry with a temperature maintained in the range of 5°C to 20°C, adding an enzyme active in the range of 5°C to 65°C to the slurry, heating the slurry to a temperature in the range of 65°C to 90°C, and adding another enzyme active in the range of 65°C to 90°C, wherein the entire process lasts for 90 to 180 minutes.

[0097] The processes used (including temperature range, pH, and enzyme treatment time) will vary depending on the plant material and the enzymes added to the slurry. Technicians will know how to determine the optimal process parameters based on, for example, the plant material and enzymes used.

[0098] The processing methods employed will also depend on the desired characteristics of the resulting plant-based food ingredients, which are largely dependent on consumer preferences and expectations. For example, viscosity is a crucial characteristic for oat-based food ingredients. Oat-based dairy alternatives typically have a viscosity similar to low-fat or skim milk. This viscosity depends in part on the beta-glucan content, with higher beta-glucan content increasing viscosity. If enzymatic treatment results in a product with too low a viscosity, it will have a bland taste, which is undesirable. However, if the viscosity is too high, the product may have a grainy texture. Furthermore, plant-based food ingredients with very high viscosity may be difficult to process in industrial manufacturing.

[0099] The resulting plant-based food ingredients also need to have the desired protein, starch, and fiber composition. To produce improved oat beverages, it is desirable to increase the protein or fiber content compared to current standards. Increasing the protein and / or fiber content in oat-based food ingredients (also referred to as "oat base") can produce oat-based beverages with an improved nutritional profile. Fiber content can be increased by increasing the amount of β-glucan in the oat base. The method of the present invention provides enzymes, including α-amylase, xylanase, optionally β-glucanase, and optionally protein deamidase, which can be used to process oat material slurries to produce oat bases with increased β-glucan and / or increased protein compared to oat bases produced by methods excluding the listed enzymes.

[0100] Enzyme inactivation can occur after treatment with the enzyme. Enzyme inactivation can occur at any step following hydrolysis. In some embodiments, enzyme inactivation is performed before or after the hydrolyzed plant material has been separated into solid and liquid streams. In other embodiments, enzyme inactivation is performed after additional food ingredients have been added to the harvested liquid stream.

[0101] In some embodiments, the enzyme is inactivated by heat treatment. In some embodiments, the heat treatment is performed at 85°C-95°C for 5-30 minutes. In another embodiment, the heat treatment is performed at 85°C-95°C for 10 minutes. In some embodiments, the heat treatment is performed at 95°C for 5, 10, 15, 20, 25, or 30 minutes.

[0102] In some embodiments, the enzyme is inactivated by ultra-high temperature (UHT) treatment. UHT treatment can be direct or indirect. In some embodiments, UHT treatment is performed at 135°C-154°C for 1-10 seconds. In other embodiments, UHT treatment is performed at 140°C-150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In still other embodiments, UHT treatment is performed at 140°C-145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, UHT treatment is performed at 143°C for 4, 5, 6, 7, or 8 seconds.

[0103] After enzyme inactivation, the hydrolyzed plant material can be cooled. For example, by centrifugation, the hydrolyzed plant material can be separated into a solid stream and a liquid stream. Centrifugation can occur in a sedimentation centrifuge. After centrifugation, the liquid stream can be harvested or collected and used as a food ingredient in dairy alternatives. The liquid stream may still contain some solid matter, also known as "dry matter." In some embodiments, the liquid stream contains 1%-80% solids. In other embodiments, the liquid stream contains 1%-10%, 5%-20%, 10%-25%, 20%-35%, 25%-40%, 30%-45%, 35%-50%, 40%-55%, 45%-60%, 50%-65%, 55%-70%, 60%-75%, or 65%-80% solids. In some embodiments, the liquid stream contains 10%-15% solids. In some embodiments, the liquid stream contains 2%-10%, 3%-8%, 4%-7%, or 5%-6% dry matter. In some embodiments, the liquid stream contains about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% dry matter.

[0104] In some embodiments, the liquid stream is further processed to remove water, also known as concentration. Concentration increases the relative amount of solids in the concentrated liquid stream. Concentration can be achieved by evaporating water from the liquid stream. In some embodiments, the concentrated liquid stream contains 10%-100% solids or dry matter. In other embodiments, the concentrated liquid stream contains 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% solids. In some embodiments, removing water will increase the viscosity of the dairy alternative food product.

[0105] In some embodiments, the liquid stream is used directly as a plant-based food ingredient. Additional food ingredients can be added to the liquid stream to produce dairy alternative food products. The liquid stream can be homogenized before or after the addition of food ingredients.

[0106] In some embodiments, the liquid stream is used directly as a dairy alternative food product. The dairy alternative food product may be UHT or ESL treated and aseptically packaged. The final product may be sold as a plant-based dairy alternative beverage, such as an oat drink.

[0107] Alternatively, the liquid stream can be further processed into other dairy alternative food products (such as fermented plant-based products, like oat-based yogurt, or plant-based ice cream), or the liquid stream can be used as an ingredient in dairy alternative food products.

[0108] In some embodiments, the end product is an oat-based beverage or an oat-based dairy alternative food product, such as oat-based creamer, oat-based yogurt, oat-based cheese, or oat-based ice cream.

[0109] In some embodiments, the plant-based food ingredients produced by the methods of the present invention have higher soluble carbohydrate, protein, and / or fiber content compared to similar methods that do not include providing α-amylase, xylanase, and optionally at least one other enzyme. In some embodiments, the plant-based food ingredients comprise 5%-8%, 7%-10%, 9%-12%, 10%-13%, or 12%-15% carbohydrates or total starch. In some embodiments, the plant-based food ingredients comprise about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% carbohydrates. In some embodiments, the plant-based food ingredients comprise 0.2%-1.2%, 0.5%-1.5%, 0.75%-1.5%, 1.0%-2.0%, 1.5%-2.5%, 1.0%-3.0%, 1.5%-3%, or 2.0%-3.0% protein. In some embodiments, the plant-based food ingredient comprises about 0.2%, about 0.5%, about 0.8%, about 1.0%, about 1.2%, about 1.5%, about 1.8%, about 2.0%, about 2.3%, about 2.5%, about 2.8%, or about 3% protein. In some embodiments, the plant-based food ingredient comprises about 0-2 g / L, about 1-3, about 2-4, about 3-5, about 4-6, about 5-7, about 6-8, or about 7-8 g / L of β-glucan. In some embodiments, the plant-based food ingredient comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8 g / L of β-glucan. In some embodiments, the plant-based food ingredient is an oat-based food ingredient comprising about 5%-15% carbohydrates, about 0.2%-3% protein, and / or about 0-8 g / L of β-glucan. In some embodiments, oat-based food ingredients comprise about 9%-13% carbohydrates, about 0.2%-1.5% protein, and / or about 0.5-3 grams of β-glucan / L. The carbohydrates, protein, and β-glucan are all measured using methods known in the art, such as those described in the examples. In some embodiments, plant-based food ingredients are oat-based food ingredients, such as oat-based feed.

[0110] In some embodiments, the method of the present invention produces liquid plant-based food ingredients and / or plant-based dairy alternative food products that offer a similar texture in terms of viscosity, protein content, and / or fiber content compared to methods in the art, but with a lower percentage of dry matter and a lower carbohydrate content. Because a lower percentage of dry matter is required to achieve the same viscosity, protein content, and / or fiber content, a lower amount of starting plant material is needed to produce plant-based food ingredients with a viscosity, protein content, and / or fiber content and texture similar to those known in the art. In other words, the method of the present invention efficiently produces more plant-based food ingredients or plant-based dairy products per unit of starting plant material compared to methods generally known in the art. This higher raw material utilization efficiency results in higher yields and lower production costs.

[0111] In another embodiment, the method of the present invention produces liquid oat-based food ingredients or oat-based dairy alternative food products containing 3%-5%, 4%-6%, 5%-7%, or 8%-10% dry matter, which have comparable texture in viscosity and equal or higher amounts of protein and / or fiber compared to liquid oat-based food ingredients or oat-based dairy alternative food products containing 10%-15% or 10%-12% dry matter produced using methods known in the art that do not include providing α-amylase, xylanase, optionally protein deamidase, and optionally β-glucanase. Compared to methods known in the art that do not include providing α-amylase, xylanase, optionally protein deamidase, and optionally β-glucanase, the method of the present invention increases the utilization rate of oat raw materials and thus reduces the production costs of oat-based food ingredients and oat-based dairy alternative food products.

[0112] The invention is further defined by the following numbered embodiments: Preferred embodiments

[0113] 1. A method for obtaining non-alcoholic plant-based food ingredients, the method comprising: a) Obtaining a pulp of plant material in water; b) Provides α-amylase, xylanase, and optionally at least one other enzyme; and c) Treat the slurry with the enzyme at 25°C-65°C for at least 10 minutes to produce hydrolyzed plant material, wherein the hydrolyzed plant material is a non-alcoholic plant-based food ingredient.

[0114] 2. The method as described in Example 1, further comprising: d) Separate the plant-based food ingredient into a solid stream and a liquid stream; e) Harvest the liquid stream as a liquid plant-based food ingredient; and f) Optionally, these enzymes may be inactivated before or after step (d) or (e).

[0115] 3. The method as described in any of the foregoing embodiments, wherein the plant material is derived from tubers, nuts, roots, stems, legume pods, fruits, seeds, or whole grains.

[0116] 4. The method as described in any of the foregoing embodiments, wherein the plant material is derived from corn, rice, barley, wheat, quinoa, oats, rye, flax, buckwheat, millet, foxtail millet, sago, cassava, tapioca, potato, sweet potato, pea, legume, broad bean, lentil, soybean, cashew, macadamia nut, almond, coconut, banana, jackfruit and / or breadfruit.

[0117] 5. The method as described in any of the foregoing embodiments, wherein the plant material is cereal flour or hulled cereal, including corn flour, rice flour, barley flour, buckwheat flour, wheat flour, millet flour, quinoa flour, oat flour, rye flour, or mixtures thereof.

[0118] 6. The method as described in any of the foregoing embodiments, wherein the plant material is oat flour, oat flakes, oat bran, hulled oat grains, or any combination thereof.

[0119] 7. A method for obtaining non-alcoholic oat-based food ingredients, the method comprising: a) Obtain a slurry of oat material in water; b) Provide amylase, xylanase, optionally β-glucanase, and optionally protein deamidase; and c) Treat the slurry with the enzyme at 25°C-65°C for at least 10 minutes to produce hydrolyzed oat material, wherein the hydrolyzed oat material is an oat-based food ingredient.

[0120] 8. The method as described in Example 7, further comprising: d) Separate the oat-based food ingredient into a solid stream and a liquid stream; e) Harvest the liquid stream as a liquid oat-based food ingredient; and f) Optionally, these enzymes may be inactivated before or after step (d) or (e).

[0121] 9. The method as described in Example 7 or 8, wherein the ratio of oat material to water in the slurry of step (a) is 1:3 to 1:8 (w / w) or 1:4 to 1:6 (w / w).

[0122] 10. The method as described in Examples 7-9, further comprising concentrating the oat-based food ingredient to reduce its moisture content.

[0123] 11. The method as described in Examples 8-10, further comprising combining the liquid oat-based food ingredient with water and optionally other food ingredients to produce an oat-based beverage comprising 2%-10%, 3%-8%, 4%-7%, 5%-8%, or 7%-10% dry oat matter.

[0124] 12. The method of Examples 8-11, further comprising combining the liquid oat-based food ingredient with water and optionally other food ingredients to produce an oat-based beverage comprising about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% dry oat matter.

[0125] 13. The method of any one of Examples 7-12, wherein the oat-based food ingredient is processed to produce a dairy alternative food product, such as an oat-based beverage, an oat-based ice cream, an oat-based creamer, an oat-based yogurt, or an oat-based cheese.

[0126] 14. The method as described in any of the foregoing embodiments, wherein the xylanase is an endogenous 1,4-xylanase.

[0127] 15. The method as described in any of the foregoing embodiments, wherein the xylanase is GH10, GH30 or GH5 xylanase.

[0128] 16. The method as described in any of the foregoing embodiments, wherein the xylanase is GH10 or GH5-21 xylanase.

[0129] 17. The method as described in any of the foregoing embodiments, wherein the xylanase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11.

[0130] 18. The method as described in any of the foregoing embodiments, wherein the xylanase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 1.

[0131] 19. The method as described in any of the foregoing embodiments, wherein the amylase in step (b) is a raw starch-degrading amylase.

[0132] 20. The method as described in any of the preceding embodiments, wherein the amylase in step (b) comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 5.

[0133] 21. The method as described in Example 19 or 20, wherein the treatment in step (c) is carried out at a temperature of 45°C-50°C, 50°C-55°C, 55°C-60°C, 58°C-62°C, or 60°C-65°C for about 30, about 60, or about 90 minutes.

[0134] 22. The method as described in Examples 19-21, wherein the treatment in step (c) is carried out at a temperature of 60°C for about 60 minutes.

[0135] 23. The method as described in any of the foregoing embodiments, further comprising, in step (b), at least one additional enzyme, wherein the additional enzyme is amylase, glucosylamylase, maltodextrin amylase, β-amylase, isoamylase, α-amylase, cyclodextrin glucantransferase, endopeptidase, protein deamidase, protease, hemicellulase, cellulase, pectinase, glucosidase, glucanase, xylanase, arabinofuranosylase, pullulanase, lipase, or any combination thereof.

[0136] 24. The method as described in any of the foregoing embodiments, wherein the additional enzyme is a protein deamidase.

[0137] 25. The method as described in any of the foregoing embodiments, wherein the additional enzyme is a protein deamidase derived from a species of the genus *Chlorobacterium*.

[0138] 26. The method as described in any of the foregoing embodiments, wherein the additional enzyme is a protein deamidase comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0139] 27. The method as described in any of the foregoing embodiments, wherein the additional enzyme is a protein deamidase comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 6.

[0140] 28. The method as described in any of the foregoing embodiments, wherein the additional enzyme is β-glucanase.

[0141] 28. The method as described in any of the foregoing embodiments, wherein the additional enzyme is β-glucanase comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 12.

[0142] 29. The method as described in any of the preceding embodiments, wherein the non-alcoholic plant-based food ingredient is combined with other food ingredients to produce a dairy alternative food product.

[0143] 30. The method as described in Example 29, wherein the dairy alternative food product is a plant-based beverage, plant-based ice cream, plant-based creamer, plant-based yogurt, or plant-based cheese.

[0144] 31. Dairy alternative food products as described in Examples 29 or 30.

[0145] 32. An alcohol-free oat-based food ingredient produced by any one of Examples 7-28.

[0146] 33. The oat-based food ingredient as described in Example 32, wherein the oat-based food ingredient comprises 5%-8%, 7%-10%, 9%-12%, 10%-13% or 12%-15% total starch; 0.2%-1.2%, 0.5%-1.5%, 0.75%-1.5%, 1.0%-2.0%, 1.5%-2.5%, 1.0%-3.0%, 1.5%-3% or 2.0%-3.0% protein; and / or 0-2, 1-3, 2-4 or 3-5 g β-glucan / L.

[0147] 34. The oat-based food ingredient as described in Example 32, wherein the oat-based food ingredient comprises about 9%-13% total starch, about 0.2%-1.5% protein and / or about 0.5-3 g β-glucan / L.

[0148] 35. Oat-based food ingredients as described in Examples 32-34, wherein the total starch, protein and / or β-glucan content is higher relative to dry matter compared to oat-based food ingredients produced by methods excluding α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase.

[0149] 36. An improved method for producing plant-based dairy alternative food products, the method comprising: a) Obtaining an alcohol-free plant-based food ingredient produced by the method of any one of the preceding claims, wherein the method comprises providing α-amylase, xylanase, and optionally at least one additional enzyme to a slurry of plant material in water, wherein the plant-based food ingredient contains more β-glucan, more protein, and / or higher viscosity compared to a plant-based food ingredient having the same amount of dry matter produced by a method not provided with providing α-amylase, xylanase, and optionally at least one additional enzyme to a slurry of plant material in water; b) Dilute the plant-based food ingredient of step a) such that the amount of β-glucan, protein, and / or viscosity is equivalent to the amount of plant-based food ingredient prepared by a method excluding the provision of α-amylase, xylanase, and optionally at least one other enzyme to a slurry of plant material in water, or reduce the amount of plant-based food ingredient added in step a) for the production of the plant-based dairy alternative food product such that the amount of β-glucan, protein, and / or viscosity used is equivalent to the amount of plant-based food ingredient prepared by a method excluding the provision of α-amylase, xylanase, and optionally at least one other enzyme to a slurry of plant material in water; and c) Add additional ingredients to create plant-based dairy alternative food products.

[0150] 37. An improved method for producing oat-based dairy alternative food products, the method comprising: a) Obtaining an oat-based food ingredient produced by the method of any one of the preceding claims, wherein the oat-based food ingredient contains more β-glucan, more protein and / or higher viscosity compared to an oat-based food ingredient having the same amount of dry matter produced by a method excluding α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase; b) Dilute the oat-based food ingredient of step a) such that the amount of β-glucan, protein, and / or viscosity is equivalent to the amount of oat-based food ingredient prepared by a method excluding α-amylase, xylanase, optionally β-glucanase, and optionally protein deamidase, or reduce the amount of the oat-based food ingredient of step a) used such that the amount of added β-glucan, protein, and / or viscosity is equivalent to the amount of oat-based food ingredient prepared by a method excluding α-amylase, xylanase, optionally β-glucanase, and optionally protein deamidase; and c) Add additional ingredients to create oat-based dairy alternative food products.

[0151] 38. Use of α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase in the production of non-alcoholic plant-based food ingredients to improve the extraction of β-glucan and / or protein from plant materials.

[0152] 39. Use of α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase in the production of non-alcoholic oat-based food ingredients to improve the extraction of β-glucan and / or protein from oat materials.

[0153] The inventions described and claimed herein are not limited to the specific embodiments disclosed herein, as these embodiments are intended to serve as illustrative examples of several aspects of the invention. Any equivalent embodiments, together with combinations of one or more of these embodiments, are intended to be included within the scope of the invention.

[0154] This document cites several references, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described through the following examples, which should not be construed as limiting the scope of the invention.

[0155] Example equipment: Saccharification unit (Lochner Labor und Technik, Germany, LB12) Centrifuge (Multifuge 3 SR, Heraeus, Thermo Scientific, Finland) Heat mixer (TM6-1, Vorwerk Elektrowerke GmbH & CoKG, Germany) LECO FP628 elemental analyzer (for protein determination via combustion method), LECO Corporation; USA Gallery Plus analyzer (Brewmaster, Thermo Fisher Scientific, Finland) HamiltonStar ViPr (Hamilton Company, USA) Spectrophotometer (Shimadzu UV-1700, Suzhou Instruments Manufacturing Co., Ltd., China)

[0156] Material Havnem oat flour (llerne, Denmark) Enzyme preparation 1 (“Preparation 1”): contains α-amylase (SEQ ID NO: 4; 480 KNU-B / g) and β-glucanase paraactivity (712 FBG / g). α-Amylase (SEQ ID NO: 4; 480 KNU-B / g), Novozymes Protein deamidase (SEQ ID NO: 6; 350 IPA (U) / g) Enzyme preparation 2 (“Preparation 2”): contains xylanase (SEQ ID NO: 1; 1500 FXU-S / g) and β-glucanase side activity (1150 FBG / g). GH10 xylanase (SEQ ID NO: 1) GH5_21 xylanase (SEQ ID NO: 2) GH30 xylanase (SEQ ID NO: 3) Sunflower seed oil (Ollineo, Germany)

[0157] Assays used to determine enzyme activity: β-glucanase activity (FBG)One fungal β-glucanase unit (FBG) is the amount of enzyme that, under the standard conditions outlined below, releases reducing oligosaccharides or reducing carbohydrates at a reducing capacity equal to 1 mol of glucose per minute. Fungal β-glucanase reacts with β-glucan to form glucose or reducing carbohydrates, which are identified as reducing sugars according to the Smoginielsen method. The standard reaction conditions are: 0.5% barley β-glucan substrate, 30°C, pH 5.0, and a reaction time of 30 min.

[0158] Xylanase (FXU(S)) Xylan degradation activity can be expressed in FXU(S) units, determined using remazol-xylan (4-O-methyl-D-glucuronic acid-D-xylan (Fluka) stained with remazol brilliant blue R) as a substrate at pH 6.0. Xylanase samples were incubated with the remazol-xylan substrate. The background of the undegraded stained substrate was precipitated by ethanol precipitation. The residual blue color in the supernatant (as determined by spectrophotometry at 585 nm) was proportional to the xylanase activity, and xylanase units were then determined relative to enzyme standards under standard reaction conditions. Standard reaction conditions were 50.0 °C, pH 6.0, 0.45% w / v substrate concentration, and 0.04–0.14 FXU(S) / ml enzyme concentration for 30 minutes. Xylanase activity (FXU(S) meter) is measured relative to the following: Novozymes FXU(S) enzyme standards (available from Novozymes), which include those derived from Aspergillus echinococcosis (… Aspergillus aculeatus Shearzyme is a single-component xylanase preparation.

[0159] Protein deamidase activity (IPA (U)) Protein deamidase activity was measured using an assay consisting of two separate parts: (1) an enzymatic step in which ammonia was formed by the catalytic action of the protein deamidase; and (2) a non-enzymatic assay step in which the ammonia formed in step (1) was derivatized into an indophenol blue compound with maximum absorption at 630 nm. One unit (expressed in indophenol assay units: IPA (U)) was defined as the amount of enzyme that produced 1 μmol of ammonia per minute at 37 °C. Activity was determined relative to known strength standards.

[0160] Quantitative determination of content: Quantitative analysis of protein The amount of nitrogen in powders, bases, precipitates, or beverages was determined by combustion on a Leco FP-528. The amount of protein was calculated as 6.25 times the amount of nitrogen.

[0161] Quantitative analysis of β-glucanThe amount of β-glucan in oat beverages or bases is quantified according to application instructions 64538 (Thermo Fisher Scientific) and measured on a Gallery Plus analyzer Brewmaster.

[0162] Quantitative analysis of total starch The total starch content in powders, bases, sediments, or beverages was analyzed using the Total Starch Assay Kit (AA / AMG) (Megazyme) and AACC Method 76-13.01. Absorbance was measured using a Shimadzu UV-1700 spectrophotometer.

[0163] Quantitative analysis of dry matter The dry matter of powders, base materials, precipitates, or beverages is determined by drying the sample at 105°C for 20 hours.

[0164] Viscosity The viscosity of the samples was measured using HamiltonStar ViPr. High negative pressure is associated with high viscosity. The method used to measure the viscosity is similar to that in WO 2011 / 107472, which is incorporated herein by reference in its entirety.

[0165] calculate: Base material yield (%) = ((g base material) / (g powder + g water + enzyme solution)) 100 Total starch yield (%) = ((% total starch in the substrate / 100) (g base material) / (g powder) (Total starch in flour / 100) 100 Dry matter yield (%) = ((% dry matter in the base material / 100) (g base material) / (g powder) (% of dry matter in powder / 100)) 100 Protein yield (%) = ((% protein in base material / 100) (g base material) / (g powder) (% protein in powder / 100)) 100 Production yield (L beverage / kg oat flour) = (100 / oat load) (% base material yield / 100) (% of base material dry matter) / (desired dry matter) Oat load = Amount of oat flour / Total amount 100

[0166] Example 1: Production of oat-based beverages The table below provides the enzymes and dosages used for each numbered sample. Percentages are based on the weight of oat flour.

[0167] Table 1: Experimental Design

[0168] Enzyme treatment 170 g of deionized water was weighed directly into the mash bath beakers. Enzymes were added according to the experimental design, and 30 g of oat flour was added to each beaker while stirring (150 rpm). The initial temperature was 22°C, and the suspension was heated to 65°C at a rate of approximately 2°C / min. When the target temperature of 65°C was reached, the stirring of the reaction mixture was reduced to 100 rpm and continued for 60 minutes. To terminate the enzyme treatment, the suspension was heated to 90°C at a rate of approximately 1.5°C / min and held at 90°C for 10 minutes. Without cooling, the hot suspension was separated into liquid matrix material and solid precipitate fractions by centrifugation using 3 x 1200 g centrifugation (centrifugation was stopped once 1200 g was reached). The liquid oat matrix material was then weighed and placed in an ice bath.

[0169] The samples were equilibrated to room temperature, and the dry matter, viscosity, protein, total starch, and β-glucan content of the base material for each sample were measured. The samples were then diluted with deionized water to prepare two different formulations containing 5% and 10% oat dry matter, respectively. Additionally, sunflower oil and sodium chloride were added to final concentrations of 1% and 0.08%, respectively. After adding the formulation ingredients, the samples were homogenized using a TM-6 thermal mixer by increasing to maximum speed (set to 10) for 10 seconds and maintaining that mixing speed for 60 seconds.

[0170] result The oat flour used in the enzyme treatment contained 9.06% protein (flour-based), 89.3% dry matter, and 72.5% total starch. As described, after enzyme incubation, the sample was separated into a liquid “base” material and a solid precipitate fraction by centrifugation. The viscosity, protein content, total starch content, and β-glucan content of the oat base were then analyzed, and the dry matter content was quantified. The results are provided in the table below.

[0171] Table 2: Oat Base Content and Viscosity

[0172] The yield was calculated from the above measurements of the oat-based feedstock and is provided in the table below.

[0173] Table 3: Yield of oat-based feed

[0174] Sample 1, containing both amylase and β-glucanase secondary activity, exhibited the highest starch yield and the lowest protein content. β-glucan was not detected in the substrate. Samples 2-5 did not contain amylase with β-glucanase secondary activity. The total starch yield in these four combinations was lower than that of Sample 1, and all four samples contained β-glucan ranging from 1.8 to 1.9 g / L. Samples 2-5 also showed increased viscosity compared to Sample 1. The addition of protein deamidase to Samples 3 and 5 showed a very high increase in protein yield. Surprisingly, Samples 4 and 5, each containing enzyme preparations with high xylanase activity and some small amounts of β-glucanase secondary activity, exhibited lower viscosity while β-glucan levels remained almost unchanged.

[0175] Oat-based beverages typically contain 8%–12% oat dry matter; therefore, this example evaluates formulations containing 5% and 10% oat dry matter. Oat base from each of the above samples was formulated to 5% and 10% oat dry matter, with deionized water added to each to a final volume of 100 ml. The amount of oat base for each formulation is provided below.

[0176] Table 4: Formulations with 5% and 10% oat dry matter

[0177] The amount (L) of oat beverage that can be produced from 1 kg of oat flour was calculated using the 15% oat load used in this experiment. This is the production yield, and the yield for each formulation is shown in the table below.

[0178] Table 5: Production Yield

[0179] The viscosity, dry matter, and protein and total starch content of the final formulation were analyzed. The β-glucan content was calculated based on the amount of added base material and the content analyzed in the base material. The results are shown in the table below.

[0180] Table 6: Content and Viscosity of Final Formulation

[0181] The dry matter from the 5% and 10% oat formulations was measured, and it was confirmed that the formulated samples successfully achieved the expected levels of dry matter calculated based on the oat base for each sample.

[0182] The amylase used in Sample 1 was chosen because it is widely used in oat beverage processing. As mentioned above, the industry standard for oat beverages contains 8%-12% oats, and therefore 10% oats was chosen for comparison with 5% oats. Oat beverage formulations containing lower oat content may be beneficial in terms of higher production yields and lower carbohydrate levels; however, the taste and texture need to be substantially the same or better to be acceptable to consumers. The example here shows that certain enzymatic treatments that increase the protein and / or fiber levels in the oat base can also increase the viscosity of the base and improve its nutritional value, thereby enabling the preparation of oat beverages containing lower oat content (such as 5% oat dry matter) that retain the desired viscosity to provide appropriate texture and mouthfeel.

[0183] In a 10% oat beverage formulation, Sample 1 provided good viscosity, while in a 5% oat beverage formulation, the sample was thin and bland. Sample 1 had a high starch content, low protein content, and was free of β-glucan.

[0184] Compared to Sample 1, Sample 2 exhibited higher viscosity in both the 5% and 10% oat dry matter formulations. However, the viscosity difference between Samples 1 and 2 was greater in the base formulation and appeared to be altered through the formulation process. In the 5% oat dry matter formulation, Sample 2 had a slightly higher viscosity than Sample 1 in the 10% oat dry matter formulation. Sample 2 also had a higher β-glucan level compared to Sample 1.

[0185] Sample 3 contains a combination of the protein deamidase of SEQ ID NO: 6 and the amylase of SEQ ID NO: 4. At both 5% and 10% oat dry matter, the viscosity levels were slightly lower than those of Sample 2. The protein levels were almost double those of Samples 1 and 2, with the protein level at 5% being higher than that at 10%. Overall, Sample 3 exhibited high viscosity at 5%, which is undesirable, although the β-glucan and protein levels in the oat beverage formulation were within acceptable limits.

[0186] Sample 4 was treated with the amylase of SEQ ID NO: 4 and enzyme preparation 2, which also contains xylanase and β-glucanase activity. Surprisingly, the viscosity was excellent at 5% oat dry matter, and the β-glucan level was the highest among all samples, which is desirable for increasing the nutritional value of oat beverages. Although the protein content was low, the oat beverage at 5% oat dry matter showed nutritional improvement and achieved the desired viscosity level.

[0187] Sample 5 contains the protein deamidase of SEQ ID NO: 6, the amylase of SEQ ID NO: 4, and enzyme preparation 2 containing xylanase and β-glucanase activities. At a 5% oat dry matter level, the viscosity of Sample 5 is very similar to that of Sample 4; however, the protein level is higher than that of Sample 4. Although enzyme preparation 2 contains β-glucanase, the β-glucan level is similar to that of Sample 3.

[0188] In summary, oat-based materials produced by methods including treating oat materials with a combination of α-amylase (such as the α-amylase of SEQ ID NO: 4) and enzyme preparations containing GH10 xylanase and β-glucanase (such as enzyme preparation 2) can be formulated into oat-based beverages containing lower oat dry matter while maintaining a good texture in terms of viscosity and offering better nutritional value with improved β-glucan content. In methods where oat materials are also treated with protein deamidases, the protein content of the resulting oat-based material can be more than doubled.

[0189] Example 2: Production of oat-based food ingredients using xylanase This example describes the production of oat “base”, a key ingredient in the production of oat-based beverages. The table below provides the enzymes and dosages used for each numbered sample. The “amylase” is the α-amylase of SEQ ID NO: 4. The percentage of amylase dosage is based on the weight of oat flour. Xylanases were heterologously expressed and harvested. GH10 xylanase contains the amino acid sequence of SEQ ID NO: 1. GH5_21 xylanase contains the amino acid sequence of SEQ ID NO: 2. GH30 xylanase contains the amino acid sequence of SEQ ID NO: 3. Xylanase amounts are provided in mg extracted protein (EP) / kg oat flour.

[0190] Table 7: Experimental Design

[0191] Enzyme treatment and results The oat flour used in the following enzyme treatment contained 8.9% protein (flour-based) and 90.5% dry matter. 170 g of deionized water was weighed directly into mash bath beakers. The enzyme was added according to the experimental design, and 30 g of oat flour was added to each beaker while stirring (150 rpm). The initial temperature was 22°C, and the suspension was heated to 65°C at a rate of approximately 2°C / min. When the target temperature of 65°C was reached, the stirring of the reaction mixture was reduced to 100 rpm and continued for 60 minutes. To terminate the enzyme treatment, the suspension was heated to 90°C at a rate of approximately 1.5°C / min and held at 90°C for 10 minutes. Then, without cooling, the hot suspension was centrifuged using 3 x 1200 g fractions to separate the liquid “base” material and the solid precipitate fraction (centrifugation was stopped once 1200 g was reached). The liquid oat base was then weighed and placed in an ice bath.

[0192] The samples were equilibrated to room temperature, and the dry matter, viscosity, protein content, and β-glucan content of the oat-based substrate were measured for each sample. The results are presented in the table below: Table 8: Oat Base Content and Viscosity

[0193] The yield was calculated from the above measurements of the oat-based feedstock and is provided in the table below: Table 9: Yield of oat-based feed

[0194] Samples 2-4 treated with purified GH10 xylanase (SEQ ID NO: 1) showed increased β-glucan levels at increased enzyme doses, with a decrease in viscosity observed. Samples 5 and 6 treated with purified GH5_21 xylanase (SEQ ID NO: 2) showed increased viscosity at increased doses, but without significantly affecting β-glucan levels. Samples 7 and 8 treated with purified GH30 xylanase (SEQ ID NO: 3) showed moderately decreased viscosity at increased dose levels, with little effect on protein or β-glucan levels.

[0195] In summary, compared to similarly prepared samples without xylanase, the addition of xylanase to the enzymatic treatment of oat materials may alter the β-glucan levels and viscosity of the oat matrix, thereby changing its texture. Increased β-glucan levels in the oat matrix improve its nutritional profile. Reduced viscosity can benefit processing efficiency.

[0196] Example 3: Production of oat-based food ingredients using xylanase and raw starch-degrading α-amylase Material Oat flour (Lantmännen batch 1000483760, 13% protein (Licco)) Amylase 5 (raw starch degrading amylase; SEQ ID NO: 5; 296 FAU(N) / g product) GH5-21 xylanase (SEQ ID NO: 2; 363 FXU-TB / g) Xylanase 11 (GH10 xylanase; SEQ ID NO: 11; 546 FXU-S / g)

[0197] Table 10: Experimental Design

[0198] Enzyme treatment Weigh out 75 g of oat flour and add 425 g of deionized water to a TM6-1 hot mixer and mix until the slurry is homogeneous. Then, aliquot 40 g of the slurry into 50 mL Falcon tubes. Add the enzyme according to the experimental design in Table 10. Heat the oat slurry to 55 °C in a FINEPCR Rotisserie incubator (Weber Scientific, Hamilton, NJ) at a heating rate of approximately 1.5 °C / min. When the target temperature of 55 °C is reached, reduce the stirring of the reaction mixture and continue stirring for 60 minutes. To terminate the enzyme treatment, heat the suspension to 90 °C and maintain it at 95 °C for 20 minutes. Then, without cooling, centrifuge using 3 x 1200 g to separate the hot suspension into liquid matrix material and solid precipitate fraction (stop centrifugation once 1200 g is reached). Then place the liquid oat matrix in an ice bath.

[0199] The samples were equilibrated to room temperature, and the viscosity and β-glucan content of the oat-based material were measured in duplicate for each sample using the method described above.

[0200] Table 11: Viscosity of Oat-Based Samples

[0201] Table 12: β-glucan content of oat-based samples

[0202] Compared to Sample 1, which contained only amylase 5 and no xylanase, all samples containing xylanase had higher β-glucan content. Xylanase 11 (GH10 xylanase, Samples 2-4) showed increased β-glucan levels at all test levels compared to Sample 1, while viscosity decreased with increasing xylanase concentration. GH5-21 xylanase (Samples 5-7) also showed increased β-glucan levels at all test levels compared to Sample 1, and additionally, Samples 5-7 had the highest viscosity of all samples (including Sample 1 (without xylanase)). These results are similar to those in Example 2, although different GH10 xylanases were used in this example and at higher concentrations.

[0203] In summary, the addition of xylanase to oat materials leads to an increase in β-glucan in the oat matrix, which is a nutritional improvement compared to oat matrix produced without the addition of xylanase. Furthermore, the use of xylanase may affect the viscosity and texture of the matrix.

Claims

1. A method for obtaining non-alcoholic oat-based food ingredients, the method comprising: a) Obtain a slurry of oat material in water; b) Provide amylase, xylanase, optionally β-glucanase, and optionally protein deamidase; and c) Treat the slurry with the enzyme composition at 25°C-65°C for at least 10 minutes to produce hydrolyzed plant material, wherein the hydrolyzed plant material is an oat-based food ingredient.

2. The method of claim 1, wherein the method further comprises d) Separate the oat-based food ingredients into a solid stream and a liquid stream; e) Harvesting the liquid stream as a liquid oat-based food ingredient; and f) Optionally, the enzyme may be inactivated before or after step (d) or (e).

3. The method as described in any of the preceding claims, wherein the oat material is oat flour, oat flakes, oat bran, hulled oat kernels, or any combination thereof.

4. The method as described in any of the preceding claims, wherein the ratio of oat material to water in the slurry of step (a) is 1:3 to 1:8 (w / w) or 1:4 to 1:6 (w / w).

5. The method of any of the preceding claims, wherein the xylanase is GH10, GH30 or GH5 xylanase.

6. The method of any of the preceding claims, wherein the xylanase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

11.

7. The method as described in any of the preceding claims, wherein the amylase in step (b) is a raw starch-degrading amylase.

8. The method of any of the preceding claims, wherein the amylase in step (b) comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO:

5.

9. The method of claim 7 or 8, wherein the treatment in step (c) is carried out at a temperature of 45°C-50°C, 50°C-55°C, 55°C-60°C, 58°C-62°C, or 60°C-65°C for about 30, about 60, or about 90 minutes.

10. The method of any of the preceding claims, wherein a protein deamidase is provided in step (b), wherein the protein deamidase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

11. The method as described in any of the preceding claims, wherein β-glucanase is provided in step (b).

12. The method of any of the preceding claims, wherein β-glucanase is provided in step (b), wherein the β-glucanase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or 100% identity with SEQ ID NO:

12.

13. The method of any of the preceding claims, wherein the oat-based food ingredients are processed to produce dairy alternative food products, such as oat-based beverages, oat-based ice cream, oat-based creamer, oat-based yogurt, or oat-based cheese.

14. The oat-based food ingredient as claimed in any of the preceding claims, wherein the total starch, protein and / or β-glucan content is higher relative to dry matter than that of an oat-based food ingredient produced by a method excluding α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase.

15. Use of α-amylase, xylanase, optionally β-glucanase and optionally protein deamidase in the production of non-alcoholic oat-based food ingredients to improve the extraction of β-glucan and / or protein from oat materials.

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