Methods for obtaining plant-based food components

Abstract

The present invention relates to a method for obtaining a plant-based food ingredient for dairy alternatives, comprising hydrolyzing a plant material at a temperature of 25°C to 60°C in the presence of raw starch-degrading alpha-amylase and optionally additional enzymes to obtain a hydrolyzed plant material for use as a plant-based food ingredient for dairy alternatives.

Description

Technical Field

[0001] Reference to Sequence Listing This application includes a sequence listing in computer-readable form. This computer-readable form is incorporated herein by reference.

[0002] The present invention relates to the use of an enzyme having alpha-amylase activity for obtaining hydrolyzed plant material.

Background Art

[0003] In recent years, due to health reasons, the number of people seeking vegan, vegetarian, or non-dairy diets has been increasing. Furthermore, animal milk, especially food products made from cows, is being increasingly recognized for its high environmental cost. These factors have led to a greater demand for dairy alternative foods for many traditional dairy-derived foods, including milk, cream, cheese, yogurt, and ice cream.

[0004] Dairy alternative foods typically originate from high starch plant materials such as grains, legumes, or potatoes. Generally, in order to convert high starch plant materials into dairy alternative foods or food ingredients contained in dairy alternative foods, starch must be hydrolyzed. The conversion of starch typically includes a gelatinization step that dissolves starch granules to form a viscous suspension, a liquefaction step that partially hydrolyzes starch to reduce viscosity, and optionally, a saccharification step that follows and involves the production of glucose and maltose by further hydrolysis.

[0005] Gelatinization is usually achieved by heating, but liquefaction and possible saccharification often involve the use of enzymes. Typically, since high temperatures are preferably used for gelatinization, liquefaction is also carried out at high temperatures. In that case, gelatinization and liquefaction are carried out at high temperatures for a long time, after which the plant material is rapidly cooled, and then saccharification is carried out as a second step at a lower temperature.

[0006] Holding starch at high temperatures for extended periods, followed by rapid cooling of the mixture, is an energy-intensive process. Given the rising global energy costs and the growing recognition among consumers and producers that improved energy efficiency is environmentally beneficial, there is a need to reduce energy consumption. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The objective of the present invention is to identify an improved, energy-efficient process for producing hydrolyzed plant-based food ingredients for the manufacture of dairy alternative foods. [Means for solving the problem]

[0008] The present invention relates to a method for obtaining a food component for the production of dairy alternative foods, wherein the food component is obtained from a slurry of plant material in water, and raw starch-degrading alpha-amylase and optionally additional enzymes are added to the slurry, which is maintained at a temperature of 25-60°C to enable hydrolysis of the plant material. The hydrolyzed plant material is a food component for dairy alternative foods. Since hydrolysis occurs at a temperature of 25-60°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.

[0009] 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 recovered as a plant-based food ingredient for dairy alternatives. The enzymes may then be inactivated, for example, by heat treatment or UHT treatment of the liquid stream, or after further treatment of the liquid stream.

[0010] The enzymes added to the slurry include raw starch-degrading alpha-amylase. This raw starch-degrading alpha-amylase may be a GH13 family amylase and may also possess a carbohydrate-binding module (CBM) that preferentially binds to starch. This CBM may be CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82, or 83.

[0011] Additional enzymes may be added to the slurry. The additional enzymes may also be raw starch-degrading enzymes. The additional enzymes may include glucoamylase, maltose-producing amylase, beta-amylase, protease, hemicellulase, cellulase, pectin-degrading enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof.

[0012] The present invention provides a method for obtaining a plant-based food component for a dairy alternative, comprising: obtaining a slurry of heat-treated oat material and water; holding the slurry at a temperature of 25-60°C in the presence of raw starch-degrading alpha-amylase and beta-glucanase; allowing the oat material to be hydrolyzed for a certain period of time; separating the hydrolyzed oat material into solid and liquid streams; and finally recovering the liquid stream as a plant-based food component for a dairy alternative. The enzymes may be inactivated before or after the separation of the hydrolyzed oat material into solid and liquid streams. In some embodiments, the enzymes are inactivated in the recovered liquid stream. The dairy alternative may be a beverage, yogurt, cheese, creamer, ice cream, or any other dairy alternative known in the art. [Modes for carrying out the invention]

[0013] The following definitions apply according to the modes of carrying out this invention. Note that the singular forms "a," "an," and "the" include multiple references unless otherwise clearly indicated by the context.

[0014] As used herein, the terms “drink” and “beverage” are interchangeable and have the same meaning.

[0015] Unless otherwise defined or clearly indicated by context, all percentages are weight percentages (percent w / w or "%(w / w)").

[0016] The term "plant-based food component" refers to a plant-based composition that can be combined with additional food components to produce food. Plant-based food components and foods containing plant-based food components can be ingested by humans or animals, including livestock such as companion animals. In some embodiments, plant-based food components may be combined with additional food components to produce dairy alternative foods. Additional food components may be any food component that is considered useful by those skilled in the art. Additional food components may be solid or liquid. Additional food components may be plant-based or not. In some embodiments, the additional food component is water.

[0017] The term "dairy substitute" refers to foods that can be used as a substitute for dairy products. Dairy substitutes are plant-based and do not contain dairy-derived food ingredients. Examples of dairy substitutes include plant-based beverages, creamers, cheeses, ice creams, yogurts, and any other dairy substitutes known in the art.

[0018] In some embodiments, plant-based food components may be directly combined with additional food components to produce ready-to-drink dairy alternative beverages. Examples of dairy alternative beverages include beverages containing oat beverages, rice beverages, barley beverages, potato beverages, pea beverages, sesame beverages, almond beverages, hemp beverages, tiger nut beverages, or any combination thereof.

[0019] In some embodiments, plant-based food components may be used as substrates for fermentation to produce dairy alternative beverages such as buttermilk, or to produce dairy alternative yogurt.

[0020] In some embodiments, the plant-based food component may be further processed. Further processing may include water removal. In some embodiments, water removal concentrates hydrolysis products, i.e., free sugars. In some embodiments, water removal increases the viscosity of the plant-based food component.

[0021] In some embodiments, the plant-based food ingredient may be optionally further processed and combined with additional food ingredients to produce dairy-alternative ice cream. In some embodiments, the plant-based food ingredient may be optionally further processed and combined with additional food ingredients to produce dairy-alternative cheese.

[0022] The plant-based food components of the present invention are derived from plant materials that are edible parts of plants or derived therefrom. In some embodiments, the plant material is derived from edible parts of plants that are also rich in starch. In some embodiments, the edible parts of plants may be tubers, roots, stems, coblets, legumes, fruits, nuts, or seeds. In some embodiments, the plant is a cereal, and the plant material is a cereal, also known as a whole grain, or derived from a cereal. In further embodiments, the cereal may be derived from corn, rice, barley, wheat, buckwheat, millet, milo, quinoa, oats, or rye. In some embodiments, the plant material is a tuber or root (including rhizome) of potatoes, sweet potatoes, cassava, tiger nuts (chuffa nuts), canna, or tapioca, or derived therefrom. In some embodiments, the plant material is a fruit such as a banana, jackfruit, or breadfruit, or derived therefrom. In some embodiments, the plant material is a nut such as an almond, macadamia, or cashew, or derived therefrom. In some embodiments, the plant material is or is derived from hemp, sago, pea, or legume.

[0023] In some embodiments, the plant material is heat-treated. In some embodiments, the plant material is dehydrated. In some embodiments, the plant material is hulled, powdered, wet-ground, and / or dry-ground. 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, hemp flour, sesame flour, nut flour (cashew, macadamia, almond flour, etc.), pea flour, bean flour, hulled oats, hulled barley, hulled wheat, hulled peas, hulled beans, or any combination thereof. In some embodiments, the plant material is powdered or ground to produce a paste.

[0024] In some embodiments, the plant material is oat material. In further embodiments, the oat material is oat flour, oat flakes, oat bran, dehulled oats, or a combination thereof. In still further embodiments, the oat material may be oat flour such as heat-treated oat flour, or may be ground oat grains such as wet-milled dehulled and heat-treated oat grains, or may be any other oat material known in the art. In some embodiments, the oat material is heat-treated oat flour, oat flakes, oat bran, or any combination thereof.

[0025] In the method of the present invention, the plant material is suspended in water to form a slurry, and the ratio of plant material to water is 1:3 to 1:8 (w / w). In some embodiments, the ratio of plant material to water is 1:4 to 1:16. In some embodiments, the ratio of plant material to water is 1:1 to 1:4.

[0026] In the method of the present invention, raw starch-degrading alpha-amylase and optionally additional enzymes are added to the slurry, and the mixture is maintained at a temperature of 25 to 60 °C to hydrolyze the plant material by the enzymes to produce a hydrolyzed plant material. In some embodiments, the slurry is maintained at a temperature lower than the gelatinization temperature of the starch in the slurry.

[0027] As used herein, "raw starch degrading enzyme" (also known as raw starch hydrolyzing enzyme) refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch. Since the gelatinization temperature of starch can vary with the onset temperature of gelatinization ranging from about 51°C to 68°C, it can be in the range of 51°C to 78°C. When using barley flour, if the onset temperature of gelatinization is about 53°C to 63°C, the raw starch degrading alpha - amylase that degrades raw starch can directly degrade raw starch. When using oat flour, if the onset temperature of gelatinization is about 55°C to 62°C, the raw starch degrading alpha - amylase that degrades raw starch can directly degrade raw starch. The raw starch degrading alpha - amylase is a raw starch degrading enzyme.

[0028] In one embodiment, the 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, and the raw 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 against gelatinized starch is measured by measuring the release of glucose produced by the enzyme in a 2% gelatinized (e.g., corn) starch reaction mixture. The activity is measured by the release of reducing sugar produced at 4 mol / hour per 1 mg of pure active enzyme. Then, using the same assay, the activity of the enzyme against raw starch can be measured, but replacing 2% of the gelatinized (e.g., corn) starch with 2% of raw (e.g., corn) starch. In both assays, the temperature is 40°C, the same pH and buffer are used, and the incubation time is 6 hours.

[0029] Raw starch-degrading enzymes are ubiquitous and produced by raw starch-degrading enzymes in plants, animals, and microorganisms, such as fungi, bacteria, and yeasts. In some embodiments, the raw starch-degrading enzyme is glucoamylase. In other embodiments, the raw starch-degrading enzyme is alpha-amylase, also called raw starch-degrading alpha-amylase. In some embodiments, the raw starch-degrading enzyme refers to alpha-amylase, glucoamylase, or a combination of one or more alpha-amylases and one or more glucoamylases. Sources of raw starch-degrading enzymes include, for example, enzymes obtained from Aspergillus spp. species such as alpha-amylases of Aspergillus oryzae, Aspergillus niger, and Aspergillus kawachii. Examples of such raw starch-degrading enzymes include those described in International Publication No. 2005 / 003311, International Publication No. 2006 / 0692, International Publication No. 2006 / 060289, and International Publication No. 2004 / 080923.

[0030] In some embodiments, the raw starch-degrading alpha-amylase is an acidic alpha-amylase. The "acidic alpha-amylase" is alpha-amylase (4-α-D-glucan glucanohydrolase, EC3.2.1.1), which, when added in an effective amount, is active at a pH in the range of 3.0 to 7.0, preferably 3.5 to 6.0, or more preferably 4.0 to 5.0. The source of the raw starch-degrading acidic alpha-amylase is an acidic alpha-amylase derived from Aspergillus niger, disclosed as "AMYA_ASPNG" in the Swiss-prot / TeEMBL database under primary accession number P56271 and described in detail in International Publication No. 1989 / 01969 (Example 3). Aspergillus niger acidic alpha-amylase is also shown as Sequence ID No. 1 in International Publication No. 2004 / 080923 (Novozymes A / S), which is incorporated herein by reference. A preferred commercially available acidic fungal alpha-amylase derived from Aspergillus niger is Product SP288 (Sequence ID No. 1 in U.S. Patent No. 7,244,597; available from Novozymes A / S). Other sources of acidic alpha-amylase include those derived from strains of the genera Rhizomucor and Meripilus, such as Rhizomucor pusillus (International Publication No. 2004 / 055178) or strains of Meripilus giganteus.In yet another embodiment, the acidic alpha-amylase is derived from Aspergillus kawachii, as 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 alpha-amylase from Aspergillus kawachii”, and further disclosed as EMBL:#AB008370.

[0031] In some embodiments, raw starch-degrading alpha-amylases have a carbohydrate-binding module (CBM) that binds to starch. In some embodiments, the CBM preferentially binds to starch, particularly thermally untreated granular starch. Such CBMs may also be called starch-binding domains (SBDs). SBDs are known to be found in 15 CBM families (i.e., CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82, and 83).

[0032] In some embodiments, the raw starch-degrading alpha-amylase may be a hybrid alpha-amylase comprising a starch-binding domain (SBD) and an alpha-amylase catalytic domain (CD). The hybrid alpha-amylase may also comprise an alpha-amylase catalytic domain (CD), a starch-binding domain (SBD), and a linker connecting the CD and the SBD, as is known in the art. In one embodiment, the catalytic domain is derived from a strain of Aspergillus kawachii. Examples of hybrid alpha-amylases are disclosed in International Publication No. 2005 / 003311, U.S. Patent Application Publication No. 2005 / 0054071 (Novozymes), and U.S. Patent No. 7,326,548 (Novozymes), which are incorporated herein by reference. Furthermore, examples include the enzymes disclosed in Tables 1-5 of the Examples in U.S. Patent No. 7,326,548 and U.S. Patent Application Publication No. 2005 / 0054071 (Table 3 on page 15), such as the Aspergillus kawachii linker and the Aspergillus niger alpha-amylase catalytic domain (CD) having a starch-binding domain (SBD).

[0033] Other acidic alpha-amylases include the enzymes disclosed in International Publication No. 2004 / 020499 and International Publication No. 2006 / 069290, as well as the enzymes disclosed in International Publication No. 2006 / 066579 as SEQ ID NO: 2 (Hybrid A. niger alpha-amylase + CBM), SEQ ID NO: 3, or SEQ ID NO: 4 (JA129). A hybrid alpha-amylase comprising an Aspergillus niger glucoamylase linker and a Rhizomucor pusillus alpha-amylase with an SBD, disclosed as V039 in Table 5 of International Publication No. 2006 / 069290.

[0034] Sequence IDs 1-4 are GH13 family raw starch-degrading alpha-amylases. In some embodiments, the raw starch-degrading alpha-amylase of the present invention is a GH13 family amylase. In some embodiments, the raw starch-degrading alpha-amylase has an amino acid sequence that is 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% identical to the amino acid sequence of Sequence ID 1, 2, 3, or 4. In some embodiments, the raw starch-degrading alpha-amylase of the present invention has the amino acid sequence of Sequence ID 1, 2, 3, or 4.

[0035] The term "identity" refers to the relationship between two amino acid sequences or two nucleotide sequences. For the purposes of this invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is preferably 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) version 3.0.0 or later. The optional parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output (obtained using the -nobrief option), labeled "longest identity," is used as % identity and is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in alignment)

[0036] Amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1 to 30 amino acids; small amino or carboxyl terminal extensions such as amino-terminal methionine residues; small linker peptides of 20 to 25 residues or less; or small extensions that facilitate purification by altering other functions, such as net charge, polyhistidine tracts, antigen epitopes, or binding domains.

[0037] Examples of conservative substitutions fall 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 low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Generally, amino acid substitutions that do not alter specific activity are known in the art, for example, as described by H. Neurath and RLHill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include 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.

[0038] In some embodiments, the raw starch-degrading alpha-amylase has at least 70% sequence identity with SEQ ID NO: 1, 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.

[0039] In another embodiment, the raw starch-degrading alpha-amylase has at least 70% sequence identity with SEQ ID NO: 2, 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.

[0040] In another embodiment, the raw starch-degrading alpha-amylase has at least 70% sequence identity with SEQ ID NO: 3, 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.

[0041] In another embodiment, the raw starch-degrading alpha-amylase has at least 70% sequence identity with SEQ ID NO: 4, 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.

[0042] Raw starch-degrading alpha-amylase may be added in a range of 500 to 25,000 parts per million (ppm) depending on the amount of plant material added. In some embodiments, raw starch-degrading alpha-amylase in concentrations of 500 to 20,000, 750 to 20,000, 750 to 18,000, 750 to 17,000, 750 to 15,000, 750 to 1,000, 750 to 7,500, or 1,000 to 5,000 ppm may be added to the slurry. In some embodiments, raw starch-degrading alpha-amylase in concentrations of 750 to 2,500 or 1,000 to 1,500 ppm may be added to the slurry.

[0043] The hydrolysis products by raw starch-degrading alpha-amylase contain malto-oligosaccharides (MOS). Malto-oligosaccharides contain glucose molecules having one or more branched links (alpha-1,4) with a typical degree of polymerization (DP) of 2 to 9. MOS can be indigestible oligosaccharides with short chain lengths (2 to 10), which may have a prebiotic effect that enhances the growth of beneficial bacteria in the human gut (see, for example, Jang et al., 2020, Molecules 25:5201, doi:10.3390 / molecules25215201). In some embodiments, dairy alternative foods containing the plant-derived food components of the present invention may have a prebiotic effect when ingested.

[0044] In some embodiments of the present invention, one or more additional enzymes are added to the slurry to enable hydrolysis of plant material. The additional enzymes may be glucoamylase, maltose-producing amylase, beta-amylase, protease, hemicellulase, cellulase, pectin-degrading enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof. The additional enzymes may be of any origin, including mammalian, plant, and microbial (bacteria, yeast, or fungal) origins.

[0045] 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 micromolar of maltose per minute under standard conditions of 37°C, pH 4.3, substrate: 23.2 mM maltose, buffer: 0.1 M acetic acid, and reaction time of 5 minutes. In some embodiments, glucoamylase may be added at concentrations of 50–1000 AGU / kg of plant material.

[0046] In some embodiments, the additional enzyme is a maltose-producing amylase. Maltose-producing alpha-amylase (EC 3.2.1.133) may be derived from the genus Bacillus. Maltose-producing alpha-amylase derived from B. stearothermophilus strain NCIB 11837 is commercially available from Novozymes A / S under the trade name Novamyl®. Maltose-producing alpha-amylase may also be a variant of maltose-producing alpha-amylase derived from B. stearothermophilus, e.g., Novamyl® 3D, as disclosed in, for example, International Publication No. 1999 / 043794, International Publication No. 2006 / 032281, or International Publication No. 2008 / 148845.

[0047] In some embodiments, the additional enzyme is xylanase. The xylanase may be of microbial origin, for example, from bacteria or fungi, such as Aspergillus, particularly A. aculeatus, A. niger, A. awamori, or A. tubigensis; from Trichoderma, for example, from strains of T. reesei; or from Humicola, for example, from strains of H. insolens. In some embodiments, the xylanase is derived from a strain of T. reeseii. Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG and PENTOPAN 500 BG (available from Novozymes A / S), GRINDAMYL POWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP 5001 (available from DSM).

[0048] In some embodiments, the additional enzyme is a protease. The protease may be derived from a Bacillus species, such as Bacillus amyloliquefaciens. A preferred protease may be Neutrase®, available from Novozymes A / S.

[0049] In some embodiments, the additional enzyme is a beta-glucanase. The beta-glucanase may have only beta-glucanase activity or may also have other enzymatic activity. In some embodiments, the beta-glucanase has at least 70% sequence identity with SEQ ID NO: 5, 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.

[0050] In some embodiments, the enzyme having beta-glucanase activity may be a preparation of endo-alpha-amylase obtained from Bacillus species such as Bacillus amyloliquefaciens, which has beta-glucanase activity. In some embodiments, the enzyme having beta-glucanase activity may be in a cellulose-degrading enzyme preparation. In further embodiments, the cellulose-degrading enzyme preparation may be obtained from Trichoderma reesei. In other embodiments, the beta-glucanase may be obtained from Aspergillus niger. Examples of enzyme preparations having beta-glucanase activity include BAN®, Celluclast®, or Ultraflo® Prime, all available from Novozymes A / S. These enzyme preparations are thought to contain beta-glucanase. Ultraflo® Prime contains both beta-glucanase and xylanase.

[0051] In some embodiments, beta-glucanase may be added in parts per million (ppm) based on the amount of plant material used. In some embodiments, 1 to 2500 ppm of beta-glucanase may be added to the slurry. In some embodiments, 5 to 2500, 20 to 2500, 50 to 2500, 50 to 2000, 50 to 1800, 50 to 1500, 50 to 1000, 50 to 750, 75 to 500, or 75 to 300 ppm of beta-glucanase may be added to the slurry. In some embodiments, 1 to 200, 1 to 150, or 1 to 100 ppm of beta-glucanase may 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 beta-glucanase may be added to the slurry.

[0052] In some embodiments, raw starch-degrading alpha-amylase and beta-glucanase are added to the slurry. In further embodiments, the slurry comprises oat material and water. In further embodiments, the slurry comprises oat flour and water. In further embodiments, the slurry comprises heat-treated oat flour and water. In some embodiments, raw starch-degrading alpha-amylase is added at 500 to 25,000 ppm and beta-glucanase is added at 1 to 2,500 ppm. In some embodiments, raw starch-degrading alpha-amylase is added at 750 to 15,000 ppm and beta-glucanase is added at 1 to 500 ppm. In some embodiments, raw starch-degrading alpha-amylase is added at 1,000 to 7,000 ppm and beta-glucanase is added at 1 to 300 ppm. In some embodiments, raw starch-degrading alpha-amylase is added at 1000-5000 ppm, and beta-glucanase is added at 1-150 ppm. In some embodiments, raw starch-degrading alpha-amylase is added at 1000-1500 ppm, and beta-glucanase is added at 1-100 ppm.

[0053] Raw starch-degrading alpha-amylase and optionally additional enzymes may be provided in any preferred form, such as a liquid, particularly a stabilized liquid, or the enzymes may be added as substantially dry powder or granules. Granules can be produced, for example, as disclosed in U.S. Patent No. 4,106,991 and U.S. Patent No. 4,661,452. Liquid enzyme preparations may be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established methods. Other enzyme stabilizers are well known in the art.

[0054] The enzyme combination may be added to a slurry containing plant material in any suitable manner, for example, by adding the individual components (separate or sequential addition of enzymes) or by adding the enzymes together in one step or one composition, or in any combination thereof.

[0055] The slurry is maintained at a temperature of 25–60°C to allow hydrolysis of the plant material. In some embodiments, the slurry is maintained at a temperature of 25–55°C, 30–55°C, 35–55°C, 40–60°C, 30–50°C, 40–55°C, 45–55°C, or 50–55°C. In further embodiments, the slurry is maintained at a temperature of approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, or approximately 60°C.

[0056] In some embodiments, the enzyme-added slurry is held at a temperature of 25–60°C for at least 10 minutes to allow enzymatic hydrolysis of the plant material. In some embodiments, the slurry is held for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, or at least about 240 minutes to allow enzymatic hydrolysis of the plant material. In some embodiments, the slurry is held for at least about 10 minutes, 30 minutes, 60 minutes, or 90 minutes. In some embodiments, the slurry is held for 30 minutes. In some embodiments, the slurry is held for 60 minutes. In some embodiments, the slurry is held for 90 minutes. Those skilled in the art will recognize that there is a relationship between the enzyme dose, the incubation temperature, and the amount of time allowed for enzymatic hydrolysis, resulting in higher doses of enzyme enabling shorter incubation times, lower doses of enzyme achieving the same level of hydrolysis with longer incubation times, and higher incubation temperatures enabling lower doses of enzyme and / or shorter incubation times.

[0057] The enzymes may be inactivated after treatment with raw starch-degrading alpha-amylase and optionally additional enzymes. The enzymes may be inactivated in any step after hydrolysis. In some embodiments, the enzymes are inactivated before or after the hydrolyzed plant material is separated into solid and liquid streams. In other embodiments, the enzymes are inactivated after additional food components are added to the recovered liquid stream.

[0058] In some embodiments, the enzyme is inactivated by heat treatment. In some embodiments, the heat treatment is performed at a temperature of 85–95°C for 5–30 minutes. In further embodiments, the heat treatment is performed at a temperature of 85–95°C for 10–15 minutes. In some embodiments, the heat treatment is performed at 95°C for 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the heat treatment is performed at a temperature of 85–95°C for 1 minute or less. In some embodiments, the heat treatment is performed at a temperature of 85–95°C for 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds.

[0059] In some embodiments, the enzyme is inactivated by ultra-high temperature (UHT) treatment. The UHT treatment may be direct or indirect. In some embodiments, the UHT treatment is 1 to 10 seconds at a temperature of 135 to 154°C. In further embodiments, the UHT treatment is 140 to 150°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In further embodiments, the UHT treatment is 140 to 145°C for 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the UHT treatment is 143°C for 4, 5, 6, 7, or 8 seconds.

[0060] Following enzymatic hydrolysis, hydrolyzed plant material is produced. The hydrolyzed plant material may also be called a plant hydrolysate, for example, oat hydrolysate. After enzymatic inactivation, the hydrolyzed plant material may be cooled. The hydrolyzed plant material can be separated into a solid flow and a liquid flow, for example, by centrifugation. Centrifugation may be performed in a decanter centrifuge. After centrifugation, the liquid flow may be harvested or recovered and used as a food ingredient in dairy alternatives. The liquid flow may still contain some solid matter. In some embodiments, the liquid flow contains 1-80% solids. In further embodiments, the liquid flow 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 called "total solids," can be measured using methods well known in the art. For example, a sample of the liquid stream may be dried, typically by heat, and then the remaining solids may be weighed.

[0061] Liquid flows, still sometimes referred to as plant hydrolysates, contain solids and sugars useful in the production of dairy alternatives. The viscosity of the liquid flow also affects its usefulness in the production of dairy alternatives. Viscosity is partially determined by the beta-glucan content, with higher beta-glucan content resulting in higher viscosity. Plant hydrolysates with very high viscosity may be difficult to process in industrial production. Furthermore, plant hydrolysates with very high viscosity may be difficult to use as food ingredients in the production of dairy alternatives, particularly dairy alternative beverages. The viscosity of plant hydrolysates also plays a role in their usefulness as food ingredients in dairy alternatives. Certain foods prefer certain levels of viscosity. For example, dairy alternative oat beverages typically have a viscosity similar to that of low-fat or skim milk.

[0062] In some embodiments, the liquid stream is further processed to remove water or to concentrate it. Concentration increases the relative amount of solids in the concentrated liquid stream. Concentration can occur by the evaporation of water in the liquid stream. In some embodiments, the concentrated liquid stream contains 10–95% solids. In further 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, water removal increases the viscosity of the dairy substitute food.

[0063] In some embodiments, the liquid stream is used directly as a plant-based food ingredient. The liquid stream may be referred to as the “base.” Additional food ingredients may be added to the liquid stream to produce dairy alternatives. In some embodiments, the liquid stream is derived from oat hydrolysate and may be referred to as the “oat base.” In further embodiments, the oat base may be formulated using, for example, sodium chloride (NaCl), oil, and optionally flavoring agents. Such formulations may be considered dairy alternatives. The oat base may be homogenized before or after the addition of food ingredients.

[0064] Dairy substitutes may be UHT or ESL treated and aseptically packaged. The final product may be marketed as a plant-based beverage that is a dairy substitute.

[0065] Alternatively, in some embodiments, the oat base may be further processed into a liquid flow or into other dairy alternative foods such as fermented plant-based products or plant-based ice cream, or used as an ingredient in dairy alternative foods.

[0066] In some embodiments, the liquid flow is an oat base that is processed into an oat-based dairy alternative food. In some embodiments, the food is an oat-based beverage, an oat-based creamer, an oat-based yogurt, an oat-based cheese, or an oat-based ice cream.

[0067] The present invention is further defined by the following numbered embodiments.

[0068] 1. A method for obtaining plant-based food ingredients for dairy product substitutes, (a) To obtain a slurry of plant material in water, (b) Add raw starch-degrading alpha-amylase and optionally additional enzymes to the slurry of step (a) and maintain at a temperature of 25-60°C to obtain hydrolyzed plant material. Includes, Hydrolyzed plant materials are plant-based food ingredients for dairy alternatives. method.

[0069] 2. (c) Separating the hydrolyzed plant material into a solid flow and a liquid flow, (d) Recovering the liquid stream as a plant-based food ingredient for dairy alternatives, (e) optionally, inactivating the enzyme before or after step (c) or (d), The method according to Embodiment 1, further comprising:

[0070] 3. The method according to either Embodiment 1 or 2, wherein the raw starch-degrading alpha-amylase is a GH13 family amylase.

[0071] 4. The method according to Embodiment 3, wherein the GH13 family amylase comprises a carbohydrate-binding module (CBM) that preferentially binds to starch.

[0072] 5. The method according to Embodiment 4, wherein the CBM is CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 68, 69, 74, 82, or 83.

[0073] 6. The method according to any one of the prior embodiments, wherein the raw starch-degrading alpha-amylase contains an amino acid sequence that 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%, at least 99%, or 100% identical to SEQ ID NOs: 1, 2, 3, or 4.

[0074] 7. The method according to any one of the preceding embodiments, wherein the additional enzyme is glucoamylase, maltose-producing amylase, beta-amylase, protease, hemicellulase, cellulase, pectin-degrading enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof.

[0075] 8. The method according to any one of the preceding embodiments, wherein the additional enzyme is a second raw starch-degrading enzyme.

[0076] 9. The method according to any one of the prior embodiments, wherein the additional enzyme is beta-glucanase.

[0077] 10. The method according to any one of the prior embodiments, wherein the additional enzyme is xylanase.

[0078] 11. The slurry of step (b) is a method according to any one of the prior embodiments, comprising beta-glucanase and xylanase.

[0079] 12. The method according to any one of the preceding embodiments, wherein raw starch-degrading alpha-amylase is added to the slurry in doses of 500-20000, 750-20000, 750-18000, 750-17000, 750-15000, 750-2500, 750-1000, 750-7500, 1000-5000, or 1000-1500 ppm.

[0080] 13. The method according to any one of Embodiments 9, 11, or 12, wherein beta-glucanase is added to the slurry in a dose of 1-200, 1-150, or 1-100 ppm.

[0081] 14. The method according to any one of the prior embodiments, wherein the temperature in step (b) is 25-55°C, 30-55°C, 35-55°C, 40-60°C, 30-50°C, or 40-55°C.

[0082] 15. The method according to any of the prior embodiments, wherein the enzyme is inactivated by heat treatment or ultra-high temperature (UHT) treatment.

[0083] 16. The method according to Embodiment 15, wherein the UHT treatment is performed at a temperature of 140-145°C for 2-8 seconds.

[0084] 17. The method according to any one of the preceding embodiments, wherein the plant material is derived from a tuber, root, stem, legume, fruit, nut, seed, or whole grain.

[0085] 18. The method according to any one of the preceding embodiments, wherein the plant material is derived from corn, rice, barley, wheat, quinoa, oat, rye, buckwheat, milo, millet, sago, cassava, tapioca, potato, sweet potato, pea, bean, almond, cashew, macadamia, banana, jackfruit and / or breadfruit.

[0086] 19. The method according to any one of the preceding embodiments, wherein the plant material is a grain flour or hulled grain comprising corn flour, rice flour, barley flour, buckwheat flour, wheat flour, millet flour, quinoa flour, oat flour, rye flour, or a mixture thereof.

[0087] 20. The method according to any one of the preceding embodiments, wherein the plant material is an oat material such as oat flour, oat flakes, oat bran, oat groat, or any combination thereof.

[0088] 21. The method according to any one of the preceding embodiments, wherein the dairy alternative is a plant-based beverage, plant-based ice cream, plant-based creamer, plant-based yogurt, or plant-based cheese.

[0089] 22. A plant-based food ingredient for dairy substitutes, manufactured by the method described in any one of the prior embodiments.

[0090] 23. The method according to any one of the preceding embodiments, wherein a plant-based food ingredient is combined with an additional food ingredient to produce a dairy substitute food.

[0091] 24. A method for obtaining oat hydrolysate food ingredients for dairy alternative foods, (a) To obtain a slurry of oat material in water, (b) Supplying raw starch-degrading alpha-amylase and beta-glucanase to the slurry of step (a), and maintaining it at a temperature of 25-60°C, 25-55°C, 45-55°C, or 50-55°C to obtain oat hydrolysate, (c) Optionally, inactivating the enzyme Includes, Oat hydrolysate is a plant-based food ingredient used as a dairy alternative. method.

[0092] twenty five. (d) Separating the oat hydrolysate into a solid stream and a liquid stream, (e) The method of Embodiment 24, further comprising recovering a liquid stream as an oat-based food ingredient for dairy alternative foods.

[0093] 26. A method for obtaining oat hydrolysate food ingredients for dairy alternative foods, (a) To obtain a slurry of oat material in water, (b) Maintain the slurry from step (a) at a temperature of 25-60°C, add raw starch-degrading alpha-amylase and beta-glucanase to obtain oat hydrolysate, (c) Separating oat hydrolysate into solid and liquid streams, (d) Recovering the liquid flow, (e) Optionally, inactivate the enzyme before or after step (c) or (d). Includes, The recovered liquid stream is an oat hydrolysate food ingredient for dairy alternative foods. method.

[0094] 27. The method according to any one of Embodiments 24 to 26, wherein the raw starch-degrading alpha-amylase contains an amino acid sequence that 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%, at least 99%, or 100% identical to SEQ ID NOs: 1, 2, 3, or 4.

[0095] 28. The method according to any one of embodiments 24 to 27, wherein the temperature of step (b) is 25-55°C, 30-55°C, 35-55°C, 40-60°C, 30-50°C, 40-55°C, 45-55°C, or 50-55°C.

[0096] 29. The method according to any one of embodiments 24 to 28, wherein the slurry of step (b) further comprises xylanase.

[0097] 30. The method according to any one of Embodiments 24 to 29, wherein raw starch-degrading alpha-amylase is added to the slurry in doses of 500-20000, 750-20000, 750-18000, 750-17000, 750-15000, 750-2500, 750-1000, 750-7500, 1000-5000, or 1000-1500 ppm.

[0098] 31. The method according to any one of Embodiments 24 to 30, wherein beta-glucanase is added to the slurry in a dose of 1 to 200, 1 to 150, or 1 to 100 ppm.

[0099] 32. A plant-based food component produced by the method of any one of the preceding embodiments, wherein the plant-based food component has a prebiotic effect when ingested.

[0100] 33. A dairy substitute food containing plant-based food components as described in Embodiment 32, which has a prebiotic effect when ingested.

[0101] 34. Oat hydrolysate food ingredient, manufactured by the method of any one of Embodiments 24 to 31, which has a prebiotic effect when ingested.

[0102] 35. A dairy substitute food containing the oat hydrolysate food component described in Embodiment 34, which has a prebiotic effect when ingested.

[0103] 36. Use of raw starch-degrading alpha-amylase in the hydrolysis of plant materials for the production of plant-based food components for dairy alternative foods.

[0104] 37. The use according to Embodiment 36, wherein the plant material is an oat material such as oat flour, oat flakes, oat bran, oat shredded oat, or any combination thereof.

[0105] 38. Use of raw starch-degrading alpha-amylase and beta-glucanase in the hydrolysis of oat material for the production of plant-based food ingredients for dairy alternative foods.

[0106] The invention described and asserted herein should not be limited to the scope of the specific embodiments disclosed herein, for these embodiments are intended to illustrate several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention, as well as combinations of one or more embodiments.

[0107] Various references are cited herein, and their disclosures are incorporated by reference in their entirety. The present invention will be further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Examples]

[0108] Example 1: Preparation of plant-based food components using raw starch-degrading alpha-amylase of SEQ ID NO: 1 Amylase encoded by Sequence ID No. 1 at 1000 ppm (based on oat flour) was added to 650 L of water at 63°C along with 50 ppm of beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) and mixed with 200 kg of oat flour. A further 150 L of water was added, and the final temperature reached was 62°C. After 10 minutes, another 50 ppm of beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) was added, and the slurry was held at 62°C for 60 minutes to allow hydrolysis.

[0109] After hydrolysis, the hydrolyzed slurry was treated in a 60°C decanter, and the separated liquid stream was cooled to 10°C. The liquid stream is a plant-based food component, also referred to here as the oat concentrate base.

[0110] The oat concentrate base was diluted with water (44% w / w), and then salt (0.08% w / w) and rapeseed oil (0.8% w / w) were added. The resulting oat beverage was subjected to UHT treatment using methods known in the art and aseptically filled. The viscosity of the oat beverage was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Tables 1 and 2 below. Table 1 shows the viscosity, total solids content, and sugar content of the oat beverage. Table 2 shows the amount of specific maltooligosaccharides (MOS) present in the oat beverage.

[0111] Example 2: Preparation of plant-based food components using the raw starch-degrading alpha-amylase of Sequence ID No. 2 Amylase encoded by Sequence ID No. 2 at 1500 ppm (based on oat flour) was added to 650 L of water at 63°C along with 100 ppm of beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) and mixed with 200 kg of oat flour. Another 150 L of water was added, and the final temperature reached was 60°C. The slurry was held at 60°C for 60 minutes to allow hydrolysis.

[0112] After hydrolysis, the hydrolyzed slurry was treated in a 60°C decanter, and the separated liquid stream was cooled to 10°C. The liquid stream is a plant-based food component, also referred to here as the oat concentrate base.

[0113] The oat concentrate base was diluted with water (47.16% w / w), and then salt (0.08% w / w) and rapeseed oil (0.8% w / w) were added. The resulting oat beverage was subjected to UHT treatment using methods known in the art and aseptically filled. The viscosity of the oat beverage was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Tables 1 and 2 below. Table 1 shows the viscosity, total solids content, and sugar content of the oat beverage. Table 2 shows the amount of specific maltooligosaccharides (MOS) present in the oat beverage.

[0114] [Table 1]

[0115] [Table 2]

[0116] Tables 1 and 2 show that an oat beverage with good viscosity and suitable amounts of total solids, total sugars, and MOS can be obtained. These results indicate that hydrolyzed oat material and oat concentrate base can serve as a source of plant-based food ingredients for dairy alternatives. The oat concentrate base produced herein, hydrolyzed in a single step at approximately 60°C, has similar viscosity and amounts of solids, sugars, and MOS to oat concentrate bases obtained by hydrolyzing oats using conventional methods including gelatinization and liquefaction at high temperatures, followed by quenching and saccharification.

[0117] Example 3: Preparation of oat hydrolysate using raw starch-degrading alpha-amylase of SEQ ID NO: 1 Heat-treated oat flour was mixed with enzyme-containing water in a ratio of 100 g oat flour to 600 g water. The assay was performed using a standard method involving amylase and beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) encoded by SEQ ID NO: 1, or two different amylases, namely BAN® 480 L (Novozymes A / S, Denmark) supplied in the dosages shown in Tables 3 and 4, and Fungamyl® 800 L (Novozymes A / S, Denmark) supplied at 1500 ppm for all samples containing BAN® 480 L. The mixture of water, enzyme, and oat flour was then heated at 25°C, 40°C, or 60°C for 30 minutes to allow liquefaction / hydrolysis, followed by inactivation of the enzymes by raising the temperature to 95°C for 15 minutes. After inactivation, the hydrolysate was cooled to 60°C for centrifugation and separated into solid and liquid phases by centrifugation at a relative centrifugal force of 1932 × g using Multifuge® 3 ​​SR (Kendro Heraeus, Hanau, Germany). The liquid phase was analyzed for viscosity using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Table 3. Table 3 shows the viscosity, total solids content, and sugar content of the oat hydrolysate. The amounts of amylase and beta-glucanase are provided in ppm on a powder basis.

[0118] [Table 3]

[0119] Table 3 shows that samples containing raw starch amylase encoded by Sequence ID No. 1 produce higher total sugar content in oat hydrolysates compared to samples containing BAN® 480L and Fungamyl® 800L prepared at temperatures of 25–60°C.

[0120] Example 4: Preparation of rice hydrolysate using the raw starch-degrading alpha-amylase of SEQ ID NO: 1 Rice flour was mixed with water containing the enzyme in a ratio of 100 g rice flour to 600 g water. The assay was performed using a standard method comprising amylase and beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) encoded by Sequence ID No. 1, or two different amylases, namely BAN® 480 L (Novozymes A / S, Denmark) supplied in the dosages shown in Tables 5 and 6, and Fungamyl® 800 L (Novozymes A / S, Denmark) supplied at 1500 ppm for all samples containing BAN® 480 L. The mixture of water, enzyme, and oat flour was then heated at 25°C, 40°C, or 60°C for 30 minutes to allow liquefaction / hydrolysis, and the enzyme was inactivated by raising the temperature to 95°C for 15 minutes. After inactivation, the hydrolysate was cooled to 60°C and centrifuged to separate the solid and liquid phases by centrifuging at 3000 RPM for 10 minutes. The viscosity of the liquid phase was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Table 4. Table 4 shows the viscosity, total solids content, and sugar content of the rice hydrolysate. The amounts of amylase and beta-glucanase are provided in ppm on a powder basis.

[0121] [Table 4]

[0122] The data in Table 4 shows that samples containing raw starch amylase encoded by Sequence ID No. 1 produce higher total sugar rice hydrolysates compared to samples containing BAN® 480L and Fungamyl® 800L, which were prepared at temperatures of 25–60°C.

[0123] Example 5: Preparation of pea hydrolysate using raw starch-degrading alpha-amylase of SEQ ID NO: 1 Pea flour was mixed with enzyme-containing water in a ratio of 100g flour to 600g water. The assay was performed using a standard method involving amylase and beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) encoded by SEQ ID NO: 1, or two different amylases, namely BAN® 480 L (Novozymes A / S, Denmark) supplied in the dosages shown in Tables 7 and 8, and Fungamyl® 800 L (Novozymes A / S, Denmark) supplied at 1500 ppm for all samples containing BAN® 480 L. The mixture of water, enzyme, and oat flour was then heated at 25°C, 40°C, or 60°C for 30 minutes to allow liquefaction / hydrolysis, followed by inactivation of the enzymes by raising the temperature to 95°C for 15 minutes. After inactivation, the hydrolysate was cooled to 60°C for centrifugation and separated into solid and liquid phases by centrifugation at a relative centrifugal force of 1932 × g using a Multifuge® 3 ​​SR (Kendro Heraeus). The liquid phase was analyzed for viscosity using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Table 5. Table 5 shows the viscosity, total solids content, and sugar content of the rice hydrolysate. The amounts of amylase and beta-glucanase are provided in ppm on a powder basis.

[0124] [Table 5]

[0125] The data in Table 5 shows that samples containing raw starch amylase encoded by Sequence ID No. 1 produce higher total sugar content in pea hydrolysates compared to samples containing BAN® 480L and Fungamyl® 800L, which were prepared at temperatures of 25–60°C.

[0126] Example 6: Preparation of oat hydrolysate using raw starch-degrading alpha-amylase of SEQ ID NO: 3 or SEQ ID NO: 4 For each assay, oat flour was mixed with enzyme-containing water in a ratio of 100g flour to 600g water. Assays were also performed using amylase encoded by SEQ ID NO: 3 or SEQ ID NO: 4, and beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark). Standard assays were also performed using two different amylases, namely BAN® 480 L (Novozymes A / S, Denmark), supplied in the dosages listed in the table below, and Fungamyl® 480 L (Novozymes A / S, Denmark), supplied at 1500 ppm for all samples, including BAN® 800 L. 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 increasing the temperature to 95°C for 15 minutes to inactivate the enzymes. After inactivation, the hydrolysate was cooled to 60°C for centrifugation and separated into solid and liquid phases by centrifugation at a relative centrifugal force of 1932 × g using Multifuge® 3 ​​SR (Kendro Heraeus). The liquid phase was analyzed for viscosity using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Tables 6 and 7 below. Tables 6 and 7 provide viscosity, total solids content, and sugar content of oat hydrolysate from assays performed using either SEQ ID NO: 3 or SEQ ID NO: 4. The amounts of amylase and beta-glucanase are provided in ppm on a powder basis.

[0127] [Table 6]

[0128] [Table 7]

[0129] The results shown in Tables 6 and 7 indicate that when hydrolysis is carried out at temperatures between 25 and 60°C, raw starch-degrading amylase produces oat hydrolysates with desirable viscosity, total solids content, and total sugars.

[0130] Example 7: Industrial testing using amylase of SEQ ID NO: 1 5700 ppm (based on oat flour) of amylase SEQ ID NO: 1 was added to 1600 L of water along with 100 ppm of beta-glucanase (Ultraflo® Prime, Novozymes A / S, Denmark) and mixed with 400 kg of oat flour. The slurry was heated to various temperatures for various minutes to allow hydrolysis, as shown in the table below.

[0131] After hydrolysis, the hydrolyzed slurry was decanted after an inactivation process at 85°C for 15 seconds. The separated liquid stream was then cooled to 10°C. The liquid stream is a plant-based food component, also referred to here as the oat concentrate base.

[0132] The oat concentrate base was diluted with water, and salt (0.08% w / w) and rapeseed oil (0.8% w / w) were added. The resulting oat beverage was subjected to UHT treatment using methods known in the art and aseptically filled. The viscosity of the oat beverage was analyzed using an Anton Paar MCR-302 (Anton Paar GmbH, Austria) equipped with a CC27 cone measuring system. Free sugars were measured using high-performance anion exchange chromatography with pulsed amperometry detection (HPAE-PAD). The results are shown in Table 8. Table 8 shows the viscosity, total solids content, and sugar content of the oat beverage.

[0133] [Table 8]

[0134] These results indicate that when using raw starch amylase, incubation temperatures of 25–60°C are sufficient to produce the desired oat beverage on an industrial scale.

[0135] Example 8: Preparation of oat hydrolysate using raw starch-degrading alpha-amylase of SEQ ID NO: 1 and 0-100 ppm of beta-glucanase. 45 g of oat flour and 255 g of deionized water were weighed directly into a 500 mL flask. The enzyme was added according to the experimental design shown in Table 9. Beta-glucanase was obtained using Ultraflo® Prime.

[0136] [Table 9]

[0137] The suspension was heated to 60°C. Once the target temperature of 60°C was reached, the reaction mixture was stirred at 300 rpm or for 120 minutes. To terminate the enzymatic activity, the suspension was heated to 90°C 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 by centrifugation at 3 × 1200 g. After separation, the oat base was weighed and placed in an ice bath.

[0138] The samples were equilibrated to room temperature, and sunflower oil and sodium chloride were added to final concentrations of 1% and 0.08%, respectively, to formulate the oat beverage. The samples were then homogenized using Thermomix™ 6 (Vorwerk, Wuppertal, Germany). The viscosity of the oat beverages was then analyzed using a Hamilton Microlab™ STAR™ Liquid Handler (Hamilton Robotics Inc. / Hamilton Bonaduz AG) and a method similar to that described in International Publication No. 2011 / 107472 (which is incorporated herein by reference in its entirety). Viscosity data were multiplied by -1 to reach positive values, and higher values ​​correlate with higher viscosity. The amount of beta-glucan in the samples was quantified according to Application Note 64538 (Thermo Scientific, Waltham, Massachusetts, USA) and measured using Gallery™ Plus Beermaster Discrete Analyzer (Thermo Scientific). Table 10 provides the viscosity and amount of beta-glucan in oat beverages.

[0139] [Table 10]

[0140] Sample 1 has a lower viscosity in the final oat beverage compared to sample 5. However, a similar viscosity to sample 1 is achieved in samples 2-4 without completely decomposing or hydrolyzing the beta-glucan. Beta-glucan cannot be detected in sample 1.

Claims

1. A method for obtaining plant-based food ingredients for dairy product substitutes, (a) To obtain a slurry of plant material in water, (b) Adding raw starch-degrading alpha-amylase and optionally additional enzymes to the slurry of step (a) and maintaining it at a temperature of 25 to 60°C to obtain hydrolyzed plant material. Includes, The hydrolyzed plant material is a plant-based food ingredient for dairy alternative foods. method.

2. (c) Separating the hydrolyzed plant material into a solid flow and a liquid flow, (d) Recovering the liquid stream as a plant-based food component for dairy alternative foods, (e) optionally inactivating the enzyme before or after step (c) or (d), The method according to claim 1, further comprising:

3. The method according to claim 1 or 2, wherein the raw starch-degrading alpha-amylase is a GH13 family amylase containing a carbohydrate-binding module (CBM) that preferentially binds to starch.

4. The method according to any one of claims 1 to 3, wherein the raw starch-degrading alpha-amylase contains 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% the same amino acid sequence as SEQ ID NOs: 1, 2, 3, or 4.

5. The method according to any one of claims 1 to 4, wherein the additional enzyme is glucoamylase, maltose-producing amylase, beta-amylase, protease, hemicellulase, cellulase, pectin-degrading enzyme, glucosidase, glucanase, xylanase, arabinofuranosidase, pullulanase, and / or lipase, or any combination thereof.

6. The method according to any one of claims 1 to 5, wherein the additional enzyme is beta-glucanase.

7. The method according to any one of claims 1 to 6, wherein the temperature in step (b) is 25 to 55°C, 30 to 55°C, 35 to 55°C, 40 to 60°C, 30 to 50°C, or 40 to 55°C.

8. The method according to any one of claims 1 to 7, wherein the plant material is derived from corn, rice, barley, wheat, quinoa, oat, rye, flax, hemp, buckwheat, milo, millet, sago, cassava, tapioca, potato, sweet potato, pea, bean, cashew, macadamia, sesame, coconut, banana, jackfruit and / or breadfruit.

9. The method according to any one of claims 1 to 8, wherein the plant material is a grain flour or hulled grain comprising 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 claims 1 to 9, wherein the plant material is oat flour, oat flakes, oat bran, oat crumbs, or any combination thereof.

11. The method according to any one of claims 1 to 10, wherein the dairy substitute is a plant-based beverage, plant-based ice cream, plant-based creamer, plant-based yogurt, or plant-based cheese.

12. Use of raw starch-degrading alpha-amylase in the hydrolysis of plant materials for the production of plant-based food components for dairy alternative foods.

13. A method for obtaining oat hydrolysate food ingredients for dairy product substitutes, (a) To obtain a slurry of oat material in water, (b) Supplying raw starch-degrading alpha-amylase and beta-glucanase to the slurry of step (a), and maintaining it at a temperature of 25-60°C, 25-55°C, 45-55°C, or 50-55°C to obtain oat hydrolysate, (c) Separating the oat hydrolysate into a solid stream and a liquid stream, (d) Recovering the liquid flow, (e) optionally inactivating the enzyme before or after step (c) or (d) Includes, The recovered liquid stream is an oat hydrolysate food ingredient for dairy alternative foods. method.

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

15. The method according to claim 13 or 14, wherein the raw starch-degrading alpha-amylase contains 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% the same amino acid sequence as SEQ ID NOs: 1, 2, 3, or 4.

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