Methods for obtaining a plant-based food ingredient

Abstract

The present invention is directed toward 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 the plant-based food ingredient produced by this method. The plant-based food ingredients produced by this method may be used to produce dairy alternative food products.

Description

[0001] METHODS FOR OBTAINING A PLANT-BASED FOOD INGREDIENT

[0002] REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to use of enzymes for an improved method of extraction of carbohydrates and protein from plant material for the production of a plant-based food ingredient.

[0006] BACKGROUND OF THE INVENTION

[0007] The number of people pursuing a vegan, vegetarian, or non-dairy diet for health reasons has increased in recent years. Further, food products made from animals’ milk, such as cows’ milk, are increasingly recognized for their high environmental costs. These factors are leading to a greater demand for dairy alternative food products for foods traditionally derived from milk, including milk, creamer, cheese, yogurt, and ice cream.

[0008] Dairy alternative food products are typically derived from high starch plant material, such as cereal grain, nuts, or legumes. In general, to convert the high-starch plant material to a dairy alternative food product or a food ingredient to be included in a dairy alternative food product, the starch must be hydrolysed. The conversion of the starch typically includes a gelatinisation step in which starch granules are dissolved to form a viscous suspension, a liquefaction step where the starch is partially hydrolysed with a concomitant loss in viscosity, and optionally followed by a saccharification step which involves the production of glucose and maltose by further hydrolysis.

[0009] An example of dairy alternative food products which have received great attention in recent years are those based on oats. Oats are perceived as healthy for a number of reasons: they are a great source of vitamins, minerals, fiber, antioxidants, and essential amino acids, and health benefits associated with consumption of oats include lower blood cholesterol levels and reduced risk of heart disease.

[0010] The use of enzymes to improve conversion of high-starch plant material into non-alcoholic plant-based food ingredients and plant-based dairy alternative food products remains a topic of interest in the industry. It is an object of the present invention to identify improved methods for producing non-alcoholic plant-based food ingredients and plant-based dairy alternative food products, particularly methods which increase raw material utilization and / or the nutritive properties of the food ingredients and food products, while maintaining or improving the organoleptic properties of the resultant plant-based dairy alternative food products. SUMMARY OF THE INVENTION

[0011] The invention relates to an improved method for producing a non-alcoholic plant-based food ingredient with increased production capacity, lower production cost, and / or better raw material utilization. This improved method comprises selective enzymes which improve solubilization of carbohydrates, protein, and / or fiber from the starting plant material.

[0012] The invention provides a method for obtaining a non-alcoholic plant-based food ingredient, comprising: a) obtaining a slurry of plant material in water; b) providing an alpha amylase, a xylanase, and optionally at least one additional enzyme; and c) treating said slurry with said enzymes to produce a hydrolyzed plant material, wherein the hydrolyzed plant material is a plant-based food ingredient.

[0013] The invention also provides a method for obtaining an oat-based food ingredient, comprising: a) obtaining a slurry of an oat material in water; b) providing an enzyme composition comprising an amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase; and c) treating said slurry with said enzyme composition to produce a hydrolyzed plant material, wherein the hydrolyzed plant material is an oat-based food ingredient.

[0014] The invention provides the plant-based food ingredient and the oat-based food ingredient obtained by these methods, which may have an improved nutritional profile compared to similar plant-based ingredients prepared using similar methods known in the art. These ingredients may further be processed to produce dairy alternative food products which may also have an improved nutritional profile.

[0015] The invention further provides the use of an alpha-amylase, a xylanase, optionally a beta- glucanase, and optionally a protein deamidase in the production of a plant-based food ingredient to improve extraction of beta-glucan and / or protein from plant material.

[0016] DEFINITIONS

[0017] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w / w or “% (w / w)”). Unless defined otherwise or clearly indicated by 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 belongs.

[0018] Alpha-Amylase: (1,4-alpha-D-glucan glucanohydrolases, EC 3.2.1.1) are a group of enzymes, which catalyze the hydrolysis of starch and other linear and branched 1 ,4glucosidic oligo and polysaccharides.

[0019] Beta-glucanase: The term “beta-glucanase” encompasses polypeptides having beta- 1,6- glucanase activity and / or exo- and / or -endo beta-1 , 3-glucanase activity.

[0020] Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0021] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0022] Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of the present invention has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0023] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified 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. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

[0024] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and / or C-terminal processing (e.g., removal of signal peptide).

[0025] Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0026] Purified: The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually 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., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0027] In one aspect, the term "purified" as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0028] Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains 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% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0029] It is, therefore, preferred that the substantially pure polypeptide 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, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form ( / .e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods. Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0030] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0031] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0032] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0033] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0034] Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0035] Variant: The term “variant” means a polypeptide having enzyme activity comprising a man-made mutation, i.e., a substitution, insertion (including extension), and / or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0036] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0037] Xylanase: The term “xylanase” means a glucuronoarabinoxylan endo-1 ,4-beta-xylanase (E.C. 3.2.1.136) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosyl links in some glucu- ronoarabinoxylans.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention is directed toward a method for producing a non-alcoholic plantbased food ingredient, comprising treating a slurry of plant material with an alpha amylase, a xylanase, and optionally at least one additional enzyme. The present invention is further directed toward a method for producing a non-alcoholic oat-based food ingredient, comprising treating a slurry of oat material with an alpha amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase. The invention further includes the non-alcoholic plant-based food ingredient produced by this method. The non-alcoholic oat-based food ingredient produced by this method may have increased amounts of beta-glucan and / or protein, thereby providing increased nutritional value, and optionally increased viscosity, compared to a method which does not treat the oat material with an alpha amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase. The oat-based food ingredient produced by the methods of the invention may be an improved oat base over methods of the art, such that less of the oat base may be needed to produce a desirable plant-based food product. The plant-based food ingredients produced by this method may be used to produce dairy alternative food products.

[0040] A “plant-based food ingredient” refers to a plant-based composition which may be combined with additional food ingredients to produce a food product. The plant-based food ingredient and the plant-based food product may be solid or liquid, such as a beverage. In some embodiments, the plant-based food ingredient is non-alcoholic. The term “non-alcoholic” means the plant-based food ingredient does not contain alcohol or does not contain a significant amount of alcohol. In some embodiments, the non-alcoholic plant-based food ingredient contains less than 0.5%, 0.4%, 0.3%, 0.2%, 0.1 %, 0.05%, or less than 0.01% alcohol by volume. In some embodiments, the plant-based food ingredient may be combined with additional food ingredients to produce a dairy alternative food product. The additional food ingredients may be any food ingredient deemed useful by a practitioner of skill in the art. The additional food ingredient may be a solid or liquid. The additional food ingredient may or may not be plantbased. In some embodiments, the additional food ingredient is water.

[0041] A “dairy alternative food product” refers to a food product which can be used as a substitute for a dairy food product. A dairy alternative food product is plant-based and does not contain milk-derived food ingredients. Dairy alternative food products include plant-based beverages, creamer, cheese, ice cream, and yogurt. In some embodiments, the dairy alternative food product is a plant-based beverage. In some embodiments, the dairy alternative food product is an oat-based beverage.

[0042] In some embodiments, the plant-based food ingredient may be used as a substrate for fermentation to produce a dairy alternative beverage, such as buttermilk, or to produce a dairy alternative yogurt.

[0043] In some embodiments, the plant-based food ingredient may be further processed. Further processing may include water removal. In some embodiments, water removal will concentrate the products of hydrolysis, namely the released carbohydrates, protein, and fiber. In some embodiments, water removal will increase the viscosity of the plant-based food ingredient.

[0044] In some embodiments, the plant-based food ingredient may be further processed and combined with additional food ingredients to produce a dairy alternative ice cream. In some embodiments, the plant-based food ingredient may be further processed and combined with additional food ingredients to produce a dairy alternative cheese.

[0045] In some embodiments, the plant-based food ingredient may be directly combined with additional food ingredients to produce a dairy alternative beverage that is ready to drink. Examples of a dairy alternative beverage include an oat beverage, cashew beverage, fava bean beverage, lentil beverage, soy beverage, rice beverage, barley beverage, quinoa beverage, flax beverage, hemp beverage, potato beverage, pea beverage, hemp beverage, almond beverage, tiger nut beverage, macadamia beverage, coconut beverage, or a beverage comprising any combination thereof.

[0046] The dairy alternative food product may be fortified with a plant-based dairy alternative powder, such as, e.g., soymilk powder, or with concentrated or isolated protein, such as, e.g., soy / pea protein isolate or soy / pea protein concentrate. In an embodiment, the dairy alternative food product is fortified, such as, e.g., an oat-based drink fortified with pea protein.

[0047] 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 at most 4% (w / w). In some embodiments, the plant-based food ingredient has a protein content of about 1 % (w / w). Additional food ingredients which may be combined with the plant-based food ingredient include, but are not limited to, e.g., lipids, such as oils, in particular plant oils; sugars, such as sucrose; proteins, various forms of synthetic amino acids, dietary fiber, salts, minerals, flavoring agents, vitamins, and any combination thereof.

[0048] In an embodiment, lipids may be a plant oil or a mixture of plant oils. The lipids may be selected from rapeseed oil, flaxseed oil, safflower oil, flaxseed 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 selection of suitable lipid depends on the type of plant-based dairy alternative food product desired.

[0049] The lipid may be added in an amount of between about 1% to about 5% (w / w), such as 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 at most 5.0% (w / w).

[0050] In some embodiments, salt is combined with the plant-based food ingredient. The salt may be sodium chloride, dicalcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium carbonate and any combination thereof.

[0051] In some embodiments, vitamins and / or minerals are combined with the plant-based food ingredient. The 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. The mineral may be calcium, phosphorous, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper and mixtures thereof.

[0052] The dairy alternative food product may be standardized and / or homogenized. The dairy alternative food product may be pasteurized or otherwise heat-treated.

[0053] The plant-based food ingredient of the invention is derived from plant material, which is or is derived from the edible portions of a plant. In some embodiments, the plant material is derived from edible portions of a plant which are also high in starch. In some embodiments, the edible portion of the plant may be tubers, roots, stems, cobs, legumes, fruits, nuts, or seeds. In some embodiments, the plant is a cereal and the plant material is or is derived from the cereal grain, also referred to as the whole grain. In further embodiments, the cereal grain may be from corn, rice, barley, wheat, flax, hemp, buckwheat, millet, milo, quinoa, oat, or rye. In some embodiments, the plant material is or is derived from a tuber or root (including rhizomes), such as a potato, sweet potato, cassava, tiger nut (chufa nut), canna, or tapioca. In some embodiments, the plant material is or is derived from a fruit or a nut, such as cashew, macadamia, almond, coconut, banana, jack fruit, or bread fruit. In some embodiments, the plant material is or is derived from a hemp, sago, pea, or bean plant, such as a soybean, fava bean, or lentil plant. In some embodiments, the plant material is heat treated. In some embodiments, the plant material is dehydrated. In some embodiments, the plant material is de-hulled, 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, hemp flour, pea flour, bean flour, de-hulled oats, de-hulled barley, de-hulled wheat, de-hulled peas, de-hulled beans, or a combination or any thereof. In some embodiments, the plant material is smashed or ground to produce a paste.

[0054] In some embodiments, the plant material is oat material. In further embodiments, the oat material is oat flour, oat flakes, oat bran, de-hulled oats, groats, or a combination thereof. In still further embodiments, the oat material may be oat flour such as heat-treated oat flour, or it may be milled oat kernels such as de-hulled and heat-treated oat kernels which have been wet-milled, or it 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 ingredient produced by the methods of the invention may also be referred to as “oat base”.

[0055] In the methods of the invention, the plant material is suspended in water to produce a slurry, where 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 where the ratio of oat material to water is 1 :3 to 1 :8 (w / w). In further embodiments, the ratio of oat material to water is 1 :4 to 1 :6 (w / w).

[0056] In the methods of the 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 at most 20% (w / w). In some embodiments, the plant material has a protein content of around 15% (w / w). In some embodiments, the plant material is oat material which has a protein content of at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (w / w).

[0057] Enzymes

[0058] In the methods of the invention, an alpha amylase, a xylanase, and optionally at least one additional enzyme is added to a slurry of plant material in water. The enzymes are allowed to act on the plant material substrate, and the resulting hydrolyzed product is a non-alcoholic plantbased food ingredient. The alpha amylase may be any alpha-amylase suitable for the methods according to the invention. In some embodiments, the alpha-amylase is a bacterial endo-alpha- amylase, preferably obtained from, or a variant of, an endo-alpha-amylase obtained from, Bacillus, preferably from Bacillus amyloliquefaciens. An example of a bacterial endo-alpha-amylase is BAN® available from Novozymes A / S. In some embodiments, the alpha-amylase is a fungal endo-alpha-amylase, preferably obtained from, or a variant of, an endo-alpha-amylase obtained from, Aspergillus, preferably from Aspergillus oryzae. An example of a fungal endo-alpha-amyl- ase is Fungamyl® available from Novozymes A / S. Another example of a suitable alpha-amylase is Termamyl® available from Novozymes A / S.

[0059] In some embodiments of the invention, the alpha-amylase comprises an amino acid sequence with 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 to SEQ ID NO: 4. In some embodiments, the alpha-amylase comprises the amino acid sequence of SEQ ID NO: 4.

[0060] In some embodiments, the alpha-amylase is a raw starch hydrolyzing alpha-amylase. A raw starch hydrolyzing alpha-amylase, also known as a raw starch degrading alpha-amylase, as used herein refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch. Sources of raw starch degrading enzymes include enzymes obtained from Aspergillus spp. such as Aspergillus oryzae, Aspergillus niger and Aspergillus kawachii. Examples of such raw starch degrading enzymes include the raw starch degrading enzymes described in WO 2005 / 003311 , WO 2006 / 0692, WO 2006 / 060289 and WO 2004 / 080923.

[0061] In some embodiments, the raw starch degrading alpha-amylase is an acid alpha-amylase. An “acid alpha-amylase” is an alpha-amylase (4-a-D-glucan glucanohydrolase, E.C. 3.2.1.1) which when added in an effective amount has activity at a pH in the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0. A source of a raw starch degrading acid alphaamylase is the acid alpha amylase from Aspergillus niger disclosed as "AMYA_ASPNG" in the Swiss-prot / TREMBL database under the primary accession no. P56271 and described in more detail in WO 1989 / 01969 (Example 3). The Aspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004 / 080923 (Novozymes A / S) which is hereby incorporated by reference. A suitable commercially available acid fungal alpha-amylase derived from Aspergillus ni- ger is the product SP288 (SEQ ID NO:1 of U.S: Patent No. 7,244,597; available from Novozymes A / S). Other sources of acid alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, such as a strain of Rhizomucor pusillus (WO 2004 / 055178) or Merip- ilus giganteus. In yet another embodiment, the acid alpha-amylase is derived from Aspergillus kawachii and is disclosed by Kaneko et al., J. Ferment. Bioeng. 81 :292-298(1996) “Molecular- cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alphaamylase from Aspergillus kawachii"', and further as EMBL:#AB008370. In some embodiments, the raw starch degrading alpha amylase possesses a carbohydrate binding module (CBM) which binds to starch. In some embodiments, the CBM binds preferentially to starch, particularly to thermally untreated, granular starch. Such a CBM may also be referred to as a starch binding domain (SBD). SBDs are known to be in 15 CBM families, namely CBM20, 21 , 25, 26, 34, 41 , 45, 48, 53, 68, 69, 74, 82, and 83.

[0062] In some embodiments of the invention, the alpha-amylase is a raw starch degrading alpha-amylase comprising an amino acid sequence with 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 to SEQ ID NO: 5. In some embodiments, the alpha-amylase is a raw starch degrading alpha-amylase comprising the amino acid sequence of SEQ ID NO: 5.

[0063] In methods of the invention, a xylanase is provided to the slurry of plant material and water. A “xylanase” refers to a glucuronoarabinoxylan endo-1 ,4-beta-xylanase (E.C. 3.2.1.136) that catalyses the endohydrolysis of 1 ,4-beta-D-xylosyl links in some glucuronoarabinoxylans. Xylanase activity can be determined with 0.2% AZCL-glucuronoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1 .0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-glucuronoxy- lan as substrate in 200 mM sodium phosphate pH 6. Examples of enzyme preparations having xylanase activity include MULTIFECT(R) Xylanase (Genencor) and HSP 6000 Xylanase (DSM).

[0064] The xylanase of the methods of the invention may be a GH3 xylanase, a GH5 xylanase, a GH8 xylanase, GH10 xylanase, a GH11 xylanase, a GH30 xylanase, a GH43 xylanase, and a GH 98 xylanase. In some embodiments, the xylanase is a GH10, GH30, or GH5 xylanase. In some embodiments, the xylanase is a GH5_21 or GH30 xylanase, which are xylanases known to act on complex xylans. In some embodiments, the xylanase may be an Aspergillus GH10 xylanase, a Talaromyces GH10 xylanase, a Chryseobacterium GH5_21 xylanase, or a Bacillus GH30 xylanase. In some embodiments, the xylanase may comprise an amino acid sequence with 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 to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11. In some embodiments, the xylanase may comprise the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11.

[0065] Xylooligosaccharides (XOS) are polymers of xylose, with a degree of polymerization (DP) typically ranging from 2 to 10. XOS may act as a prebiotic, selectively stimulating the growth of microorganisms in a human or mammalian gut, such as Bifidobacteria or Lactobacilli, which are beneficial for gut health. In some embodiments, methods of the invention may produce a plantbased food ingredient that comprises XOS and that may act as a prebiotic when consumed.

[0066] In the methods of the invention, a slurry of plant material in water may be treated with a protein-modifying enzyme, such as a deamidase, transglutaminase, or peptidase. In further embodiments, the slurry of plant material may be treated with a protein deamidase. In the present invention, a “protein deamidase”, or “deamidase”, refers to an enzyme having an effect of directly acting on an amide group of a side chain of an amino acid that constitutes a protein to cause deamidation and release of ammonia without cleaving a peptide bond of the protein and / or crosslinking proteins.

[0067] The term “deamidase” means a protein-glutamine glutaminase (also known as glutami- nylpeptide glutaminase) activity, as described in EC 3.5.1.44, which catalyzes the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or both the alpha-amino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L- glutaminylglycine. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues and are also referred to as protein glutamine deamidases. Deamidases comprise 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 pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belong to the InterPro entry IPR041325.

[0068] Another example of a protein deamidase is a protein asparaginase that directly acts on an amide group of the side chain of an asparagine residue contained in a protein, releasing ammonia and thus converting the asparagine residue into an aspartate residue. In the present invention, as a protein deamidase, any one of the protein glutaminase and the protein asparaginase can be used, or both can be used in combination. In some embodiments, the protein deamidase used in the present invention is a protein glutaminase.

[0069] Deamidase activity may be measured using an assay consisting of two separate decoupled parts: (1) an enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and (2) a non-enzymatic detection step, wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm. The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (given in Indophenol Assay Unit: IPA(U)). The activity may be determined relative to a standard of declared strength.

[0070] A protein deamidase to be used in a method of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0071] The types or origins of the protein deamidase used in the present invention are not particularly limited. Examples of the protein deamidase include protein deamidases derived from Chryseobacterium genus, Flavobacterium genus, Empedobacter genus, Sphingobacterium genus, Aureobacterium genus, or Myroides genus. Protein deamidases can be obtained from a culture broth of the above-described microorganisms and used in the present invention. In some embodiments, the protein deamidase may be derived from Chryseobacterium genus, such as Chryseobacterium sp-62563, C. gambrini, C. culicis, C. defluvii, or C. proteolyticum. In some embodiments, the deamidase in the methods of the invention is derived from or obtained from Chryseobacterium sp-62563. EP1839491 discloses cloning of a protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum.. A protein glutaminase derived from Chryseobacterium proteolyticum is commercially available as, for example, "Amano" 500 (manufactured by Amano Enzyme Inc.)

[0072] In some embodiments of the invention, an alpha amylase, xylanase, protein deamidase, and optionally additional enzymes are added to the 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 which is 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 to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In further 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.

[0073] In some embodiments of the invention, an alpha amylase, xylanase, and a protein deamidase are added to the slurry of plant material in water and allowed to act on the plant material substrate to produce a plant-based food ingredient. In further embodiments, the alpha amylase is a raw starch degrading alpha amylase. In some embodiments, the xylanase is a GH10 xylanase. In some embodiments, the xylanase is a GH5_21 xylanase.

[0074] In further embodiments, an alpha amylase, xylanase, and a protein deamidase are added to the slurry of oat material in water and held at 50-65°C for 30-90 minutes to produce a nonalcoholic plant-based food ingredient, wherein:

[0075] -the alpha amylase is a raw starch degrading alpha amylase comprising an amino acid sequence with 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 to SEQ ID NO: 5;

[0076] -the xylanase is a GH10 xylanase or a GH5_21 comprising an amino acid sequence with 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 to SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 11 ; and

[0077] -the protein deamidase comprises an amino acid sequence which is 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 to SEQ ID NO: 6.

[0078] In some embodiments, the enzymes and slurry are held at 60°C or less for 60 minutes or less.

[0079] In some embodiments, the resulting oat-based food ingredient comprises more soluble protein compared to a food ingredient produced by a similar method which does not comprise a protein deamidase. In some embodiments, the resulting oat-based food ingredient comprises more soluble beta glucan compared to a food ingredient produced by a similar method which does not comprise a GH10 or GH5_21 xylanase.

[0080] In some methods of the invention, the oat-based food ingredient is separated into a solid and liquid stream, and the liquid stream is harvested as a oat-based food ingredient, namely an oat base. In some embodiments, the resulting oat base comprises more soluble protein compared to a liquid stream produced by a similar method which does not comprise a protein deamidase. In some embodiments, the resulting oat base comprises more soluble beta glucan compared to a food ingredient produced by a similar method which does not comprise a GH10 or GH5_21 xylanase.

[0081] In some embodiments of the invention, one or more additional enzyme(s) is added to the slurry comprising plant material. The additional enzymes may be an alpha-amylase, a glucoamylase, maltogenic amylase, beta-amylase, iso-amylase, cyclodextrin glucanotransferase, endopeptidase, protein deamidase, protease, hemicellulase, cellulase, pectolytic enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof. The additional enzyme(s) may be of any origin, including mammalian, plant, and microbial (bacterial, yeast or fungal) origin.

[0082] In some embodiments of the invention, an alpha amylase, xylanase, beta-glucanase, and optionally additional enzymes are added to the slurry of plant material in water and allowed to act on the plant material substrate to produce a non-alcoholic plant-based food ingredient. A beta- glucanase may possess beta-1 ,6-glucanase activity and / or exo- and / or -endo beta-1 ,3-glucanase activity, and may possess other enzymatic activities as well. The enzymes having beta-1 , 6-glu- canase activity and / or exo- and / or -endo beta-1 , 3-glucanase activity are members of a glycoside hydrolase family selected from GH16, GH64, and GH5. In some embodiments, the beta-glucanase is from GH5.

[0083] In some embodiments, the enzyme having beta-glucanase activity may be a preparation of an endo-alpha-amylase obtained from Bacillus, such as for example from Bacillus amylolique- faciens, which has beta-glucanase side activity. In some embodiments, the enzyme having beta-glucanase activity may be in a cellulolytic enzyme preparation. In further embodiments, the cellulolytic enzyme preparation may be obtained from Trichoderma reesei. Examples of enzyme preparations having beta-glucanase activity include BAN®, Celluclast®, or Ultraflo® Prime, each available from Novozymes A / S. These enzyme preparations are considered to comprise a beta- glucanase. Ultraflo® Prime comprises a beta-glucanase and a GH10 xylanase.

[0084] In some embodiments of the invention, an alpha amylase, xylanase, beta-glucanase, and optionally additional enzymes are added to the 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 beta-glucanase comprises an amino acid sequence which is 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 to SEQ ID NO: 12. In further embodiments, the beta-glucanase comprises the amino acid sequence of SEQ ID NO: 12.

[0085] In some embodiments of the invention, an alpha amylase, xylanase, beta-glucanase, and protein deamidase are added to the slurry of oat material in water and allowed to act on the plant material substrate to produce a plant-based food ingredient. In further embodiments, the xylanase is a GH10 or GH5_21 xylanase comprising an amino acid sequence with 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 to SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 11. In some embodiments, the protein deamidase In some embodiments, the protein deamidase comprises an amino acid sequence which is 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 to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, the beta-glucanase comprises an amino acid sequence which is 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 to SEQ ID NO: 12. In some embodiments, the alpha amylase is a raw starch degrading alpha amylase and the enzymes act on the plant material substrate at a temperature of 60°C or less for 60 minutes or less. In some embodiments, the alpha amylase is not a raw starch degrading alpha amylase.

[0086] In some embodiments, the additional enzyme is a glucoamylase (also known as amyloglu- cosidase). A glucoamylase, for example an exo-glucoamylase, hydrolyzes maltose into glucose. A suitable glucoamylase may be Amylase® AG, available from Novozymes A / S.

[0087] In some embodiments, the additional enzyme is a maltogenic amylase. The maltogenic alpha-amylase (EC 3.2.1.133) may be from Bacillus. A maltogenic alpha-amylase from B. stea- rothermophilus strain NCIB 11837 is commercially available from Novozymes A / S under the tradename Novamyl®. The maltogenic alpha-amylase may also be a variant of the maltogenic alpha-amylase from B. stearothermophilus as disclosed in, e.g., WO1999 / 043794; W02006 / 032281 ; or W02008 / 148845, e.g., Novamyl® 3D. Another suitable maltogenic amylase may be Maltogenase®, also available from Novozymes A / S.

[0088] In some embodiments, the additional enzyme is a hemicellulolytic enzyme, or hemicellulase. The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Microbial hemicellulases, Current Opinion 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, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates of these enzymes, the hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families marked by numbers. Some families, with overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). An informative and updated classification of these and other carbohydrate active enzymes is available on the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chern. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C.

[0089] In one embodiment, the hemicellulase comprises a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME™ (Novozymes A / S), CELLIC® HTec (Novozymes A / S), CELLIC® HTec2 (Novozymes A / S), VISCOZYME® (Novozymes A S), UL- TRAFLO® (Novozymes A / S), PULPZYME(R) HC (Novozymes A / S), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK). In some embodiments, the additional enzyme is a protease. The protease may be from Bacillus, e.g., B. amyloliquefaciens. A suitable protease may be Neutrase® or Formea®, each available from Novozymes A / S.

[0090] In some embodiments, the additional enzyme is a cyclodextrin glucanotransferase (CGTase). A suitable CGTase may be the cyclodextrin glucanotransferase “Amano” (manufactured by Amano Enzyme Inc.) or Toruzyme® (Novozymes A / S).

[0091] Conventions for Designation of Variants:

[0092] In the context of the present invention, the term “variant” means a polypeptide having enzymatic activity comprising an alteration, i.e., a substitution, insertion, and / or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding one or more (e.g., several) amino acids, e.g., 1-5 amino acids, adjacent to and immediately following the amino acid occupying a position.

[0093] The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0094] Examples of conservative substitutions are within the groups of 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 generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0095] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may affect the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0096] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for enzymatic activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

[0097] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0098] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0099] Enzyme Treatment

[0100] The enzymes used in the methods of the invention may be added to the slurry comprising the plant material in any suitable form, such as in the form of a liquid, in particular a stabilized liquid, or it may be added as a substantially dry powder or granulate. Granulates may be produced, e.g., as disclosed in US Patent No. 4,106,991 and US Patent No. 4,661 ,452. Liquid enzyme preparations may, for instance, be stabilized by adding a sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well-known in the art.

[0101] The enzymes may be added to the slurry comprising the plant material in any suitable manner, such as individual components (separate or sequential addition of the enzymes) or addition of the enzymes together in one step or one composition, or any combination thereof.

[0102] The enzymes are added to the slurry and the mixture is held at 20-65°C to allow for hydrolysis of the plant material. The step of incubating the slurry with enzymes and holding the mixture at a temperature and amount of time sufficient to allow for hydrolysis of the plant material may also be referred to as an enzyme treatment, or treating the slurry with enzymes.

[0103] In some embodiments, the slurry is held at a temperature between 25-40°C, 30-45°C, 35- 50°C, 40-55°C, 50°C-60°C, or 50°C-65°C. In further embodiments, the slurry is held at a temperature of about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, or about 65°C. In some embodiments, the slurry is held at a temperature between 50-55°C, 55-60°C, 58-62°C, or 60-65°C.

[0104] In some embodiments, the slurry with the added enzymes is held at 25-65°C for at least 10 minutes to allow for enzymatic hydrolysis of the plant material. In some embodiments, the slurry is held 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 for enzymatic hydrolysis of the plant material. In some embodiments, the slurry is held for at least about 10, 30, or 60 minutes. In some embodiments, the slurry is held for about 60 minutes.

[0105] A raw starch degrading enzyme can directly degrade raw starch granules below the gelatinization temperature of starch. The gelatinization temperature of starch can range from 51°C to 78°C as the gelatinization initiation temperature can vary from about 51°C to 68°C. When oat flour is used, the raw starch degrading alpha-amylase can directly degrade raw starch when the gelatinization temperature is about 55°C to 62°C. In some methods of the invention, a raw starch degrading amylase and additional enzymes are provided to a slurry of plant material, and the enzyme treatment of the slurry may be performed at 50-55°C, 55-62°C, 58-62°C, about 50°C, about 55°C, or about 60°C for at least 20, 30, 40, or 60 minutes.

[0106] In some embodiments, the slurry is held at different temperatures depending on the enzymes added to the slurry. In some embodiments, the enzymes treatment is carried out in two steps with different temperatures, referred to hereinafter as a two-step process. In the context of the present invention, a two-step process comprises holding the slurry at different temperatures for certain time periods, wherein the temperature in a given step is set to match the enzyme(s) added to the slurry according to optimal temperature for enzymatic activity. For example, the slurry may, in one step, be heated to a temperature in the range of 70-90°C, one or more first enzyme(s) may be added and the slurry kept at this temperature range for a time period, such as, e.g., for 30 minutes. The slurry may then be cooled to a temperature in the range of 20-60°C, such as, e.g., 50-60°C, followed by the addition of one or more second enzyme(s) different from the first enzyme(s), and the slurry be kept at the temperature range for, e.g., 30 minutes. Alternatively, in the context of the present invention, a process referred to hereinafter as a one-step process may be carried out, comprising the steps of providing a slurry held a temperature in the range of 5-20°C, adding to the slurry an enzyme having activity in the range of 5-65°C, heating the slurry to a temperature in the range of 65-90°C, adding a further enzyme having enzymatic activity in the temperature range 65-90°C, wherein the entire process lasts 90-180 minutes.

[0107] The process used, including temperature ranges, pH and the length of enzymatic treatment, will vary depending on the plant material and the enzymes added to the slurry. The skilled person will know how to determine the best process parameters based, e.g., on the plant material and enzymes used. The process used will also depend on the desired characteristics of the resulting plantbased food ingredient, which is heavily dependent upon the consumer’s preferences and expectations. For example, for the production of an oat-based food ingredient, the viscosity is an important property of the product. An oat-based dairy alternative beverage typically has a viscosity similar to that of low-fat or skim milk. The viscosity is determined in part by the beta-glucan content, where a higher beta-glucan content increases viscosity. If the enzyme treatment results in a product with very low viscosity, it will have a watery mouthfeel, which is undesirable. However, if the viscosity is too high, the product can have a sandy mouthfeel. Additionally, a plantbased food ingredient which has very high viscosity may be difficult to process in industrial manufacturing.

[0108] The resulting plant-based food ingredient also needs to have a composition of protein, starch, and fiber which are desirable. For the production of an improved oat beverage, it is desirable to raise the protein content or the fiber content compared to the current standard. Increasing protein and / or fiber content in an oat-based food ingredient, which may also be referred to as the “oat base”, can produce an oat-based beverage with an improved nutritional profile. Fiber content may be increased by increasing the amount of beta-glucan in the oat base. The methods of the invention provide for enzymes, including an alpha-amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase, which may be used to treat a slurry of oat material to produce an oat base with an increase in beta-glucan and / or an increase in protein compared to an oat base produced by a method that does not include the recited enzymes.

[0109] After treatment with the enzymes, the enzymes may be inactivated. The enzymes may be inactivated at any step after hydrolysis. In some embodiments, the enzymes are inactivated before or after the hydrolyzed plant material has been separated into solid and liquid streams. In other embodiments, the enzymes are inactivated after additional food ingredients have been added to the harvested liquid stream.

[0110] In some embodiments, the enzymes are inactivated by a heat treatment. In some embodiments, the heat treatment is 85-95°C for 5-30 minutes. In further embodiments, the heat treatment is 85-95°C for 10 minutes. In some embodiments, the heat treatment is 95°C for 5,

[0111] 10, 15, 20, 25, or 30 minutes.

[0112] In some embodiments, the enzymes are inactivated by an Ultra High Temperature (UHT) treatment. The UHT treatment may be direct or indirect. In some embodiments, the UHT treatment is 135-154°C for 1-10 seconds. in further embodiments, the UHT treatment is 140-150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds, In further embodiments, the UHT treatment is 140-145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is 143°C for 4,

[0113] 5, 6, 7, or 8 seconds.

[0114] After enzyme inactivation, the hydrolyzed plant material may be cooled. The hydrolyzed plant material may be separated into a solid and a liquid stream, for example by centrifugation. Centrifugation may occur in a decanter centrifuge. Following centrifugation, the liquid stream may be harvested or collected and used as a food ingredient for dairy alternative foods. The liquid stream may still comprise some solid matter, also referred to as “dry matter”. In some embodiments, the liquid stream comprises 1-80% solids. In further embodiments, the liquid stream comprises 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 comprises 10-15% solids. In some embodiments, the liquid stream comprises 2-10%, 3-8%, 4-7%, or 5-6% dry matter. In some embodiments, the liquid stream comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% dry matter.

[0115] In some embodiments, the liquid stream is further processed to remove water, also referred to as concentrated. Concentration increases the relative amount of solids in the concentrated liquid stream. Concentration may occur by evaporation of the water in the liquid stream. In some embodiments, the concentrated liquid stream comprises 10-100% solids, or dry matter. In further embodiments, the concentrated liquid stream comprises 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80%-90%, or 90-100% solids. In some embodiments, water removal will increase the viscosity of the dairy alternative food product.

[0116] In some embodiments, the liquid stream is used directly as a plant-based food ingredient. Additional food ingredients may be added to the liquid stream to produce a dairy alternative food product. The liquid stream may be homogenized before or after the addition of food ingredients.

[0117] 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 packed. The final product may be sold as a plant-based dairy alternative beverage, such as an oat beverage.

[0118] Alternatively, a liquid stream may be further processed into other dairy alternative food products, such as a fermented plant-based product, for example oat-based yogurt, or a plantbased ice cream, or it may be used as an ingredient in a dairy alternative food product.

[0119] In some embodiments, the final 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 oatbased ice cream.

[0120] In some embodiments, the methods of the invention produce a plant-based food ingredient which has a higher soluble carbohydrate content, protein content, and / or fiber content compared to a similar method which does not comprise providing an alpha amylase, a xylanase, and optionally at least one additional enzyme. In some embodiments, the plant-based food ingredient comprises 5-8%, 7-10%, 9-12%, 10-13%, or 12-15% carbohydrates or total starch. In some embodiments, the plant-based food ingredient comprises 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 ingredient comprises 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 grams of beta-glucan / L, about 1-3, about 2-4, about 3-5, about 4-6, about 5-7, about 6-8, or about 7-8 g beta-glucan / L. 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 grams beta-glucan / L. In some embodiments, the plant-based food ingredient is an oat-based food ingredient which comprises about 5-15% carbohydrates, about 0.2-3% protein, and / or about 0-8 grams beta-glu- can / L. In some embodiments, the oat-based food ingredient comprises about 9-13% carbohydrates, about 0.2-1.5% protein, and / or about 0.5-3 grams beta-glucan / L. Carbohydrates, protein, and beta-glucan are all measured using methods known in the art, for example the methods described in the Examples. In some embodiments, the plant-based food ingredient is an oatbased food ingredient, such as oat base.

[0121] In some embodiments, the methods of the invention produce a liquid plant-based food ingredient and / or plant-based dairy alternative food product which provides 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 needed to achieve the same viscosity, protein content, and / or fiber content, a lower amount of starting plant material is needed to produce a plant-based food ingredient with viscosity, protein content, and / or fiber content and texture similar to what is known in the art. In other words, methods of the invention effectively produce more plant-based food ingredient or plant-based dairy product per unit of starting plant material compared to methods generally known in the art. This greater efficiency in utilization of raw materials results in a higher yield and a lower cost of production.

[0122] In further embodiments, the methods of the invention produce a liquid oat-based food ingredient or oat-based dairy alternative food product comprising 3-5%, 4-6%, 5-7%, or 8-10% dry matter which is comparable in texture in terms of viscosity and has equal or greater amounts of protein and / or fiber compared to a liquid oat-based food ingredient or oat-based dairy alternative food product comprising 10-15% or 10-12% dry matter using methods known in the art which do not comprise providing an alpha amylase, a xylanase, optionally a protein deamidase and optionally a beta-glucanase. Methods of the invention increase raw oat material utilization and therefore lower production costs of oat-based food ingredients and also oat-based dairy alternative food products compared to methods known in the art which do not comprise providing an alpha amylase, a xylanase, optionally a protein deamidase and optionally a beta-glucanase.

[0123] The invention is further defined by the following numbered embodiments: PREFERRED EMBODIMENTS

[0124] 1 . A method for obtaining a non-alcoholic plant-based food ingredient, comprising: a) obtaining a slurry of plant material in water; b) providing an alpha amylase, a xylanase, and optionally at least one additional enzyme; and c) treating said slurry with said enzymes at 25-65°C for at least 10 minutes to produce a hydrolyzed plant material, wherein the hydrolyzed plant material is a non-alcoholic plant-based food ingredient.

[0125] 2. The method of embodiment 1 , further comprising: d) separating the plant-based food ingredient into solid and liquid streams; e) harvesting the liquid stream as a liquid plant-based food ingredient; and f) optionally inactivating the enzymes before or after steps (d) or (e).

[0126] 3. The method of any of the preceding embodiments, wherein the plant material is derived from tubers, nuts, roots, stems, legumes, fruits, seeds, or whole grains.

[0127] 4. The method of any one of the preceding embodiments, wherein the plant material is derived from corn, rice, barley, wheat, quinoa, oat, rye, flax, hemp, buckwheat, milo, millet, sago, cassava, tapioca, potatoes, sweet potatoes, peas, beans, fava bean, lentil, soy, cashew, macadamia, almond, coconut, banana, jack fruit, and / or bread fruit.

[0128] 5. The method of any one of the preceding embodiments, wherein the plant material is a cereal flour or de-hulled grains, including corn flour, rice flour, barley flour, buckwheat flour, wheat flour, millet flour, quinoa flour, oat flour, rye flour, or a mixture thereof.

[0129] 6. The method of any one of the preceding embodiments, wherein the plant material is oat flour, oat flakes, oat bran, groats, or any combination thereof.

[0130] 7. A method for obtaining a non-alcoholic oat-based food ingredient, comprising: a) obtaining a slurry of an oat material in water; b) providing an amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase; and c) treating said slurry with said enzymes at 25-65°C for at least 10 minutes to produce a hydrolyzed oat material, wherein the hydrolyzed oat material is an oat-based food ingredient.

[0131] 8. The method of embodiment 7, further comprising d) separating the oat-based food ingredient into solid and liquid streams; e) harvesting the liquid stream as a liquid oat-based food ingredient; and f) optionally inactivating the enzymes before or after steps (d) or (e).

[0132] 9. The method of embodiment 7 or 8, wherein the slurry of step (a) comprises a ratio of oat material to water of 1 :3 to 1 :8 (w / w), or 1 :4 to 1 :6 (w / w).

[0133] 10. The method of embodiments 7-9, further comprising concentrating the oat-based food ingredient to reduce water content.

[0134] 11. The method of embodiments 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% oat dry matter.

[0135] 12. The method of embodiments 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% oat dry matter.

[0136] 13. The method of any of embodiments 7-12, where the oat-based food ingredient is processed to produce a dairy alternative food product, such as an oat-based beverage, oat-based ice cream, oat-based creamer, oat-based yogurt, or oat-based cheese.

[0137] 14. The method of any one of the preceding embodiments, wherein the xylanase is an endo- 1 ,4-xylanase.

[0138] 15. The method of any one of the preceding embodiments, wherein the xylanase is a GH10, GH30, or GH5 xylanase.

[0139] 16. The method of any one of the preceding embodiments, wherein the xylanase is a GH10 or GH5-21 xylanase.

[0140] 17. The method of any one of the preceding embodiments, wherein the xylanase comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11. 18. The method of any one of the preceding embodiments, wherein the xylanase comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 1.

[0141] 19. The method of any one of the preceding embodiments, wherein the amylase of step (b) is a raw starch degrading amylase.

[0142] 20. The method of any one of the preceding embodiments, wherein the amylase of step (b) comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 5.

[0143] 21. The method of embodiments 19 or 20, wherein the treatment of step (c) is at a temperature of 45-50°C, 50-55°C, 55-60°C, 58-62°C, or 60-65°C for about 30, about 60, or about 90 minutes.

[0144] 22. The method of embodiments 19-21 , wherein the treatment of step (c) is at a temperature of 60°C for about 60 minutes.

[0145] 23. The method of any one of the preceding embodiments, further providing at least one addition enzyme in step (b), wherein the additional enzyme is an amylase, glucoamylase, maltogenic amylase, beta-amylase, isoamylase, alpha-amylase, cyclodextrin glucanotransferase, endopeptidase, protein deamidase, protease, hemicellulase, cellulase, pectolytic enzyme, glucosidase, glu- canase, xylanase, arabinofuranosidase, pullulanase, lipase, or any combination thereof.

[0146] 24. The method of any one of the preceding embodiments, wherein the additional enzyme is a protein deamidase.

[0147] 25. The method of any one of the preceding embodiments, wherein the additional enzyme is a protein deamidase derived from a Chryseobacterium spp.

[0148] 26. The method of any one of the preceding embodiments, wherein the additional enzyme is a protein deamidase which comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0149] 27. The method of any one of the preceding embodiments, wherein the additional enzyme is a protein deamidase which comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 6.

[0150] 28. The method of any one of the preceding embodiments, wherein the additional enzyme is a beta-glucanase.

[0151] 28. The method of any one of the preceding embodiments, wherein the additional enzyme is a beta-glucanase which comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 12.

[0152] 29. The method of any one of the preceding embodiments, wherein the non-alcoholic plantbased food ingredient is combined with additional food ingredients to produce a dairy alternative food product.

[0153] 30. The method of embodiment 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.

[0154] 31. The dairy alternative food product of embodiment 29 or 30.

[0155] 32. A non-alcoholic oat-based food ingredient produced by the method of any of embodiments 7-28.

[0156] 33. The oat-based food ingredient of embodiment 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 beta- glucan / L. 34. The oat-based food ingredient of embodiment 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 grams beta-glu- can / L.

[0157] 35. The oat-based food ingredient of embodiment 32-34, wherein the total starch, protein, and / or beta-glucan content is higher relative to dry matter compared to an oat-based food ingredient produced by a method which did not comprise an alpha amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase.

[0158] 36. An improved method of producing a plant-based dairy alternative food product, comprising: a) obtaining a non-alcoholic plant-based food ingredient produced by the method of any of the preceding claims, wherein the method comprises providing an alpha amylase, a xylanase, and optionally at least one additional enzyme to a slurry of plant material in water, wherein the plant-based food ingredient comprises more beta-glucan, more protein, and / or higher viscosity compared to a plant-based food ingredient with the same amount of dry matter not produced by a method which did not comprise providing an alpha amylase, a xylanase, and optionally at least one additional enzyme to a slurry of plant material in water; b) diluting the plant-based food ingredient of step a) so that the amount of beta-glucan, protein, and / or viscosity is comparable to that of a plant-based food ingredient prepared by a method which did not comprise providing an alpha amylase, a xylanase, and optionally at least one additional enzyme to a slurry of plant material in water, or reducing the amount of the plantbased food ingredient of step a) added to produce the plant-based dairy alternative food product, so that the amount of beta-glucan, protein, and / or viscosity used is comparable to that of a plantbased food ingredient prepared by a method which did not comprise providing an alpha amylase, a xylanase, and optionally at least one additional enzyme to a slurry of plant material in water; and c) adding additional ingredients to produce a plant-based dairy alternative food product.

[0159] 37. An improved method of producing an oat-based dairy alternative food product, comprising: a) obtaining a oat-based food ingredient produced by the method of any of the preceding claims, wherein the oat-based food ingredient comprises more beta-glucan, more protein, and / or higher viscosity compared to an oat-based food ingredient with the same amount of dry matter produced by a method which did not comprise an alpha amylase, a xylanase, optionally a beta- glucanase, and optionally a protein deamidase; b) diluting the oat-based food ingredient of step a) so that the amount of beta-glucan, protein, and / or viscosity is comparable to that of an oat-based food ingredient prepared by a method which did not comprise an alpha amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase, or reducing the amount of the oat-based food ingredient of step a) used, so that the amount of beta-glucan, protein, and / or viscosity added is comparable to that of a oat-based food ingredient prepared by a method which did not comprise an alpha amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase; and c) adding additional ingredients to produce an oat-based dairy alternative food product.

[0160] 38. Use of an alpha-amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase in the production of a non-alcoholic plant-based food ingredient to improve extraction of beta-glucan and / or protein from plant material.

[0161] 39. Use of an alpha-amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase in the production of a non-alcoholic oat-based food ingredient to improve extraction of beta-glucan and / or protein from oat material.

[0162] The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention as well as combinations of one or more of the embodiments.

[0163] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0164] EXAMPLES

[0165] Equipment:

[0166] Mashing device (Lochner Labor und Technik, LB12, Germany)

[0167] Centrifuge (Multifuge 3 S-R, Heraeus, Thermo Scientific, Finland)

[0168] Thermomix (TM6-1 , Vorwerk Elektrowerke GmbH & Co KG; Germany)

[0169] LECO FP628 Elemental Analysis by combustion for protein determination, LECO Corporation; USA

[0170] Gallery Plus Analyzer (Brewmaster, Thermo Scientific, Finland)

[0171] HamiltonStar ViPr (Hamilton Company, USA)

[0172] Spectrophotometer (Shimadzu UV-1700, Suzhou Instruments Manufacturing Co LtD, China) Materials

[0173] Oat flour (Havnemollerne, Denmark)

[0174] Enzyme Preparation 1 (“Prep 1”): comprises alpha-amylase (SEQ ID NO: 4; 480 KNU-B / g) and beta-glucanase side activity (712 FBG / g)

[0175] Alpha-amylase (SEQ ID NO: 4; 480 KNU-B / g,), Novozymes A / S

[0176] Protein deamidase (SEQ ID NO: 6; 350 IPA(U) / g

[0177] Enzyme Preparation 2 (“Prep 2”): comprises xylanase (SEQ ID NO: 1 ; 1500 FXU-S / g) and beta- glucanase side activity (1150 FBG / g)

[0178] GH10 Xylanase (SEQ ID NO: 1)

[0179] GH5_21 Xylanase (SEQ ID NO: 2)

[0180] GH30 Xylanase (SEQ ID NO: 3)

[0181] Sunflower oil (Ollineo, Germany)

[0182] Assays for determining enzymatic activity: fi-glucanase activity (FBG): One fungal beta glucanase unit (FBG) is the amount of enzyme, which, according to the standard conditions outlined below, releases reducible oligosaccharides or reduces carbohydrate with a reduction capacity equivalent to 1 mol glucose per minute. Fungal beta glucanase reacts with beta-glucan to glucose or reducing carbohydrate that is determined as reducing sugar according to the Somogyi Nelson method. The standard reaction conditions were: 0.5% barley beta-glucan substrate, 30°C, pH 5.0, and reaction time of 30 min.

[0183] Xylanase (FXU(S)): The xylanolytic activity can be expressed in FXU(S)-units, determined at pH 6.0 with remazol-xylan (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka) as substrate. A xylanase sample is incubated with the remazol-xylan substrate. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant (as determined spectrophotometrically at 585 nm) is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions. Standard reaction conditions are a 0.45% w / v substrate concentration and an enzyme concentration of 0.04 - 0.14 FXU(S) / ml at 50.0°C, pH 6.0, with a 30 minute reaction time. Xylanase activity in FXU(S) is measured relative to a Novozymes FXU(S) enzyme standard (obtainable from Novozymes), comprising the monocomponent xylanase preparation Shearzyme from Aspergillus aculeatus.

[0184] Protein deamidase activity (IPA(U)): Protein deamidase activity was measured using an assay consisting of two separate de-coupled parts: (1) an enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and (2) a non-enzymatic detection step, wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm. The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (given in Indophenol Assay Unit: IPA(U)). The activity may be determined relative to a standard of declared strength.

[0185] Quantification of content:

[0186] Quantification of protein’. The amount of nitrogen in the flour, base, pellet or drink was determined by a combustion method on a Leco FP-528. The amount of protein was calculated as 6.25 times the amount of nitrogen.

[0187] Quantification of beta-glucan: The amount of beta-glucan in oat drink or base was quantified according to the Application Note 64538 (Thermo Scientific) and measured on Gallery Plus Analyzer Brewmaster.

[0188] Quantification of total starch: The amount of total starch in the flour, base, pellet or drink was analyzed using the Total Starch Assay Kit (AA / AMG) (Megazyme) and the AACC Method 76- 13.01. The absorbance was measured using a Shimadzu UV-1700 spectrophotometer.

[0189] Quantification of dry matter. Dry matter of flour, base, pellet or drink were determined by drying the samples at 105°C for 20 hours.

[0190] Viscosity: The viscosity of the samples was measured using the HamiltonStar ViPr. A high negative pressure correlates to a high viscosity. Methods for measuring viscosity are similar to those in WO 2011 / 107472, herein incorporated by reference in its entirety.

[0191] Calculations:

[0192] Base yield (%) = ((g base) / (g flour+g water+enzyme solution))*100

[0193] Total starch yield (%) = ((%total starch in base / 100)*(g base)) / (g flour*(%total starch in flour / 100))*100

[0194] Dry matter yield (%) = ((%dry matter in base / 100)*(g base)) / (g flour*(%dry matter in flour / 100))*100

[0195] Protein yield (%) = ((%protein in base / 100)*(g base)) / (g flour*(%protein in flour / 100))*100

[0196] Production yield (L drink / kg oat flour) = (100 / oat load)*(%Base yield / 100)*(%Dry matter of base) / (Desired dry matter)

[0197] Oat load = amount of oat flour / total amount*100

[0198] 1 : Production of an oat-based beverage The following table provides the enzymes and the dosage used for each numbered sample. Percentages are based on the oat flour weight.

[0199] Table 1 : Experimental design

[0200] Enzyme treatment

[0201] 170 g deionized water was weighed directly into a mash bath beaker. Enzymes were added according to the experimental design and 30 g of oat flour was added to each beaker while stirring (150 rpm). Starting temperature was 22°C and the suspension was heated to 65°C at a heating rate of about 2°C / min. When the target temperature of 65° was reached, 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 heating rate of about 1.5°C / min. and held at 90°C for 10 minutes. Without cooling, the hot suspension was separated into a liquid base material and a solid pellet fraction using centrifugation at 3x1200 g (centrifugation was switched off as soon as 1200 g was reached). The liquid oat base was then weighed and placed in an ice bath.

[0202] The samples were equilibrated to room temperature and dry matter, viscosity, protein, total starch, and beta-glucan content of the base material was measured for each sample. The samples were then diluted with deionized water to prepare two different formulations which contained 5% and 10% oat dry matter, respectively. In addition, sunflower oil and sodium chloride were added to a final concentration of 1% and 0.08%, respectively. After addition of formulation ingredients the samples were homogenized using Thermomix TM-6 by increasing to max speed (setting of 10) over 10 seconds and holding this mixing speed for 60 seconds.

[0203] Results

[0204] The oat flour used in the described enzyme treatments contained 9.06% protein (on flour basis), 89.3% dry matter, and 72.5% total starch. As described, following enzymatic incubation the samples were separated via centrifugation into a liquid “base” material and a solid pellet fraction. The oat base was then analysed for viscosity and protein content, total starch content, and beta-glucan content, and dry matter was quantified. The results are provided in the table below.

[0205] Table 2: Oat base content and viscosity

[0206] Yields were calculated from the above measurements of the oat base and are provided in the table below.

[0207] Table 3: Yield in oat base

[0208] Sample 1, which contained an amylase and beta-glucanase side activity, had in the highest starch yield and the lowest protein content. No beta-glucan could be detected in the base. Samples 2-5 did not contain the amylase with beta-glucanase side activity. Total starch yield in these four combinations were lower than Sample 1, and all four samples contained beta-glucan ranging from 1.8-1.9 g / L. Samples 2-5 also had increased viscosity compared to Sample 1. The addition of a protein deamidase in samples 3 and 5 showed a very high increase in protein yield. Unexpectedly, for samples 4 and 5, which each contained an enzyme preparation with high xylanase activity and also some small amount of beta-glucanase side activity, viscosity was lower while beta-glucan levels were almost unaltered.

[0209] Oat-based beverages typically contain 8-12% oat dry matter, so formulations comprising 5% and 10% oat dry matter were evaluated for this example. The oat base from each of the samples described above were formulated to 5% and 10% oat dry matter, each to a final volume of 100 ml with the addition of deionized water. Amounts of oat base for each formulation are provided below.

[0210] Table 4: Formulation with 5% and 10% oat dry matter

[0211] The amount of oat drink (L) which can be produced from 1 kg of oat flour were calculated based on the 15% oat load used in this experiment. This is the production yield and is shown for each formulation in the table below. Table 5: Production yield

[0212] The final formulations were analysed for viscosity, dry matter, and protein and total starch content. Beta-glucan content was calculated based on the amount of base added and the content analysed in the base. Results are shown in the table below. Table 6: Content and viscosity of final formulations

[0213] Dry matter from the 5% and 10% oat formulations was measured, and it was confirmed that the formulated samples successfully reached the expected levels based on dry matter calculated from the oat base of each sample. The amylase applied in sample 1 was selected as being widely used in oat drink processing. As stated above, industry standard for oat drink products contains 8-12% oat and therefore comparing 10% oat with 5% oat was chosen. An oat drink formulation which comprises a lower oat content may be beneficial both in terms of higher production yield and lower level of carbohydrates, however taste and texture need to be essentially the same or better for consumer acceptance. The examples here show that certain enzymatic treatments that increase the level of protein and / or fiber in an oat base can also increase the viscosity and improve the nutritional value of the base, thereby enabling the preparation of an oat beverage comprising a lower oat content, such as 5% oat dry matter, which retains the desired viscosity to provide a proper texture and mouthfeel.

[0214] Sample 1 in a 10% oat drink formulation delivered good viscosity while in a 5% oat drink formulation it was thin and watery. The nutritional profile of sample 1 was higher in starch, lower in protein and contained no beta-glucan.

[0215] Sample 2 exhibited higher viscosity in both 5% and 10% oat dry matter formulations compared to sample 1 , however, the difference in viscosity between sample 1 and 2 was higher in the base and seemed to be changed by the formulation process. The viscosity of sample 2 in the 5% oat dry matter formulation was somewhat higher than sample 1 in the 10% oat dry matter formulation. Sample 2 had a high beta-glucan level compared to Sample 1 .

[0216] Sample 3 contained the protein deamidase of SEQ ID NO: 6 in combination with the amylase of SEQ ID NO: 4. The viscosity level was slightly below sample 2 both at 5% and 10% oat dry matter. Protein levels are almost doubled compared to sample 1 and 2, with the protein level at 5% higher than the protein level of samples 1 and 2 at 10%. Overall, sample 3 at 5% had high viscosity, which is undesirable, although beta-glucan and protein levels for oat drink formulation were within acceptable ranges.

[0217] Sample 4 was treated with the amylase of SEQ ID NO: 4 and also with Enzyme Prep 2, which comprises xylanase and beta-glucanase activities. Surprisingly, the viscosity was very good at 5% oat dry matter, and the level of beta-glucan was the highest of all samples, which is desirable for increased nutrition in an oat drink. The protein content was low, however the oat drink at 5% oat dry matter was nutritionally improved and had a desirable level of viscosity.

[0218] Sample 5 contained the protein deamidase of SEQ ID NO: 6, the amylase of SEQ ID NO: 4, and Enzyme Prep 2, which comprises xylanase and beta-glucanase activities. The viscosity of sample 5 at 5% oat dry matter level was very similar to that of Sample 4, however the protein level is higher compared to Sample 4. Beta-glucan levels are similar to that of Sample 3, despite the presence of the beta-glucanase in Enzyme Prep 2.

[0219] In summary, a method of producing oat base comprising treating the oat material with a combination of an alpha-amylase, such as the alpha-amylase of SEQ ID NO: 4, and an enzyme preparation comprising a GH10 xylanase and a beta glucanase, such as Enzyme Prep 2, produces an oat base which can be formulated to make an oat-based beverage comprising lower oat dry matter while retaining good texture in terms of viscosity, and also with better nutrition in terms of improved beta-glucan content. In methods where the oat material is also treated with a protein deamidase, the protein content of the resultant oat base may be more than doubled.

[0220] Example 2: Production of an oat-based food ingredient using a xylanase

[0221] This example describes the production of an oat “base”, which is the main ingredient in the production of an oat-based beverage. The following table provides the enzymes and the dosage used for each numbered sample. The “Amylase” is the alpha-amylase of SEQ ID NO: 4. Percentages of amylase dosage are based on the oat flour weight. Xylanases were expressed heterologously and harvested. The GH10 xylanase comprises the amino acid se- guence of SEQ ID NO: 1. The GH5_21 xylanase comprises the amino acid seguence of SEQ ID NO: 2. The GH30 xylanase comprises the amino acid seguence of SEQ ID NO: 3. Xylanase amounts are provided as mg extracted protein (EP) per kg oat flour.

[0222] Table 7: Experimental design

[0223] Enzyme treatment and Results

[0224] The oat flour used in the following enzyme treatments contained 8.9% protein (on flour basis) and 90.5% dry matter. 170 g deionized water was weighed directly into a mash bath beaker. Enzymes were added according to the experimental design and 30 g of oat flour was added to each beaker while stirring (150 rpm). Starting temperature was 22°C and the suspension was heated to 65°C at a heating rate of about 2°C / min. When the target temperature of 65° was reached then stirring of the reaction mixture was reduced to 100 rpm and continued stirred for 60 min. To terminate the enzyme treatment, the suspension was heated to 90°C at a heating rate of about 1.5°C / min. and held at 90°C for 10 min. Without cooling, the hot suspension then was separated into a liguid “base” material and a solid pellet fraction using centrifugation at 3x1200 g (centrifugation was switch off as soon as 1200 g was reached). The liguid oat base was then weighed and placed in an ice bath. The samples were equilibrated to room temperature and dry matter, viscosity, protein content, and beta-glucan content of the oat base were measured for each sample. The results are provided in the table below: Table 8: Oat base content and viscosity

[0225] Yields were calculated from the above measurements of the oat base and are provided in the table below:

[0226] Table 9: Yield in oat base

[0227] Samples 2-4, treated with purified GH10 xylanase (SEQ ID NO: 1), showed increased beta-glucan levels at increasing enzyme dose, while a reduction in viscosity was observed. Samples 5 and 6, treated with purified GH5_21 xylanase (SEQ ID NO: 2), had increased viscosity at increasing dose but did not greatly impact beta-glucan levels. Samples 7 and 8, treated with purified GH30 xylanase (SEQ ID NO: 3), showed moderately decreased viscosity at increasing dose levels, while protein or beta-glucan levels were not greatly impacted.

[0228] In summary, it was possible to alter the beta-glucan levels and the viscosity, and thereby the texture, of an oat base with the addition of a xylanase to the enzymatic treatment of oat material, compared to a similarly prepared sample which did not comprise a xylanase. An increase in beta-glucan levels in the oat base improves the nutritional profile of the oat base. A reduction in viscosity may beneficial to improve processing efficiency.

[0229] Example 3: Production of an oat-based food ingredient using a xylanase and a raw starch degrading alpha amylase

[0230] Materials

[0231] Oat flour (Lantmannen batch 1000483760, 13% protein (LEGO))

[0232] Amylase 5 (raw starch degrading amylase; SEQ ID NO: 5; 296 FAU(N) / g product)

[0233] GH5-21 xylanase (SEQ ID NO: 2; 363 FXU-TB / g)

[0234] Xylanase 11 (GH 10 xylanase; SEQ ID NO: 11 ; 546 FXU-S / g)

[0235] Table 10: Experimental design

[0236] Enzyme treatment

[0237] 75 g of oat flour was weighed out and 425 g deionized water was added to the Thermomix TM6-1 and mixed until the slurry was homogenous, then 40 g of the slurry was aliquoted into 50m L Falcon tubes. Enzymes were added according to the experimental design of Table 10. The oat slurry was heated to 55°C at a heating rate of about 1.5°C / min in a FINEPCR Rotisserie incubator (Weber Scientific, Hamilton, NJ). When the target temperature of 55° was reached the stirring of the reaction mixture was reduced and stirring continued for 60 minutes. To terminate the enzyme treatment, the suspension was heated to 90°C and held at 95°C for 20 min. Without cooling, the hot suspension was then separated into a liquid base material and a solid pellet fraction using centrifugation at 3x1200 g (centrifugation was switch off as soon as 1200 g was reached). The liquid oat base was then placed in an ice bath. The samples were equilibrated to room temperature and viscosity and beta-glucan of the oat base material was measured in duplicates on each sample using methods described above.

[0238] Table 11 : Viscosity of oat base samples

[0239] Table 12: Beta-glucan content of oat base samples

[0240] All samples containing a xylanase had higher beta-glucan content compared to sample 1 , which only had Amylase 5 and no added xylanases. Xylanase 11 (a GH10 xylanase, samples 2-4) showed increased beta-glucan levels at all amounts tested compared to sample 1 , while viscosity decreased with increasing xylanase amounts. GH5-21 xylanase (samples 5-7) also showed increased beta-glucan levels at all amounts tested compared to sample 1 , and additionally the viscosity of samples 5-7 were the highest of all samples, including sample 1 (no xylanase). These results are similar to those in Example 2, although in this example a different GH10 xylanase is used and the enzymes are at higher concentrations.

[0241] In summary, the addition of a xylanase to the oat material results in an oat base with an increase in beta-glucan, which is a nutritional improvement compared to an oat base produced without xylanase added. Further, it was possible to impact viscosity and texture of the base using xylanases.

Claims

CLAIMS1 . A method for obtaining a non-alcoholic oat-based food ingredient, comprising: a) obtaining a slurry of an oat material in water; b) providing an amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase; and c) treating said slurry with said enzyme composition at 25-65°C for at least 10 minutes to produce a hydrolyzed plant material, wherein the hydrolyzed plant material is an oat-based food ingredient.

2. The method of claim 1 , further comprising d) separating the oat-based food ingredient into solid and liquid streams; e) harvesting the liquid stream as a liquid oat-based food ingredient; and f) optionally inactivating the enzymes before or after steps (d) or (e).

3. The method of any one of the preceding claims, wherein the oat material is oat flour, oat flakes, oat bran, groats, or any combination thereof.

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

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

6. The method of any one of the preceding claims, wherein the xylanase comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11.

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

8. The method of any one of the preceding claims, wherein the amylase of step (b) comprises an amino acid sequence which 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 least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or is 100% identical to SEQ ID NO: 5.

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

10. The method of any one of the preceding claims, wherein a protein deamidase is provided in step (b), wherein the protein deamidase comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

11. The method of any one of the preceding claims, wherein a beta-glucanase is provided in step (b).

12. The method of any one of the preceding claims, wherein a beta-glucanase is provided in step (b), wherein the beta-glucanase comprises an amino acid sequence which 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, or is 100% identical to SEQ ID NO: 12.

13. The method of any one of the preceding claims, where the oat-based food ingredient is processed to produce a dairy alternative food product, such as an oat-based beverage, oat-based ice cream, oat-based creamer, oat-based yogurt, or oat-based cheese.

14. The oat-based food ingredient of any one of the preceding claims, wherein the total starch, protein, and / or beta-glucan content is higher relative to dry matter compared to an oat-based food ingredient produced by a method which did not comprise an alpha amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase.

15. Use of an alpha-amylase, a xylanase, optionally a beta-glucanase, and optionally a protein deamidase in the production of a non-alcoholic oat-based food ingredient to improve extraction of beta-glucan and / or protein from oat material.

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