Method for obtaining plant-based food ingredients

Abstract

The present invention relates to a process for obtaining a plant-based food ingredient for a dairy alternative food product, in which a plant material is hydrolyzed at a temperature between 25 DEG C and 60 DEG C in the presence of a raw starch degrading alpha-amylase and optionally a further enzyme to obtain a hydrolyzed plant material, use as a plant-based food ingredient for dairy alternative food products.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a Sequence Listing in computer-readable form. The computer-readable form is hereby incorporated by reference into this application. Technical Field

[0003] The present invention relates to the use of an enzyme having α-amylase activity for obtaining hydrolyzed plant material. Background Art

[0004] In recent years, the number of people pursuing a vegan, vegetarian or non-dairy diet for health reasons has increased. Additionally, food products made from the milk of animals (especially cows) are increasingly being looked at due to their high environmental cost. These factors are driving an increasing demand for dairy alternative food products (including milk, creamers, cheese, yogurt and ice cream) for many foods that are traditionally derived from milk.

[0005] Dairy alternative food products typically originate from high-starch plant materials such as cereal grains, peas or potatoes. Generally, in order to convert high-starch plant materials into dairy alternative food products or food ingredients included in dairy alternative food products, the starch must be hydrolyzed. The conversion of starch typically includes a gelatinization step, in which the starch granules are dissolved to form a viscous suspension; a liquefaction step, in which the starch is partially hydrolyzed with a concomitant loss of viscosity; and optionally a subsequent saccharification step, which involves the production of glucose and maltose by further hydrolysis.

[0006] Gelatinization is generally achieved by heating, while liquefaction and possible saccharification often involve the use of enzymes. Typically, since gelatinization preferably uses high temperatures, liquefaction is also carried out at high temperatures. In this case, gelatinization and liquefaction are carried out at high temperatures for an extended period of time, and then the plant material is rapidly cooled, and then saccharification is carried out as a second step at a lower temperature.

[0007] Maintaining the starch at high temperature for an extended period of time and then rapidly cooling the mixture is an energy-intensive process. There is a need to reduce energy consumption, both because of the rising global energy costs and because both consumers and producers recognize the environmental benefits of increased energy efficiency.

[0008] The object of the present invention is to determine an improved energy-saving process for the production of hydrolyzed plant-based food ingredients (for the production of dairy alternative food products). Temperature

[0009] Temperature Summary of the Invention

[0010] The present invention relates to a method for obtaining a food ingredient for use in producing a dairy alternative food product, wherein the food ingredient is obtained from a slurry of plant material in water, and a raw starch-degrading α-amylase and optionally additional enzymes are added to the slurry, and the slurry is maintained at a temperature between 25°C and 60°C to allow hydrolysis of the plant material. The hydrolyzed plant material is a food ingredient for dairy alternative foods. Since the hydrolysis occurs at a temperature between 25°C and 60°C, this method is an improved and more energy-efficient process compared to known methods in the art that use higher temperatures for enzymatic hydrolysis.

[0011] In some methods of the present invention, the hydrolyzed plant material is then separated into a solid stream and a liquid stream, and the liquid stream is harvested as a plant-based food ingredient for a dairy alternative food product. The enzymes can then be inactivated, for example, by heat treatment or UHT treatment of the liquid stream or the further processed liquid stream.

[0012] The enzymes added to the slurry include a raw starch-degrading α-amylase. The raw starch-degrading α-amylase can be an amylase of the GH13 family and can also have a carbohydrate-binding module (CBM) that preferentially binds to starch. This CBM can be CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82, or 83.

[0013] Additional enzymes can also be added to the slurry. These additional enzymes can also be raw starch-degrading enzymes. These additional enzymes can include glucoamylase, maltogenic amylase, β-amylase, protease, hemicellulase, cellulase, pectinolytic enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof.

[0014] The method of the present invention includes a method for obtaining a plant-based food ingredient for a dairy alternative food product, the method including obtaining a slurry of heat-treated oat material and water, maintaining the slurry at a temperature between 25°C and 60°C in the presence of a raw starch-degrading α-amylase and a β-glucanase, allowing the enzymes to hydrolyze the oat material for a certain period of time, separating the hydrolyzed oat material into a solid stream and a liquid stream, and finally harvesting the liquid stream as a plant-based food ingredient for a dairy alternative food product. The enzymes can be inactivated before or after the hydrolyzed oat material is separated into a solid stream and a liquid stream. In some embodiments, the enzymes are inactivated in the harvested liquid stream. The dairy alternative food product can be a beverage, yogurt, cheese, creamer, ice cream, or any other dairy alternative food product known in the art. Detailed Description

[0015] For the purposes of this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural referents unless the context clearly dictates otherwise.

[0016] As used herein, the terms “drink” and “beverage” are used interchangeably and have the same meaning.

[0017] Unless otherwise defined or clearly indicated by the context, all percentages are weight percentages (percent w / w or “%(w / w)”).

[0018] The term “plant-based food ingredient” refers to a plant-based composition that can be combined with additional food ingredients to produce a food product. Plant-based food ingredients and food products containing plant-based food ingredients are edible by humans or animals (including domestic animals such as companion animals). In some embodiments, the plant-based food ingredient can be combined with additional food ingredients to produce a dairy alternative food product. The additional food ingredients can be any food ingredients that are considered useful by those skilled in the art. The additional food ingredients can be solid or liquid. The additional food ingredients can be or can not be plant-based. In some embodiments, the additional food ingredient is water.

[0019] The term “dairy alternative food product” refers to a food product that can be used as an alternative to a dairy food product. Dairy alternative food products are plant-based and do not contain dairy-derived food ingredients. Dairy alternative food products include plant-based beverages, creamers, cheeses, ice creams, yogurts, and any other dairy alternative food products known in the art.

[0020] In some embodiments, the plant-based food ingredient can be combined with additional food ingredients to produce a ready-to-drink dairy alternative beverage. Examples of dairy alternative beverages include oat beverages, rice beverages, barley beverages, potato beverages, pea beverages, sesame beverages, almond beverages, tiger nut beverages, or beverages comprising any combination thereof.

[0021] In some embodiments, the plant-based food ingredient can be used as a substrate for fermentation to produce a dairy alternative beverage (such as buttermilk) or to produce a dairy alternative yogurt.

[0022] In some embodiments, the plant-based food ingredient can be further processed. The further processing can include removing water. In some embodiments, removing water will concentrate the hydrolysis products, i.e., the released sugars. In some embodiments, removing water will increase the viscosity of the plant-based food ingredient.

[0023] In some embodiments, the plant-based food ingredient may optionally be further processed and combined with additional food ingredients to produce dairy alternative ice cream. In some embodiments, the plant-based food ingredient may optionally be further processed and combined with additional food ingredients to produce dairy alternative cheese.

[0024] The plant-based food ingredient of the present invention is derived from plant material that is or is derived from an edible part of a plant. In some embodiments, the plant material is derived from an edible part of a plant that also has a high starch content. In some embodiments, the edible part of the plant can be a tuber, root, stem, spike, legume, fruit, nut, or seed. In some embodiments, the plant is a cereal and the plant material is or is derived from a cereal grain (also known as a whole grain). In further embodiments, the cereal grain can be from corn, rice, barley, wheat, buckwheat, millet, sorghum, quinoa, oats, or rye. In some embodiments, the plant material is or is derived from a tuber or root (including a rhizome), such as a potato, sweet potato, cassava, tiger nut, canna, or yuca. In some embodiments, the plant material is or is derived from a fruit, such as a banana, jackfruit, or breadfruit. In some embodiments, the plant material is or is derived from a nut, such as an almond, macadamia nut, or cashew nut. In some embodiments, the plant material is or is derived from sago, peas, or legumes.

[0025] In some embodiments, the plant material is heat-treated. In some embodiments, the plant material is dehydrated. In some embodiments, the plant material is dehulled, ground, wet-milled, and / or dry-milled. In some embodiments, the plant material is corn flour, rice flour, barley flour, wheat flour, buckwheat flour, millet flour, quinoa flour, oat flour, rye flour, potato flour, sweet potato flour, cassava flour, tiger nut flour, yuca flour, sesame flour, nut flour (such as cashew, macadamia, or almond flour), pea flour, legume flour, dehulled oats, dehulled barley, dehulled wheat, dehulled peas, dehulled legumes, or any combination thereof. In some embodiments, the plant material is crushed or ground to produce a paste.

[0026] In some embodiments, the plant material is oat material. In further embodiments, the oat material is oat flour, oatmeal, oat bran, dehulled oats, or a combination thereof. In still further embodiments, the oat material can be oat flour (such as heat-treated oat flour), or the oat material can be milled oat groats (such as dehulled and heat-treated oat groats that have been wet-milled), or the oat material can be any other oat material known in the art. In some embodiments, the oat material is heat-treated oat flour, oatmeal, oat bran, or any combination thereof.

[0027] In the method of the present invention, plant material is suspended in water to produce a slurry, wherein the ratio of plant material to water is from 1:3 to 1:8 (w / w). In some embodiments, the ratio of plant material to water is from 1:4 to 1:16. In some embodiments, the ratio of plant material to water is from 1:1 to 1:4.

[0028] In the method of the present invention, raw starch-degrading α-amylase and optionally additional enzymes are added to the slurry, and the mixture is maintained at a temperature between 25°C and 60°C such that the plant material is hydrolyzed by these enzymes to produce hydrolyzed plant material. In some embodiments, the slurry is maintained at a temperature below the gelatinization temperature of the starch in the slurry.

[0029] As used herein, a "raw starch-degrading enzyme" (also referred to as a raw starch-hydrolyzing enzyme) refers to an enzyme that can directly degrade raw starch granules at a temperature below the gelatinization temperature of the starch. The gelatinization temperature range of starch can be from 51°C to 78°C, as the onset temperature of gelatinization can vary between about 51°C and 68°C. When using barley flour, the raw starch-degrading α-amylase can directly degrade raw starch at a gelatinization temperature of about 53°C to 63°C. When using oat flour, the raw starch-degrading α-amylase can directly degrade raw starch at a gelatinization temperature of about 55°C to 62°C. The raw starch-degrading α-amylase is a raw starch-degrading enzyme.

[0030] In one embodiment, a raw starch-degrading enzyme is defined as an enzyme having a raw starch-degrading index of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, wherein the raw starch-degrading index is the ratio (Ra / Ga) of the activity of degrading raw starch to the activity of degrading gelatinized starch. Preferably, the raw starch-degrading enzyme is defined as an enzyme having a raw starch-degrading index higher than 1. The activity towards gelatinized starch is measured by measuring the glucose release produced by the enzyme for a 2% gelatinized (e.g., corn) starch reaction mixture. The activity is measured by the release of reducing sugar produced by pure active enzyme at 4 mol / hour / mg. Then, the same assay can be used to measure the activity of the enzyme towards raw starch, but with 2% raw (e.g., corn) starch replacing 2% gelatinized (e.g., corn) starch. In both assays, the temperature is 40°C, and the same pH and buffer solution are used, and the incubation time is 6 hours.

[0031] Raw starch degrading enzymes are widely present and are produced by plants, animals, and microorganisms such as fungi, bacteria, and yeast raw starch degrading enzymes. In some embodiments, the raw starch degrading enzyme is glucoamylase. In other embodiments, the raw starch degrading enzyme is α-amylase, also known as raw starch degrading α-amylase. In some embodiments, the raw starch degrading enzyme refers to α-amylase, glucoamylase, or a combination of one or more α-amylases and one or more glucoamylases. Sources of raw starch degrading enzymes include enzymes obtained from the genus Aspergillus, such as Aspergillus oryzae, Aspergillus niger, and Aspergillus kawachii α-amylase. Examples of such raw starch degrading enzymes include those described in WO 2005 / 003311, WO 2006 / 0692, WO 2006 / 060289, and WO 2004 / 080923.

[0032] In some embodiments, the raw starch degrading α-amylase is an acidic α-amylase. An "acidic α-amylase" is an α-amylase (4-α-D-glucan glucanohydrolase, E.C. 3.2.1.1) that is active in the pH range of 3.0 to 7.0, preferably 3.5 to 6.0, or more preferably 4.0 - 5.0 when added in an effective amount. The source of the raw starch degrading acidic α-amylase is the α-amylase from Aspergillus niger that is disclosed as "AMYA_ASPNG" in the Swiss-prot / TeEMBL database under accession number P56271 and is described in more detail in WO1989 / 01969 (Example 3). The Aspergillus niger acidic α-amylase is also shown as SEQ ID NO:1 in WO2004 / 080923 (Novozymes A / S), which is hereby incorporated by reference. A suitable commercially available acidic fungal α-amylase derived from Aspergillus niger is product SP288 (SEQ ID NO:1 of U.S. Patent No. 7,244,597, available from Novozymes). Other sources of acidic α-amylase include those derived from strains of Rhizomucor and Meripilus, such as Rhizomucor pusillus (WO2004 / 055178) or strains of Meripilus giganteus. In yet another embodiment, the acidic α-amylase is derived from Aspergillus kawachii and is disclosed by Kaneko et al. J. Ferment. Bioeng. [Journal of Fermentation and Bioengineering] 81:292 - 298 (1996) "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii [Molecular cloning and determination of the nucleotide sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii]"; and is further disclosed as EMBL:#AB008370.

[0033] In some embodiments, the raw starch degrading α-amylase has a carbohydrate-binding module (CBM) that binds to starch. In some embodiments, the CBM preferentially binds to starch, especially to unheat-treated granular starch. Such a CBM can also be referred to as a starch-binding domain (SBD). The SBD is known to be in 15 CBM families, namely CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82, and 83.

[0034] In some embodiments, the raw starch-degrading α-amylase can be a hybrid α-amylase comprising a starch-binding domain (SBD) and an α-amylase catalytic domain (CD). The hybrid α-amylase can also comprise an α-amylase catalytic domain (CD), a starch-binding domain (SBD), and a linker connecting the CD and the SBD as known in the art. In an embodiment, the catalytic domain is derived from a strain of Aspergillus kawachii. Examples of the hybrid α-amylase include those disclosed in WO 2005 / 003311, US Patent Publication No. 2005 / 0054071 (Novozymes A / S), and US Patent No. 7,326,548 (Novozymes A / S), which are hereby incorporated by reference. Examples also include those enzymes disclosed in Tables 1 to 5 of the examples in US Patent No. 7,326,548 and those disclosed in US Patent Publication No. 2005 / 0054071 (Table 3 on page 15), such as the Aspergillus niger α-amylase catalytic domain (CD) together with the Aspergillus kawachii linker and starch-binding domain (SBD).

[0035] Other acidic α-amylases include the enzymes disclosed in WO 2004 / 020499 and WO 2006 / 069290 and the enzymes disclosed in WO2006 / 066579 as SEQ ID NO:2 (hybrid Aspergillus niger α-amylase + CBM), SEQ ID NO:3, or SEQ ID NO:4 (JA129). A hybrid α-amylase consisting of a Rhizopus microsporus α-amylase with an Aspergillus niger glucoamylase linker and SBD is disclosed as V039 in Table 5 of WO 2006 / 069290.

[0036] SEQ ID NOs: 1-4 are GH13 family raw starch-degrading α-amylases. In some embodiments, the raw starch-degrading α-amylase of the present invention is a GH13 family amylase. In some embodiments, the raw starch-degrading α-amylase has an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:1, 2, 3, or 4. In some embodiments, the raw starch-degrading α-amylase of the present invention has the amino acid sequence of SEQ ID NO:1, 2, 3, or 4.

[0037] The term "identity" is the relatedness between two amino acid sequences or two nucleotide sequences. For the purposes of the present invention, 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 in Genetics 16:276-277) (preferably version 3.0.0 or later), is used to determine the degree of identity between two amino acid sequences. The optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -non - simplified option) is used as the percent identity and is calculated as follows:

[0038] (Identical residues x 100) / (Alignment length - total number of gaps in the alignment)

[0039] Amino acid changes can have minor properties, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino - terminal or carboxyl - terminal extensions, such as a methionine residue at the amino - terminus; small linker peptides of up to 20 - 25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a poly - histidine segment, an epitope, or a binding domain).

[0040] Examples of conservative substitutions are within the following groups: 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 generally do not 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 / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0041] In some embodiments, the raw starch-degrading α-amylase has at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or even 100% sequence identity to SEQ ID NO:1.

[0042] In another embodiment, the raw starch-degrading α-amylase has at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or even 100% sequence identity to SEQ ID NO:2.

[0043] In another embodiment, the raw starch-degrading α-amylase has at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or even 100% sequence identity to SEQ ID NO:3.

[0044] In another embodiment, the raw starch-degrading α-amylase has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or even 100% sequence identity to SEQ ID NO:4.

[0045] Based on the amount of plant material added, the raw starch-degrading α-amylase can be added in the range of 500 - 25000 parts per million (ppm). In some embodiments, 500 - 20000, 750 - 20000, 750 - 18000, 750 - 17000, 750 - 15000, 750 - 1000, 750 - 7500, or 1000 - 5000 ppm of the raw starch-degrading α-amylase can be added to the slurry. In some embodiments, 750 - 2500 or 1000 - 1500 ppm of the raw starch-degrading α-amylase can be added to the slurry.

[0046] The products hydrolyzed by the raw starch-degrading α-amylase contain maltooligosaccharides (MOS). Maltooligosaccharides contain glucose molecules and have one or more branched linkages (α-1,4), with a typical degree of polymerization (DP) of 2 - 9. MOS can be indigestible oligosaccharides with short chain lengths (2 - 10), which can have a prebiotic effect of enhancing the growth of beneficial bacteria in the human intestine (see, for example, Jang et al., 2020, Molecules 25:5201, doi:10.3390 / molecules25215201). In some embodiments, the dairy alternative food product containing the plant-derived food ingredient of the present invention can have a prebiotic effect when consumed.

[0047] In some embodiments of the present invention, one or more additional enzymes are added to the slurry to allow hydrolysis of the plant material. These additional enzymes can be glucoamylase, maltogenic amylase, β-amylase, protease, hemicellulase, cellulase, pectinase, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof. One or more additional enzymes can be from any source, including mammalian, plant, and microbial (bacterial, yeast, or fungal) sources.

[0048] In some embodiments, the additional enzyme is glucoamylase (also known as amyloglucosidase). One glucoamylase unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under the following standard conditions: 37 °C, pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes. In some embodiments, glucoamylase can be added at a concentration of 50 - 1000 AGU / kg of plant material.

[0049] In some embodiments, the additional enzyme is maltogenic amylase. Maltogenic α - amylase (EC 3.2.1.133) can be from the genus Bacillus. Maltogenic α - amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes under the trade name and can also be a variant of maltogenic α - amylase from Bacillus stearothermophilus, as disclosed in, for example, WO 1999 / 043794, WO 2006 / 032281, or WO 2008 / 148845, such as 3D.

[0050] In some embodiments, the additional enzyme is xylanase. Xylanase can be of microbial origin, for example, from strains of bacteria or fungi such as Aspergillus (especially Aspergillus aculeatus, Aspergillus niger, Aspergillus awamori, or Aspergillus tubigensis), Trichoderma (e.g., Trichoderma reesei), or Humicola (e.g., Humicola insolens). In some embodiments, the xylanase is from a strain of Trichoderma reesei. Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG, and PENTOPAN 500BG (available from Novozymes), GRINDAMYLPOWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP5001 (available from DSM).

[0051] In some embodiments, the additional enzyme is protease. Protease can be from the genus Bacillus, such as Bacillus amyloliquefaciens. Suitable proteases can be

[0052] In some embodiments, the additional enzyme is a β-glucanase. The β-glucanase can have only β-glucanase activity or can also have other enzyme activities. In some embodiments, the β-glucanase has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or even 100% sequence identity with SEQ ID NO:5.

[0053] In some embodiments, the enzyme having β-glucanase activity can be a preparation of an endo-α-amylase having β-glucanase side activity obtained from the genus Bacillus (e.g., obtained from Bacillus amyloliquefaciens). In some embodiments, the enzyme having β-glucanase activity can be in a cellulase preparation. In additional embodiments, the cellulase preparation can be obtained from Trichoderma reesei. In other embodiments, the β-glucanase is obtained from Aspergillus niger. Examples of enzyme preparations having β-glucanase activity include or Prime, each of which is available from Novozymes. These enzyme preparations are believed to contain β-glucanase. Prime contains β-glucanase and xylanase.

[0054] In some embodiments, the β-glucanase can be added at parts per million (ppm) based on the amount of plant material used. In some embodiments, 1 - 2500 ppm of β-glucanase can be added to the slurry. In some embodiments, 5 - 2500, 20 - 2500, 50 - 2500, 50 - 2000, 50 - 1800, 50 - 1500, 50 - 1000, 50 - 750, 75 - 500, or 75 - 300 ppm of β-glucanase can be added to the slurry. In some embodiments, 1 - 200, 1 - 150, or 1 - 100 ppm of β-glucanase can be added to the slurry. In some embodiments, 1 - 200, 1 - 100, 1 - 50, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ppm of β-glucanase can be added to the slurry.

[0055] In some embodiments, raw starch degrading α - amylase and β - glucanase are added to the slurry. In additional embodiments, the slurry comprises oat material and water. In still further embodiments, the slurry comprises oat flour and water. In still further embodiments, the slurry comprises heat - treated oat flour and water. In some embodiments, the raw starch degrading α - amylase is added at 500 - 25000 ppm, and the β - glucanase is added at 1 - 2500 ppm. In some embodiments, the raw starch degrading α - amylase is added at 750 - 15000 ppm, and the β - glucanase is added at 1 - 500 ppm. In some embodiments, the raw starch degrading α - amylase is added at 1000 - 7000 ppm, and the β - glucanase is added at 1 - 300 ppm. In some embodiments, the raw starch degrading α - amylase is added at 1000 - 5000 ppm, and the β - glucanase is added at 1 - 150 ppm. In some embodiments, the raw starch degrading α - amylase is added at 1000 - 1500 ppm, and the β - glucanase is added at 1 - 100 ppm.

[0056] The raw starch degrading α - amylase and optionally one or more additional enzymes can be provided in any suitable form, such as in liquid (especially stabilized liquid) form, or the one or more enzymes can be added as a substantially dry powder or granule. For example, the granules can be produced as disclosed in U.S. Patent No. 4,106,991 and U.S. Patent No. 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well - known in the art.

[0057] The enzyme combination can be added to the slurry containing plant material in any suitable manner, such as adding as separate components (adding the enzymes separately or sequentially), or adding the enzymes together in one step or one composition, or any combination thereof.

[0058] The slurry is maintained at a temperature between 25°C and 60°C to allow hydrolysis of the plant material. In some embodiments, the slurry is maintained at a temperature between 25°C - 55°C, 30°C - 55°C, 35°C - 55°C, 40°C - 60°C, 30°C - 50°C, 40°C - 55°C, 45°C - 55°C or 50°C - 55°C. In additional embodiments, the slurry is maintained 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 or about 60°C.

[0059] In some embodiments, the slurry with added enzyme is maintained at a temperature between 25°C and 60°C for at least 10 minutes to allow enzymatic hydrolysis of the plant material. In some embodiments, the slurry is maintained for about 10, about 15, about 20, about 25, about 30, about 60, about 120, about 180, about 240, or at least about 240 minutes to allow enzymatic hydrolysis of the plant material. In some embodiments, the slurry is maintained for at least about 10, 30, 60, or 90 minutes. In some embodiments, the slurry is maintained for 30 minutes. In some embodiments, the slurry is maintained for 60 minutes. In some embodiments, the slurry is maintained for 90 minutes. Those skilled in the art will recognize that there is a relationship between the enzyme dosage, incubation temperature, and the amount of time allowed for enzymatic hydrolysis such that a higher dosage of enzyme will allow a shorter incubation time, a lower dosage of enzyme can achieve the same level of hydrolysis over a longer incubation time, and a higher incubation temperature can allow a lower dosage of enzyme and / or a shorter incubation time.

[0060] After treatment with raw starch-degrading α-amylase and optionally additional enzymes, one or more enzymes can be inactivated. The enzymes can 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 a solid stream and a liquid stream. In other embodiments, the enzymes are inactivated after additional food ingredients have been added to the harvested liquid stream.

[0061] In some embodiments, the enzymes are inactivated by heat treatment. In some embodiments, the heat treatment is at a temperature between 85°C and 95°C for 5 - 30 minutes. In additional embodiments, the heat treatment is at a temperature between 85°C and 95°C for 10 - 15 minutes. In some embodiments, the heat treatment is at 95°C for 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the heat treatment is at a temperature between 85°C and 95°C for one minute or less. In some embodiments, the heat treatment is at a temperature between 85°C and 95°C for 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds.

[0062] In some embodiments, the enzymes are inactivated by ultra-high temperature (UHT) treatment. The UHT treatment can be direct or indirect. In some embodiments, the UHT treatment is at a temperature between 135°C and 154°C for 1 - 10 seconds. In additional embodiments, the UHT treatment is at a temperature between 140°C and 150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In additional embodiments, the UHT treatment is at a temperature between 140°C and 145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is at 143°C for 4, 5, 6, 7, or 8 seconds.

[0063] After enzymatic hydrolysis, hydrolyzed plant material is produced. This hydrolyzed plant material can also be referred to as a plant hydrolysate, such as an oat hydrolysate. After enzyme inactivation, the hydrolyzed plant material can be cooled. The hydrolyzed plant material is separated into a solid stream and a liquid stream, for example by centrifugation. The centrifugation can occur in a decanter centrifuge. After centrifugation, the liquid stream can be harvested or collected and used as a food ingredient for dairy alternative foods. The liquid stream can still contain some solid matter. In some embodiments, the liquid stream contains 1% - 80% solids. In additional embodiments, the liquid stream contains 1% - 10%, 5% - 20%, 10% - 25%, 20% - 35%, 25% - 40%, 30% - 45%, 35% - 50%, 40% - 55%, 45% - 60%, 50% - 65%, 55% - 70%, 60% - 75% or 65% - 80% solids. In some embodiments, the liquid stream contains 10% - 15% solids. The solids in the liquid stream (also referred to as "total solids") can be measured using methods well known in the art. For example, a sample of the liquid stream can be dried (typically by heating), and then the remaining solids can be weighed.

[0064] The liquid stream (which can still be referred to as a plant hydrolysate) contains solids and sugars that can be used to produce dairy alternative food products. The viscosity of the liquid stream also affects its usefulness for producing dairy alternative food products. This viscosity depends in part on the β-glucan content, where a higher β-glucan content increases the viscosity. A plant hydrolysate with a very high viscosity may be difficult to process in industrial manufacturing. Additionally, a plant hydrolysate with a very high viscosity may be difficult to use as a food ingredient for producing dairy alternative food products (especially for producing dairy alternative beverages). The viscosity of the plant hydrolysate also plays a role in its usefulness as a food ingredient in dairy alternative food products. Certain food products have a preference for certain viscosity levels. For example, dairy alternative oat beverages typically have a viscosity similar to that of low-fat or skim milk.

[0065] In some embodiments, the liquid stream is further processed to remove water or to be concentrated. The concentration increases the relative amount of solids in the concentrated liquid stream. The concentration can be achieved by evaporating the water in the liquid stream. In some embodiments, the concentrated liquid stream contains 10% - 95% solids. In additional embodiments, the concentrated liquid stream contains 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90% or 90% - 95% solids. In some embodiments, removing water will increase the viscosity of the dairy alternative food product.

[0066] In some embodiments, the liquid stream is directly used as a plant-based food ingredient. This liquid stream may be referred to as the "base material". Additional food ingredients can be added to the liquid stream to produce dairy alternative food products. In some embodiments, the liquid stream is derived from an oat hydrolyzate and may be referred to as an "oat base material". In additional embodiments, the oat base material can be formulated with, for example, sodium chloride (NaCl), oil, and optionally flavorings. Such formulations can be considered dairy alternative food products. The oat base material can be homogenized before or after adding the food ingredients.

[0067] The dairy alternative food products can be UHT or ESL treated and aseptically packaged. The final product can be sold as a plant-based beverage, which is a dairy alternative food product.

[0068] Alternatively, the liquid stream (in some embodiments, the oat base material) can be further processed into other dairy alternative food products (such as fermented plant-based products or plant-based ice cream), or the liquid stream can be used as an ingredient in dairy alternative food products.

[0069] In some embodiments, the liquid stream is an oat base material that is processed into an oat-based dairy alternative food product. In some embodiments, the food product is an oat-based beverage, oat-based creamer, oat-based yogurt, oat-based cheese, or oat-based ice cream.

[0070] The present invention is further defined by the following numbered embodiments:

[0071] 1. A method for obtaining a plant-based food ingredient for a dairy alternative food product, the method comprising:

[0072] (a) obtaining a slurry of plant material in water; and

[0073] (b) providing a raw starch-degrading α-amylase and optionally one or more additional enzymes to the slurry of step (a) and maintaining at a temperature between 25°C and 60°C to obtain a hydrolyzed plant material;

[0074] wherein the hydrolyzed plant material is a plant-based food ingredient for a dairy alternative food product.

[0075] 2. The method according to embodiment 1, the method further comprising

[0076] (c) separating the hydrolyzed plant material into a solid stream and a liquid stream;

[0077] (d) harvesting the liquid stream as a plant-based food ingredient for a dairy alternative food; and

[0078] (e) Optionally inactivate the enzyme before or after step (c) or (d).

[0079] 3. The method according to any one of Examples 1 or 2, wherein the raw starch-degrading α-amylase is an amylase of the GH13 family.

[0080] 4. The method according to Example 3, wherein the GH13 family amylase comprises a carbohydrate-binding module (CBM) that preferentially binds to starch.

[0081] 5. The method according to Example 4, wherein the CBM is CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82 or 83.

[0082] 6. The method according to any one of the foregoing examples, wherein the raw starch-degrading α-amylase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3 or 4.

[0083] 7. The method according to any one of the foregoing examples, wherein the additional enzyme is glucoamylase, maltogenic amylase, β-amylase, protease, hemicellulase, cellulase, pectinolytic enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase and / or lipase, or any combination thereof.

[0084] 8. The method according to any one of the foregoing examples, wherein the additional enzyme is a second raw starch-degrading enzyme.

[0085] 9. The method according to any one of the foregoing examples, wherein the additional enzyme is β-glucanase.

[0086] 10. The method according to any one of the foregoing examples, wherein the additional enzyme is xylanase.

[0087] 11. The method according to any one of the foregoing examples, wherein the slurry of step (b) comprises β-glucanase and xylanase.

[0088] 12. The method according to any one of the foregoing embodiments, wherein the raw starch-degrading α-amylase is added to the slurry at a dose between 500 - 20000, 750 - 20000, 750 - 18000, 750 - 17000, 750 - 15000, 750 - 2500, 750 - 1000, 750 - 7500, 1000 - 5000 or 1000 - 1500 ppm.

[0089] 13. The method according to any one of embodiments 9, 11 or 12, wherein the β-glucosidase is added to the slurry at a dose between 1 - 200, 1 - 150 or 1 - 100 ppm.

[0090] 14. The method according to any one of the foregoing embodiments, wherein the temperature of step (b) is a temperature between 25°C - 55°C, 30°C - 55°C, 35°C - 55°C, 40°C - 60°C, 30°C - 50°C or 40°C - 55°C.

[0091] 15. The method according to any one of the foregoing embodiments, wherein the enzyme is inactivated by heat treatment or ultra-high temperature (UHT) treatment.

[0092] 16. The method according to embodiment 15, wherein the UHT treatment is carried out at a temperature between 140°C - 145°C for 2 - 8 seconds.

[0093] 17. The method according to any one of the foregoing embodiments, wherein the plant material is derived from tubers, roots, stems, legume pods, fruits, nuts, seeds or whole grains.

[0094] 18. The method according to any one of the foregoing embodiments, wherein the plant material is derived from corn, rice, barley, wheat, quinoa, oats, rye, buckwheat, sorghum, millet, sago, cassava, yuca, potato, sweet potato, peas, beans, almonds, cashews, macadamia nuts, bananas, jackfruit and / or breadfruit.

[0095] 19. The method according to any one of the foregoing embodiments, wherein the plant material is a cereal flour or dehulled grain, including corn flour, rice flour, barley flour, buckwheat flour, wheat flour, millet flour, quinoa flour, oat flour, rye flour, or a mixture thereof.

[0096] 20. The method according to any one of the foregoing embodiments, wherein the plant material is an oat material, such as oat flour, oatmeal, oat bran, dehulled oats, or any combination thereof.

[0097] 21. The method according to any one of the foregoing embodiments, 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.

[0098] 22. A plant-based food ingredient for a dairy alternative food product produced by a method as described in any of the foregoing embodiments.

[0099] 23. A method as described in any of the foregoing embodiments, wherein the plant-based food ingredient is combined with additional food ingredients to produce a dairy alternative food product.

[0100] 24. A method for obtaining an oat hydrolyzate food ingredient for a dairy alternative food product, the method comprising

[0101] (a) obtaining a slurry of oat material in water;

[0102] (b) providing raw starch-degrading α-amylase and β-glucanase to the slurry of step (a) and maintaining at a temperature between 25°C - 60°C, 25°C - 55°C, 45°C - 55°C or 50°C - 55°C to obtain an oat hydrolyzate; and

[0103] (c) optionally inactivating the enzymes;

[0104] wherein the oat hydrolyzate is a plant-based food ingredient for a dairy alternative food.

[0105] 25. The method according to embodiment 24, the method further comprising

[0106] (d) separating the oat hydrolyzate into a solid stream and a liquid stream; and

[0107] (e) harvesting the liquid stream as an oat-based food ingredient for a dairy alternative food product.

[0108] 26. A method for obtaining an oat hydrolyzate food ingredient for a dairy alternative food product, the method comprising

[0109] (a) obtaining a slurry of oat material in water;

[0110] (b) maintaining the slurry of step (a) at a temperature between 25°C - 60°C and adding raw starch-degrading α-amylase and β-glucanase to obtain an oat hydrolyzate;

[0111] (c) separating the oat hydrolyzate into a solid stream and a liquid stream;

[0112] (d) harvesting the liquid stream; and

[0113] (e) optionally inactivating the enzymes before or after step (c) or (d);

[0114] wherein the harvested liquid stream is an oat hydrolyzate food ingredient for a dairy alternative food product.

[0115] 27. The method according to any one of embodiments 24 - 26, wherein the raw starch-degrading α-amylase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, or 4.

[0116] 28. The method according to any one of embodiments 24 - 27, wherein the temperature of step (b) is between 25°C - 55°C, 30°C - 55°C, 35°C - 55°C, 40°C - 60°C, 30°C - 50°C, 40°C - 55°C, 45°C - 55°C, or 50°C - 55°C.

[0117] 29. The method according to any one of embodiments 24 - 28, wherein the slurry of step (b) further comprises xylanase.

[0118] 30. The method according to any one of embodiments 24 - 29, wherein the raw starch-degrading α-amylase is added to the slurry at a dosage between 500 - 20000, 750 - 20000, 750 - 18000, 750 - 17000, 750 - 15000, 750 - 2500, 750 - 1000, 750 - 7500, 1000 - 5000, or 1000 - 1500 ppm.

[0119] 31. The method according to any one of embodiments 24 - 30, wherein the β-glucosidase is added to the slurry at a dosage between 1 - 200, 1 - 150, or 1 - 100 ppm.

[0120] 32. A plant-based food ingredient produced by the method according to any one of the foregoing embodiments, wherein the plant-based food ingredient has a prebiotic effect when consumed.

[0121] 33. A dairy alternative food product comprising the plant-based food ingredient according to embodiment 32, wherein the dairy alternative food product has a prebiotic effect when consumed.

[0122] 34. An oat hydrolyzate food ingredient produced by the method according to any one of embodiments 24 - 31, wherein the oat hydrolyzate food ingredient has a prebiotic effect when consumed.

[0123] 35. A dairy alternative food product comprising an oat hydrolyzate food ingredient as described in Example 34, wherein the dairy alternative food product has a prebiotic effect when consumed.

[0124] 36. Use of a raw starch-degrading α-amylase in the hydrolysis of plant-based materials for the production of plant-based food ingredients for dairy alternative food products.

[0125] 37. Use as described in Example 36, wherein the plant-based material is an oat material such as oat flour, oatmeal, oat bran, dehulled oats, or any combination thereof.

[0126] 38. Use of a raw starch-degrading α-amylase and a β-glucanase in the hydrolysis of oat materials for the production of plant-based food ingredients for dairy alternative food products.

[0127] The inventions described and claimed herein are not limited to the scope of the specific embodiments disclosed herein, as these embodiments are intended to be illustrative of several aspects of the invention. Any equivalent embodiments, together with combinations of one or more of these embodiments, are intended to be included within the scope of the invention.

[0128] Multiple references are cited herein, the disclosures of which are incorporated by reference in their entireties. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0129] Examples

[0130] Example 1: Preparation of a plant-based food ingredient using the raw starch-degrading α-amylase of SEQ ID NO:1

[0131] 1000 ppm (based on oat flour) of the amylase encoded by SEQ ID NO:1 was added to 650 L of water at 63 °C together with 50 ppm of β-glucanase ( Prime, Novozymes A / S, Denmark) and mixed with 200 Kg of oat flour. An additional 150 L of water was added and the final temperature reached 62 °C. After 10 minutes, an additional 50 ppm of β-glucanase ( Prime, Novozymes A / S, Denmark) was added and the slurry was held at 62 °C for 60 minutes to allow hydrolysis.

[0132] After hydrolysis, the hydrolyzed slurry was processed in a decanter at 60 °C and the separated liquid stream was cooled to 10 °C. The liquid stream is a plant-based food ingredient, now also referred to as an oat concentrate base.

[0133] Dilute the oat concentrate base with water (44% w / w), then add salt (0.08% w / w) and rapeseed oil (0.8% w / w). The resulting oat beverage is subjected to UHT treatment and aseptic packaging using methods known in the art. The viscosity of the oat beverage is analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 conical cone measurement system. The released sugars are measured using high performance anion exchange chromatography - pulsed amperometric detection (HPAE-PAD). The results are shown in Tables 1 and 2 below. Table 1 provides the viscosity, total solids, and sugar content of the oat beverage. Table 2 shows the amounts of certain malt oligosaccharides (MOS) present in the oat beverage. Example 2: Preparation of a plant-based food ingredient using a raw starch-degrading α-amylase of SEQ ID NO:2

[0134] 1500 ppm (based on oat flour) of the amylase encoded by SEQ ID NO:2 is added to 650 L of water at 63 °C together with 100 ppm β-glucanase ( Prime, Novozymes A / S, Denmark) and mixed with 200 Kg of oat flour. An additional 150 L of water is added and the final temperature reaches 60 °C. The slurry is held at 60 °C for 60 minutes to allow hydrolysis.

[0135] After hydrolysis, the hydrolyzed slurry is processed in a decanter at 60 °C and the separated liquid stream is cooled to 10 °C. The liquid stream is a plant-based food ingredient, now also known as oat concentrate base.

[0136] Dilute the oat concentrate base with water (47.16% w / w), then add salt (0.08% w / w) and rapeseed oil (0.8% w / w). The resulting oat beverage is subjected to UHT treatment and aseptic packaging using methods known in the art. The viscosity of the oat beverage is analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 conical cone measurement system. The released sugars are measured using high performance anion exchange chromatography - pulsed amperometric detection (HPAE-PAD). The results are shown in Tables 1 and 2 below. Table 1 provides the viscosity, total solids, and sugar content of the oat beverage. Table 2 shows the amounts of certain malt oligosaccharides (MOS) present in the oat beverage.

[0137] Table 1: Viscosity and released sugars in oat beverages

[0138]

[0139] Table 2: MOS in oat beverages

[0140]

[0141] Tables 1 and 2 show that an oat beverage having good viscosity and containing a suitable amount of total solids, total sugars, and MOS was obtained. These results show that hydrolyzed oat material and oat concentrate base can be sources of plant-based food ingredients for dairy alternative food products. The oat concentrate base produced herein (wherein the oats have been hydrolyzed in one step at about 60 °C) has a viscosity similar to that of an oat concentrate base (wherein the oats have been hydrolyzed using a conventional method), as well as amounts of solids, sugars, and MOS, the conventional method including gelatinization and liquefaction at high temperature, followed by rapid cooling and saccharification.

[0142] Example 3: Preparation of Oat Hydrolysates Using Raw Starch-Degrading α-Amylase of SEQ ID NO:1

[0143] The heat-treated oat flour was mixed with water containing the enzyme at a ratio of 100 g of oat flour to 600 g of water. The amylase and β-glucanase encoded by SEQ ID NO:1 ( Prime, Novozymes A / S, Denmark), or a standard method was used for determination, the standard method including two different amylases, namely 480L (Novozymes A / S, Denmark), provided at the doses described in Tables 3 and 4, and also 800L (Novozymes A / S, Denmark), for all samples containing 480L was provided at 1500 ppm. Then the mixture of water, enzyme, and oat flour was heated to 25 °C, 40 °C, or 60 °C for 30 minutes to allow liquefaction / hydrolysis, and then the enzyme was inactivated by raising the temperature to 95 °C for 15 minutes. After inactivation, the hydrolysate was cooled to 60 °C for centrifugation, and a 3S-R (Kendro Heraeus, Hanau, Germany) was used to centrifuge at a relative centrifugal force of 1932 x g to separate the solid phase and the liquid phase. The viscosity of the liquid phase was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 cone calorimetry measurement system. The released sugars were measured using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD). The results are shown in Table 3. Table 3 provides the viscosity, total solids, and sugar content of the oat hydrolysates. The amounts of amylase and β-glucanase are provided in ppm based on the flour.

[0144] Table 3: Viscosity and Released Sugars in Oat Hydrolysates

[0145]

[0146]

[0147] Table 3 shows that, compared with samples containing 480L and 800L at temperatures between 25°C and 60°C, samples containing the raw starch amylase encoded by SEQ ID NO:1 produced oat hydrolysates with a higher total sugar content.

[0148] Example 4: Preparation of rice hydrolysates using the raw starch-degrading α-amylase of SEQ ID NO:1

[0149] Rice flour was mixed with water containing the enzyme at a ratio of 100 g of rice flour to 600 g of water. The amylase and β-glucanase ([[]] Prime, Novozymes A / S, Denmark) encoded by SEQ ID NO:1 were used, or measurements were carried out using a standard method that included two different amylases, namely 480L (Novozymes A / S, Denmark), provided at the dosages described in Tables 5 and 6, and also 800L (Novozymes A / S, Denmark), which was provided at 1500 ppm for all samples containing 480L. The mixture of water, enzyme, and rice flour was then heated to 25°C, 40°C, or 60°C for 30 minutes to allow liquefaction / hydrolysis, and then the enzyme was inactivated by raising the temperature to 95°C for 15 minutes. After inactivation, the hydrolysate was cooled to 60°C for centrifugation, and centrifuged at 3000 RPM for 10 minutes to separate the solid and liquid phases. The viscosity of the liquid phase was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 cone viscometry system. The released sugars were measured using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD). The results are shown in Table 4. Table 4 provides the viscosity, total solids, and sugar content of the rice hydrolysates. The amounts of amylase and β-glucanase are provided in ppm based on the flour.

[0150] Table 4: Viscosity and released sugars in rice hydrolysates

[0151]

[0152]

[0153] The data in Table 4 show that, compared with samples containing 480L and Compared with the sample of 800L, the sample containing the raw starch amylase encoded by SEQ ID NO:1 produced a rice hydrolysate with a higher total sugar content. Example 5: Preparation of pea hydrolysate using the raw starch-degrading α-amylase of SEQ ID NO:1

[0154] Mix pea flour with water containing the enzyme at a ratio of 100 g of flour to 600 g of water. Use the amylase and β-glucanase encoded by SEQ ID NO:1 ( Prime, Novozymes A / S, Denmark), or use a standard method for determination, which includes two different amylases, namely 480L (Novozymes A / S, Denmark), provided at the doses described in Tables 7 and 8, and also 800L (Novozymes A / S, Denmark), for all samples containing 480L were provided at 1500 ppm. Then, heat the mixture of water, enzyme, and oat flour to 25 °C, 40 °C, or 60 °C for 30 minutes to allow liquefaction / hydrolysis, and then inactivate the enzyme by raising the temperature to 95 °C for 15 minutes. After inactivation, cool the hydrolysate to 60 °C for centrifugation, and use 3S-R (Kendro Laboratory Products) to centrifuge at a relative centrifugal force of 1932 x g to separate the solid and liquid phases. Analyze the viscosity of the liquid phase using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 conical calorimetric measurement system. Measure the released sugars using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD). The results are shown in Table 5. Table 5 provides the viscosity, total solids, and sugar content of the rice hydrolysate. The amounts of amylase and β-glucanase are provided in ppm based on the flour.

[0155] Table 5: Viscosity and released sugars in pea hydrolysate

[0156]

[0157]

[0158] The data in Table 5 show that compared with the samples containing 480L and 800L at temperatures between 25 °C and 60 °C, the sample containing the raw starch amylase encoded by SEQ ID NO:1 produced a pea hydrolysate with a higher total sugar content. Example 6: Preparation of oat hydrolysate using the raw starch-degrading α-amylase of SEQ ID NO:3 or SEQ ID NO:4

[0159] For each determination, oat flour was mixed with water containing enzymes at a ratio of 100 g of flour to 600 g of water. An amylase encoded by SEQ ID NO:3 or SEQ ID NO:4 was used, as well as a β-glucanase ( Prime, Novozymes A / S, Denmark) for the determination. A standard determination was also carried out, which included two different amylases, namely 480L (Novozymes A / S, Denmark), provided at the doses described in the following table, as well as 800L (Novozymes A / S, Denmark), provided at 1500 ppm for all samples containing 480L. The mixture of water, enzyme, and oat flour was then heated to 25 °C, 40 °C, or 60 °C for 30 minutes to allow liquefaction / hydrolysis, followed by inactivating the enzyme by raising the temperature to 95 °C for 15 minutes. After inactivation, the hydrolysate was cooled to 60 °C for centrifugation, and a 3S-R (Kendro Laboratory Products) was used to centrifuge at a relative centrifugal force of 1932 x g to separate the solid and liquid phases. The viscosity of the liquid phase was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 cone rheometry system. The released sugars were measured using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD). The results are shown in Tables 6 and 7 below. Tables 6 and 7 provide the viscosity, total solids, and sugar content of the oat hydrolysates from the determinations carried out with SEQ ID NO:3 or SEQ ID NO:4. The amounts of amylase and β-glucanase are provided in ppm based on the flour.

[0160] Table 6: Viscosity and released sugars in oat hydrolysates from determinations carried out with SEQ ID NO:3

[0161]

[0162] Table 7: Viscosity and released sugars in oat hydrolysates from determinations carried out with SEQ ID NO:4

[0163]

[0164]

[0165] The results shown in Tables 6 and 7 indicate that when hydrolysis is carried out at temperatures between 25 °C and 60 °C, the raw starch-degrading amylase produces oat hydrolysates with the desired viscosity, total solids, and total sugars.

[0166] Example 7: Industrial-scale test using the amylase of SEQ ID NO:1

[0167] 5700 ppm (based on oat flour) of amylase SEQ ID NO:1 was added to 1600 L of water together with 100 ppm of β-glucanase ( Prime, Novozymes A / S, Denmark) and mixed with 400 Kg of oat flour. The slurry was heated to different temperatures and held for different minutes to allow hydrolysis as shown in the table below.

[0168] After hydrolysis, the hydrolyzed slurry underwent an inactivation step at 85 °C for 15 seconds and then was processed in a decanter. The separated liquid stream was then cooled to 10 °C. The liquid stream is a plant-based food ingredient and is now also referred to as an oat concentrate base.

[0169] The oat concentrate base was diluted with water and salt (0.08% w / w) and canola oil (0.8% w / w) were added. The resulting oat beverage was subjected to UHT treatment and aseptically packaged using methods known in the art. The viscosity of the oat beverage was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) with a CC27 cone calorimetry measurement system. The released sugars were measured using high performance anion exchange chromatography - pulsed amperometric detection (HPAE-PAD). The results are shown in Table 8. Table 8 provides the viscosity, total solids, and sugar content of the oat beverage.

[0170] Table 8: Viscosity and released sugars in the oat beverage

[0171]

[0172] These results indicate that when using raw starch amylase, an incubation temperature between 25 °C and 60 °C is sufficient to produce the desired oat beverage on an industrial scale.

[0173] Example 8: Preparation of Oat Hydrolysate Using Raw Starch-Degrading α-Amylase of SEQ ID NO:1 and 0 - 100 ppm of β-Glucanase

[0174] 45 g of oat flour and 255 g of deionized water were directly weighed into a 500 mL flask. Enzymes were added according to the experimental design shown in Table 9. β-Glucanase was provided by Prime.

[0175] Table 9: Experimental design

[0176]

[0177]

[0178] Heat the suspension to 60 °C. Once the target temperature of 60 °C is reached, stir the reaction mixture at 300 rpm for 120 min. To terminate the enzyme action, heat the suspension to 90 °C and hold at 90 °C for 10 min. Without cooling, separate the hot suspension into a liquid base material and a solid precipitate fraction by centrifugation at 3 x 1200 g. After separation, weigh the oat base and place it in an ice bath.

[0179] Equilibrate the sample to room temperature and add sunflower oil and sodium chloride to final concentrations of 1% and 0.08%, respectively, to formulate the oat beverage. Then use (Vorwerk, Wuppertal, Germany) to homogenize the sample. Then use a Hamilton STAR TM Liquid Handler (Hamilton Robotics Inc. / Hamilton Bonaduz AG) and methods similar to those in WO 2011 / 107472 (incorporated herein by reference in its entirety) to analyze the viscosity of the oat beverage. The viscosity data have been multiplied by –1 to obtain positive values; higher values are associated with higher viscosities. Quantify the amount of β-glucan in the sample according to Application Note 64538 (Thermo Scientific, Waltham, MA, United States) and use a Gallery TM Plus Beermaster discrete analyzer (Thermo Scientific) for the measurement. Table 10 provides the viscosity and amount of β-glucan of the oat beverage.

[0180] Table 10: Viscosity and β-glucan in oat beverage

[0181]

[0182]

[0183] Compared with Sample No. 5, Sample No. 1 has a lower viscosity in the final oat beverage. However, similar viscosities to those in Sample No. 1 were achieved in Samples No. 2 - 4 without complete degradation or hydrolysis of β-glucan. β-Glucan was not detected in Sample No. 1.

Claims

1. A method for obtaining a plant-based food ingredient for a dairy alternative food product, the method comprising: (a) obtaining a slurry of plant material in water; and (b) providing a raw starch-degrading α-amylase and optionally one or more additional enzymes to the slurry of step (a), and maintaining at a temperature between 25°C and 60°C to obtain a hydrolyzed plant material; wherein the hydrolyzed plant material is a plant-based food ingredient for a dairy alternative food product.

2. The method according to claim 1, the method further comprising (c) separating the hydrolyzed plant material into a solid stream and a liquid stream; (d) harvesting the liquid stream as a plant-based food ingredient for a dairy alternative food product; and (e) optionally inactivating the one or more enzymes before or after step (c) or (d).

3. The method according to any one of claims 1 or 2, wherein the raw starch-degrading α-amylase is an amylase of the GH13 family comprising a carbohydrate-binding module (CBM) that preferentially binds starch.

4. The method according to any one of the preceding claims, wherein the raw starch-degrading α-amylase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, or 4.

5. The method according to any one of the preceding claims, wherein the additional enzyme is glucoamylase, maltogenic amylase, β-amylase, protease, hemicellulase, cellulase, pectinolytic enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof.

6. The method according to any one of the preceding claims, wherein the additional enzyme is β-glucanase.

7. The method according to any one of the preceding claims, wherein the temperature of step (b) is between 25°C and 55°C, 30°C and 55°C, 35°C and 55°C, 40°C and 60°C, 30°C and 50°C, or 40°C and 55°C.

8. The method according to any one of the preceding claims, wherein the plant material is derived from corn, rice, barley, wheat, quinoa, oats, rye, flax, buckwheat, sorghum, millet, sago, cassava, tapioca, potato, sweet potato, pea, bean, cashew nut, macadamia nut, sesame, coconut, banana, jackfruit, and / or breadfruit.

9. The method according to any one of the preceding claims, wherein the plant material is a cereal flour or dehulled grain, including corn flour, rice flour, barley flour, buckwheat flour, wheat flour, millet flour, quinoa flour, oat flour, rye flour, or a mixture thereof.

10. The method according to any one of the preceding claims, wherein the plant material is oat flour, oatmeal, oat bran, dehulled oats, or any combination thereof.

11. The method according to any one of the preceding claims, 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.

12. Use of a raw starch-degrading α-amylase in the hydrolysis of plant-based materials for the production of plant-based food ingredients for dairy alternative food products.

13. A method for obtaining an oat hydrolyzate food ingredient for a dairy alternative food product, the method comprising (a) obtaining a slurry of oat material in water; (b) providing a raw starch-degrading α-amylase and a β-glucanase to the slurry of step (a) and maintaining at a temperature between 25°C - 60°C, 25°C - 55°C, 45°C - 55°C or 50°C - 55°C to obtain an oat hydrolyzate; (c) separating the oat hydrolyzate into a solid stream and a liquid stream; (d) harvesting the liquid stream; and (e) optionally inactivating the enzyme before or after step (c) or (d); wherein the harvested liquid stream is an oat hydrolyzate food ingredient for a dairy alternative food product.

14. The method according to claim 13, wherein the raw starch-degrading α-amylase is a GH13 family amylase comprising a carbohydrate-binding module (CBM) that preferentially binds starch.

15. The method according to claim 13 or 14, wherein the raw starch-degrading α-amylase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3 or 4.

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