Method for increasing output of plant products

Abstract

FIELD: food industry.SUBSTANCE: method for producing a plant-based beverage is proposed, comprising: wet milling in an aqueous medium of plant-based raw material to obtain a raw material suspension; sifting the raw material suspension to obtain primary milk and a fibrous suspension; grinding the fibrous suspension; treating the fibrous suspension with a protease selected from the group consisting of a metalloendoprotease and a trypsin selected from the group consisting of bacterial trypsin and fungal trypsin; treating the fibrous suspension specified protease at a temperature below the specified operating temperature for the specified protease to obtain a processed fibre suspension; and sifting the processed fibre suspension to obtain secondary milk and clean fibre. A method is proposed for reducing the viscosity of the fibrous suspension.EFFECT: preservation of the quality of the extracted material, including beta-glucan and protein, by using low temperatures and minimal protease activity and digestion time during extraction.22 cl, 10 dwg, 28 tbl, 24 ex

Description

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 151,321, filed February 19, 2021, which is incorporated herein in its entirety.TECHNICAL FIELD

[0002] The present invention relates to a method for increasing the yield of plant nutrients in the production of plant-based foods and beverages, including plant-based milk.BACKGROUND

[0003] When producing plant-based milks from grains, nuts, or seeds, a certain percentage of the plant material may be discarded as waste. This waste may consist of a viscous, insoluble, fibrous concentrate or slurry, which, even after dilution, is difficult or impossible to pass through a mesh filter during plant-based milk processing. The fibrous slurry consists primarily of fibrous cell wall material, such as bran and seed coats, which often contain valuable nutrients. This material may contain beta-glucan, protein, and bioactive phenols and antioxidants, which are present in large quantities in the cell wall. Therefore, it is highly desirable to utilize the fibrous slurry rather than discard it.

[0004] Currently, bran or bran-like material is primarily used as a low-value ingredient for human and animal consumption. This relatively low use as a food ingredient is due to the sensory properties and texture of bran and bran extracts, as well as the low efficiency of nutrient extraction methods. For example, bran extracts can have a bitter taste due to the presence of certain protein or lipid breakdown products that become rancid upon oxidation and incompatibility with some food matrices.

[0005] To utilize fiber material from grain, it is often necessary to further process the material to disrupt the interactions between fibers to reduce viscosity and improve texture. These interactions can be disrupted by treatment with enzymes that break down and separate cell wall material. For example, in the processing of wheat, enzymes including cellulase, hemicellulase, lipase, protease, amylase, and xylanase were used to facilitate the separation of grain material (WO2008132238A1). Cellulase, hemicellulase, and xylanase are known to act directly on the main components of cell walls, promoting disintegration. Amylase, lipase, and protease act on starch, fat, and proteins, which are not the main components of the cell wall, but their enzymatic degradation can disrupt some interactions between cell wall components.

[0006] In the brewing industry, xylanase is used to break down the cell wall material of grains used in brewing to improve processing and increase yield (Novozymes®, 2013). Xylanase breaks down xylan, the main component of the cell wall material of grains. The protease Neutrase® can be used in combination with xylanase during the brewing process to increase the amount of free amino nitrogen (FAN) released during protein hydrolysis so that yeast can utilize FAN to promote growth (Novozymes®, 2013). Neutrase® can also be hypothesized to break down proteins that are part of the matrix, which may contribute to cell wall stability (Novozymes®, 2013). Although xylanase, cellulase and hemicellulase are effective in reducing the viscosity of fibrous grain waste, they can have undesirable side effects in the final food product and during further processing, including the production of sugar as a by-product.Lipase, amylase, and protease can also have undesirable effects on the final product, but these effects may differ from those caused by xylanase, cellulase, and hemicellulase.

[0007] Proteases themselves are not typically used to reduce the viscosity of grain material. In a publication for the brewing industry, Novozymes®, a leading manufacturer of commercial enzymes, lists xylanases, cellulases, hemicellulases, and beta-glucanases, as well as alpha-amylases, for use in reducing viscosity, and Neutrase® is listed for use in enhancing fermentation through protein digestion (Novozymes® Brewing Manual, p. 40). Novozymes® markets Neutrase® for use in oat processing under its product “Neutrase® for Oats,” stating that “Neutrase® is a high-quality, broad-spectrum endoprotease that provides gentle hydrolysis. It can be used to improve protein solubility.” (Novozymes®, 2021). Novozymes® lists the operating temperature range of Neutrase® for oats as 30-65°C and an operating pH range of 6-9 (Novozymes®, 2021).BIOCAT, an enzyme supplier, discloses in its product information sheet that NEUTRAL PROTEASE L (a variant of Neutrase®) “reduces the viscosity of fish or chicken by-products” (BIOCAT, 2019). BIOCAT discloses a temperature range of 30°C to 70°C, with an optimum temperature of 55°C. BIOCAT discloses a pH range of 5.5-9.0, with an optimum pH of 6.5. BIOCAT discloses that the application rate for typical hydrolysis varies depending on the application, with a typical range of 0.1% to 1.0% (BIOCAT, 2019).

[0008] While the use of protease to reduce the viscosity of grain material is not common, protease extraction, which separates and purifies nutrients, is a well-known method for increasing nutrient yields from fibrous plant material, including grain. Protease extraction typically involves endoproteases, which cleave peptide bonds in proteins. However, peptide bond cleavage during protease extraction can compromise the functionality of the protein and other nutrients. For example, native proteins or proteins close to their native state may have improved organoleptic properties and foaming ability, among other properties.

[0009] The effect of protein hydrolysis on protein functionality depends largely on the hydrolysis conditions, including pH, temperature, hydrolysis duration, enzyme choice, and enzyme and substrate concentration. (Wouters et al. 2016). Some studies show that protein hydrolysis has a negative effect on gel strength compared to intact protein (Lamsal et al., 2007; Fan et al., 2005; Pinterits and Arntfield, 2007).

[0010] The flavor and aroma aspects of protein hydrolysates influence the quality of finished products. In the case of soy proteins, a problematic flavor is the beany or grassy taste of hydrolysates (Rackis et al., 1979; Wansink and Chan, 2001; Wansink and Park, 2002; Damodaran and Arora 2013). Furthermore, protein hydrolysis often causes bitterness (Guigoz and Solms, 1976; Maehashi and Huang, 2009).

[0011] Protease treatment is typically carried out at temperatures between approximately 30°C and 65°C, where enzyme activity is optimal or near optimal, although this can vary depending on the enzyme, enzyme source, and application. As disclosed above, Novozymes® specifies the operating temperature range of Neutrase® for Oats™ as 30-65°C and an operating pH range of 6-9 (Novozymes®, 2021), with the optimal temperature for activity being approximately 42°C. According to Novozymes® guidance, the optimal pH for Neutrase® treatment is approximately 6, with activity rapidly dropping to 0 at a pH of approximately 4.3.

[0012] Regarding the temperature used during Neutrase® grain processing, U.S. Patent No. 4,377,602 issued to Conrad describes a method for producing a hydrolyzed product from milled whole grain using protease. Conrad's method produced a product containing protein and sugar from a grain slurry by converting water-insoluble proteins into water-soluble products. After 1 hour at 50°C, according to Conrad, all the protein had been converted into water-soluble products. These conditions resulted in a product with a relatively high degree of hydrolysis and protein denaturation. This resulted in milk with a lower viscosity, which had an inferior texture and flavor.

[0013] Although protease extraction of protein and other nutrients from fibrous material has been effective, it has limitations. Protease treatment typically hydrolyzes protein to a certain extent, breaking down intact protein into smaller fragments. This can affect protein functionality, imparting a bitter taste and affecting its emulsifying properties, digestibility, and viscosity. Furthermore, protease extraction from bran and other insoluble material is carried out at relatively high temperatures, typically ranging from approximately 30°C to 65°C, and typically for extended periods of time. Proteases, like most enzymes, have an optimal time and temperature range within which they are effective. For proteases used to extract nutrients from bran, the time and temperature typically range from 30°C to 65°C and from 1 to 24 hours.

[0014] U.S. Patent Publication No. 20150257411 to Janse also discloses the use of a Neutrase®-like protease for extracting protein from fibrous rice bran. Janse increased the protein yield from rice bran while limiting the degree of protein hydrolysis to maintain the overall molecular weight of the hydrolysate above 500 kDa. Janse proposes incubating the protease with rice bran for approximately 1-4 hours at a temperature of 45°C to 65°C or, more preferably, at an incubation temperature of 48°C to 55°C, with optimal metalloendoprotease extraction at a pH of 7.0 and at 50°C. Janse claimed a relatively low degree of hydrolysis (DH) of 10 to 16% for the claimed process.

[0015] Similarly, U.S. Patent No. 8,575,310 to Hettiarachchy describes the extraction of limited hydrolysis protease from rice bran, where reaction conditions were optimized at 50°C for 1 hour at a pH of 8.0. Janse and Hettiarachchy reported relatively low DH, typically between approximately 10 and 25%. U.S. Patent No. 5,716,801 to Nielsen describes the use of protease to produce flavorful and organoleptically acceptable protein hydrolysates from plant proteins. Nielsen discloses a DH of 15 to 35% and suggests protease treatment at 55°C for 18 hours, where the pH is 8.5 for the Alcalase treatment and optimally 7.0 for the Neutraze treatment. The protease was inactivated to stop proteolysis after 18 h by lowering the pH to 4.2 with 30% HCl.

[0016] In the article entitled "Protease technology for obtaining a soy pulp extract enriched in bioactive compounds: isoflavones and peptides," Orts describes a method for extracting bioactive components from soy pulp, which is typically discarded as waste (Orts et al. 2019). Orts developed a process for extracting specific nutrients, including protein fragments and isoflavones, from soy pulp using protease. Orts specifies optimal protease extraction conditions of approximately 2 hours at 55°C.

[0017] Hanmoungjai et al. (2001) in the article entitled “Enzymatic Process for Extracting Oil and Protein from Rice Bran” disclose a method of enzymatic extraction of oil and protein using a commercial protease (Alcalase). Hanmoungjai indicates the extraction conditions: 1-3 hours and 40-60°C, respectively. In the article entitled “Effect of hydrolyzing enzymes on wheat bran cell wall integrity and protein solubility”, Arte et al. (2016) described a method of treating wheat bran with protease to extract proteins. Arte proposes an optimized protease treatment for 3 hours at 35°C. Santo Domingo et al. (2015) disclose a method of treating insoluble plant fiber waste with protease, which extracts fiber. Santo Domingo offers optimal protease conditions at 40°C for 5 hours.

[0018] Abdulkarim et al. (2006) in an article entitled "Use of Enzymes to Enhance Oil Recovery During Aqueous Extraction of Moringa Oleifera Seed Oil" disclose a method for extracting protease from Moringa Oleifera seeds. Abdulkarim describes optimal protease extraction using Neutrase at 45°C for 2 hours of incubation at pH 6.8.

[0019] In a literature review on plant nutrient protease extraction, Yussof et al. (2014) described the general optimum reaction conditions: According to Rui et al. (2009), the optimum temperature range for enzymatic hydrolysis is 40–55°C, so many authors use AEE (aqueous enzymatic extraction) temperatures that fall within this range. In practice, it is often preferred to use the lowest possible temperature that ensures adequate activity (Passos et al. 2009). In the case of olive fruit, a lower temperature of 30°C has been shown to be beneficial, especially for preserving oil quality (Aliakbarian et al. 2008; De Faveri et al. 2008; Ranalli et al. 2003; Garcia et al. 2001; Ranalli et al. 1999). Gros et al. (2003) also used a temperature of 34°C for the same reason when extracting flaxseed oil. A significant effect of temperature on oil yield was reported by Sharma et al.(2002), where the highest peanut oil yield was observed at 40°C, but it decreased significantly as the temperature decreased to 37°C.

[0020] Yussof et al. (2014) also noted that incubation time is another factor that can limit the enzymatic extraction of nutrients from plant material, as longer incubation times can negatively affect the quality of nutrients extracted from the plant material: According to Jiang et al. (2010), Abdulkarim et al. (2006), Santos and Ferrari (2005), and Dominguez et al. (1996), the degradation of cell wall components can be enhanced by increasing the incubation time. Passos et al. (2009) also reported that the application of a mixture of cellulase, protease, xylanase, and pectinase enzymes for 120 hours resulted in a 3.8% increase in yield compared to 24 hours of incubation. However, this time duration (i.e. 120 h) is too long to be acceptable in practice (Passos et al. 2009) and may result in a reduction in oil quality (Jiang et al.2010), resulting in high energy consumption and production of undesirable products (Abdulkarim et al. 2006).

[0021] In a review, Mwaurah et al. (2020) compared known methods for extracting oil from plant material, including seeds. Regarding enzymatic extraction, Mwaurah states that "research shows that enzyme-to-substrate ratios of 1% to 8%, temperatures of 40 to 55°C, and pH of 4 to 8 are typical for enzymatic oil extraction from various oilseed crops." (Mwaurah et al. 2020).

[0022] According to Mwaurah, oil extraction from grain depends on proteolytic activity, and proteolytic activity is sensitive to temperature and pH. Mwaurah writes that due to the temperature sensitivity of proteases, "[t]emperature is one of the critical factors for any oil extraction method."

[0023] Protease extraction has also been used specifically to extract beta-glucan from grains such as oats and barley, as well as other sources of beta-glucan. Beta-glucans are found in grains, including oats and barley, as well as in bacteria, fungi, yeast, algae, and lichens. Beta-glucan is used in several areas, especially in functional foods. Beta-glucan has been shown to have medicinal benefits, particularly in relation to immunity and cholesterol reduction.

[0024] Beta-glucan is an important structural component of the cell wall of cereal grains and is typically difficult to extract from these plant products. Conventional beta-glucan extraction methods involve the use of acid or alkaline solutions, which often leads to the breakdown of the beta-glucan polymer, thereby reducing its biological activity and, consequently, health benefits. “To avoid alkaline-acid methods, which are believed to destroy beta-glucans, some researchers have introduced enzymatic extraction as an alternative to treatment with strong chemical solvents.” (Avramia and Amariei, 2021). However, protease treatment to extract beta-glucan from cell wall material typically uses long processing times and high temperatures, such as treating the cell walls for 5 hours at pH 10.5 and 45°C, followed by successive washes of the precipitate with acetone or ethanol. (Avramia and Amariei, 2021).However, these conditions will lead to significant protein degradation, and are only acceptable for beta-glucan extraction purposes, as protein degradation is not a concern. In cases where the extraction of native beta-glucan and native protein, such as plant milk, is desired, conventional protease-based beta-glucan extraction methods are undesirable.

[0025] The above references demonstrate that extracting nutrients from plant material using protease under either optimal or standard conditions can negatively impact the quality of the extracted nutrients. Protease treatment under either optimal or standard conditions can cause protein degradation, which can reduce protein functionality. Furthermore, protease treatment under standard conditions uses temperatures that promote rapid microbial growth. Furthermore, standard protease reaction incubation conditions increase lipase activity, causing oil oxidation and negatively impacting flavor. Furthermore, protein structure can be altered by heat or high DH levels under standard protease reaction conditions, affecting protein functionality.Thus, when extracting protease nutrients from plant material, lower temperatures and shorter incubation times are desirable, especially for use in applications such as plant milk.SUMMARY OF THE INVENTION.

[0026] The present invention solves the problems associated with traditional protease extraction methods by dramatically reducing the temperature, incubation time, and proteolysis during protease extraction. The present invention relates to protease treatment for increasing the yield of plant or other material by extracting nutrients from a portion of the fibrous waste of ground plant material while maintaining the nutritional and functional qualities of the extracted material for use as a food product. The said method preserves the quality of the extracted material, including beta-glucan and protein, by using low temperatures and minimal protease activity and digestion time during extraction. In some embodiments, the method of the present invention is used in combination with wet milling in an aqueous medium to produce milk or liquids based on plant or microbial origin.In some embodiments of the present invention, the overall nutrient yield from raw grain can be increased by about 5-10% or more, and for specific desired nutrients, including beta-glucan in oats, can result in an increase in nutrient yield by about 80% or more, thereby providing a yield close to about 80% or more for total beta-glucan from the grain in the final product.

[0027] In one embodiment, oat grain is wet-milled in an aqueous medium and filtered at a low temperature to produce primary plant milk. After filtration, a fiber suspension or retentate is separated from the primary milk. The fiber suspension has a viscosity and texture that, even after dilution, prevents the material from passing through a mesh filter. After filtration, which may also be referred to herein as sifting, the fiber suspension, which may contain approximately 40% total solids, can then be diluted to a total solids content of approximately 5-15% and briefly milled. In one embodiment, the diluted fiber retentate or fiber suspension is maintained at a low temperature, just above 0°C, throughout the process. The fiber suspension can then be transferred to a tank and maintained at a low temperature.The diluted fiber suspension can then be treated with a protease, which may preferably be Neutrase® or its equivalent, or microbial trypsin, for a reaction at low temperatures of approximately 0° to 5°C. The low-temperature reaction, in which proteolytic activity is insignificant or undetectable, protects the native protein structure, thereby preserving the functionality of the native protein.

[0028] Surprisingly, the addition of Neutrase® to the fiber suspension results in a rapid and significant reduction in viscosity. The viscosity reduction achieved with Neutrase®, which can be added at or below standard application rates, is significant and unexpected given the low temperature of the substrate fiber suspension. In some embodiments, the viscosity reduction after the enzymatic reaction in less than ten minutes is sufficient to enable filtration of the retentate, yielding a commercially viable secondary plant milk. Without enzyme treatment, the diluted fiber suspension remains highly viscous and slimy, making it impractical for most applications, including plant milk production.

[0029] Neutrase® treatment followed by filtration produces secondary milk. In some embodiments, secondary milk may contain approximately 10% of the total dry matter of the raw grain material. Given the low cost of the present method, 10% represents a significant and commercially significant increase in yield.

[0030] Before recycled milk can be packaged, it must first undergo heat treatment to inactivate the protease or any other enzymes that may be used during processing in addition to the protease. Heat treatment typically involves rapid heating to a temperature to denature the enzyme, which in this case can be approximately 75°C to 90°C. Rapid heating during enzyme inactivation prevents significant protease activity and proteolysis during the deactivation step and limits thermal denaturation of protein and microbial growth. Heat treatment for enzyme inactivation may in some cases be followed by a second heat treatment to prevent microbial growth in the final product after packaging.

[0031] During the heat inactivation step, the viscosity of the cereal product typically increases due to starch gelatinization or other interactions. Surprisingly, in the present invention, heating the Neutrase®-treated fiber slurry to inactivate the enzyme did not cause a significant increase in viscosity. Gelatinization occurs when products containing starch granules are heated to temperatures that disrupt the molecular bonds in the starch, leading to water absorption and an increase in viscosity. Although the amount of starch in the oat fiber slurry of the present invention is relatively low compared to virgin oat milk, gelatinization was expected to cause a significant increase in viscosity, leading to this unexpected result.

[0032] The unexpected lack of a significant increase in viscosity during protease inactivation has important implications for the further processing of secondary milk according to the present invention. Amylase is typically added to plant-based milks during liquefaction. Amylase is typically needed to break down starch during gelatinization, thereby reducing viscosity and liquefying the product. Liquefaction allows plant-based milks to be processed at high temperatures without clogging the processor pipes.

[0033] The relatively low viscosity of heat-inactivated recycled milk after treatment with Neutrase® and enzyme deactivation allows for complete reprocessing of recycled milk without the use of amylase or other liquefaction enzymes. Eliminating or reducing the need for amylase in the product has significant advantages in terms of cost and consumer demand. Treatment with amylase results in sugar production, as do treatments with many other viscosity-reducing enzymes, which may be undesirable for some products. Amylase treatment can also, in some cases, have a negative impact on taste. Furthermore, there is growing consumer demand for clean-label products, and eliminating an ingredient such as enzymes can improve consumer perception of the product, particularly for plant-based products such as oat milk.

[0034] In some embodiments, the increase in overall milk yield from grains such as oats is approximately 9-10% and from nuts such as almonds, approximately 5%. The method increases the yield of protein, fat, fiber, carbohydrates, and ash from the grain or nut. For grains such as oats, the fiber suspension is particularly rich in beta-glucan, a nutrient with well-known health benefits. In some embodiments, the method of the present invention can increase the yield of beta-glucan by up to 80% or more, while generally preserving the native structure of beta-glucan. Preserving the native structure of beta-glucan is important for maintaining the full functionality and health benefits of the molecule.

[0035] The low-temperature treatment of the present invention also prevents microbial growth, particularly during protease treatment of the fiber suspension. Low temperatures also minimize or eliminate protein proteolysis during protease treatment. Although the chemical mechanism causing the rapid decrease in fiber suspension viscosity is unclear, the degree of hydrolysis after protease treatment is unexpectedly virtually zero or very close to it. The advantages of the present process, including increased raw material yield from plant materials, prevention of microbial growth, minimal proteolysis or alteration of nutrient structure, and elimination of the need for amylase or other enzymes during high-temperature treatment, are significant improvements over existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Fig. 1 is a block diagram illustrating one embodiment of a method according to the present invention.

[0037] Fig. 2 is a diagram illustrating the relative activity of neutral protease L™ as a function of temperature according to one embodiment of the present invention.

[0038] Fig. 3 is a diagram illustrating the relative activity of neutral protease L™ as a function of pH according to one embodiment of the present invention.

[0039] Fig. 4 shows a reducing SDS-PAGE gel showing the size of the protein and the degree of hydrolysis, according to one embodiment of the present invention.

[0040] Fig. 5A is a non-reducing SDS-PAGE gel showing protein size and degree of hydrolysis, according to one embodiment of the present invention.

[0041] Fig. 5B is a reducing SDS-PAGE gel showing the protein size and degree of hydrolysis, according to one embodiment of the present invention.

[0042] Fig. 6 shows a graph of the increase in viscosity of an oat fiber suspension at a low temperature according to one embodiment of the present invention.

[0043] Fig. 7 shows a graph of the change in viscosity of an oat fiber suspension at a low temperature after enzyme treatment according to one embodiment of the present invention.

[0044] Fig. 8 is a graph showing the change in viscosity of an oat fiber suspension at low temperature after treatment with neutral protease L™ at different enzyme concentrations according to one embodiment of the present invention.

[0045] Fig. 9 is a graph showing the change in viscosity of an oat fiber suspension at low temperature after treatment with trypsin at different enzyme concentrations according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0046] In the following description, various embodiments of the present invention will be described in detail. However, such details are included to facilitate an understanding of the present invention and to describe illustrative embodiments of the present invention. Such details should not be used to limit the invention to the specific embodiments described, since other variations and embodiments are possible within the scope of the invention. The contents of all references, patents, and published patent applications cited in this application are expressly incorporated herein by reference. Results may vary slightly from experiment to experiment, as would be expected by one skilled in the art.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this description, including definitions, prevails. Furthermore, the materials, methods, and examples are illustrative and not limiting. All references to percentages are by weight unless otherwise indicated. The details of one or more embodiments are set forth in the accompanying drawings and the description below.Other features, objectives and advantages of the invention will be apparent from the description and graphic materials and from the claims.

[0048] Furthermore, although numerous details are set forth to ensure a thorough understanding of the present invention, it will be apparent to one skilled in the art that these specific details are not required for the practical application of the present invention. In other cases, details such as well-known methods, data types, protocols, procedures, processes, etc., are not described in detail.

[0049] The present invention relates to protease treatment for increasing the yield of plant material by extracting nutrients from a portion of the fiber waste or fiber suspension of ground plant material while maintaining the nutritional and functional qualities of the extracted material for use as a food product. This method preserves the quality of the extracted material by using low temperatures and minimal protease activity and digestion time during extraction. In some embodiments, the method of the present invention is used in combination with a wet milling process in an aqueous medium to produce plant milk. In some embodiments of the method of the present invention, the overall yield of nutrients from wet-milled grain can be increased by approximately 8-10%.

[0050] The method produces a secondary milk product that can be added to a primary milk product, thereby, in some embodiments, increasing the overall milk yield from grains, such as oats, by approximately 9-10%, and from nuts, such as almonds, by approximately 5%. It is believed that these figures can be increased in an industrial setting, where commercial application of this method can be achieved using industrial filtration and milling systems. The method increases the yield of protein, fat, fiber, carbohydrates, and ash from grains or nuts.

[0051] In one embodiment of the method 10, as shown in Fig. 1, wet-milled oat grain is filtered at a low temperature to obtain primary plant milk. This step involves grinding grains, nuts, or seeds by wet milling in cold water to form a primary slurry, followed by sifting the resulting primary slurry through a sieve. The fiber slurry 150 remains on the filter as a retentate. The fiber slurry may also be referred to as a fiber retentate or a fiber fraction. During processing in accordance with the present invention, the fiber slurry 150 can be maintained at a low temperature, slightly above 0°C. The fiber slurry 150 can then be transferred to a tank and maintained at a low temperature.The diluted fiber suspension 150 or retentate is then treated with a protease, which may preferably be NEUTB, which can then be added to the diluted fiber suspension for a reaction at low temperatures of approximately 0° to 5°C. The low-temperature reaction, in which proteolytic activity is insignificant or undetectable, protects the native protein structure, thereby preserving the functionality of the native protein.

[0052] As shown in Fig. 1, raw material 100, which may include grains, nuts, or seeds, is added to cold water 102, the temperature of which may be 7°C, and crushed or ground 104 to reduce the size of the raw material 100. In some embodiments, the size may be reduced to <1 mm at 7°C. Grinding 104 provides a suspension 106 of the raw material. After filtration, the fibrous suspension 150 is separated from the primary milk. The fibrous suspension has a viscosity and texture that prevents the material from passing through a mesh filter.The raw material slurry 106 may be sieved 108 through a No. 60-400 mesh screen, or more preferably through a No. 80-160 mesh screen, or more preferably through a No. 100-140 mesh screen, or more preferably through a No. 120 mesh screen at approximately 10°C to separate the primary milk fraction 110, which may consist primarily of starchy, white, soft endosperm components, from the fibrous slurry fraction 150.

[0053] The sieving step separates the primary milk (filtrate) from the viscous, coarse-grained, and generally insoluble fraction of the primary suspension (retentate). Sieving may also be referred to interchangeably with filtration in the present invention. The primary milk consists primarily of starchy, white, soft components of the endosperm. The viscous retentate likely consists primarily of fibrous-protein aggregates and structural components of the seeds from the aleurone and subaleurone layers, or bran, and portions of the hard, transparent endosperm.

[0054] The fiber suspension, which may contain approximately 40% total solids, can then be diluted with cold water to a total solids content of approximately 5-15% and briefly mixed or ground before enzymatic treatment. The grinding or milling of the raw material 104 of the fiber suspension 150 and the sifting of the raw material 108 are typically carried out at temperatures below the protein denaturation temperature. As a result of sifting the raw material 108, primary milk 110 and fiber suspension 150 are obtained.

[0055] Primary milk 110 may, in some embodiments, be produced and processed in accordance with known methods, examples of which are described in U.S. Patent No. 7,678,403 issued to Mitchell. As shown in Fig. 1, primary milk 110 can be heated 112 to 99°C at a rate of 6°C per minute. In the next step, primary milk 110 can then be rapidly cooled 114 to 71°C. Cooling 114 produces processed primary milk 116.

[0056] In addition to the primary milk 110, sifting the raw material suspension 108, as described previously, results in the formation of a retentate of the fiber suspension 150. The fiber suspension 150, which in some embodiments may consist primarily of bran, is subjected to enzymatic extraction to extract nutrients from the fiber suspension 150. Before treatment with protease, the fiber suspension 150 may be diluted. Extraction with protease 154 in accordance with the present invention involves treating the fiber suspension 150 by adding protease 154, which in some embodiments may be a bacterial or fungal neutral metalloendoprotease or abbreviated "neutral protease" (neutral protease may be interchangeable herein with Neutrase® or neutral protease L™).

[0057] For extraction with protease, cold water 152, typically at a temperature of about 0 to 25°C, can be added to the fiber suspension 150, followed by the addition of neutral protease 154. Then, the fiber suspension 150 containing the protease can be stirred at low speed 156. It is important to note that the extraction with protease 154 is carried out under suboptimal conditions, which are typically below the established operating temperature or pH range for the protease, preferably from 0°C to 15°C, or more preferably from 0°C to 5°C. In addition, in some embodiments, the extraction with protease 154 from the fiber suspension 150 can be carried out in a short time, which in some embodiments can be as little as 10 minutes at 10°C.

[0058] As discussed previously, protease-based nutrient extraction is typically performed under optimal or near-optimal conditions for protease activity. However, optimal protease activity conditions are not optimal for preserving nutrients and plant-based dairy products in their ideal state. Higher temperatures and longer incubation times destroy nutrients, thereby reducing their quality.

[0059] Figure 2 shows the effect of temperature on the activity of neutral protease L (NEUTB) from BIOCAT. As shown in Figure 2, NEUTB is expected to be minimally active at 10°C. In conjunction with this graph, BIOCAT lists a temperature range of 30°C to 70°C with an optimum temperature of 55°C. Figure 3, also published by BIOCAT, shows that NEUTB is expected to be essentially inactive at pH<5.0. In conjunction with this graph, BIOCAT lists a pH range of 5.5–9.0 with an optimum of pH 6.5. Novozymes® has published similar activity data for Neutrase®. Thus, based on the data presented, and without being bound by theory, it can be hypothesized that significant atypical protease activity may occur, causing extraction and a corresponding increase in nutrient yields under extremely suboptimal conditions.This atypical activity may include the destruction of cellular structures by means other than the hydrolysis of large protein molecules into smaller molecules via protease activity.

[0060] Figure 4 shows the effect of protease extraction according to the present invention on the molecular structure of oat protein from a fiber suspension at high and low temperatures. These temperature conditions correspond to the conditions under which the samples shown in the SDS-PAGE gel in Figure 4 were processed, as shown and further described in detail in Example 6 and Table 10.

[0061] Figures 5A and 5B also show the effect that protease extraction according to the present invention has on the molecular structure of oat protein from fiber suspension under different conditions. The data in Table 13 are taken from the data in Figures 5A and 5B, which show SDS-PAGE of oat fiber suspension samples digested with protease according to the present invention. Shown are test samples and a control, where the test samples were treated with different proteases or alpha-amylases under reducing and non-reducing conditions. Lane 162 was treated with ALKP, lane 264 was treated with NEUTB, lane 391 was treated with TRY1, lane 527 was treated with PAPN, lane 650 is an enzyme-free control, and lane 903 was treated with AAMY. The data in Figure 5A and 5B are discussed in more detail in Example 9, and the data are shown in Table 13.The degree of hydrolysis (DH) was calculated as described previously and SDS-PAGE was performed generally as described earlier in this paper.

[0062] Figure 6 shows the changes in the viscosity of an oat fiber suspension depending on the storage time at 2°C. After wet grinding or wet milling and initial filtration through a mesh, the fiber suspension, which is the retentate, becomes more viscous over time during storage. The data shown in Figure 6 relates to an oat fiber suspension stored at 2°C, since this temperature is the preferred temperature for preventing microbial growth and maintaining the nutrient structure before and during protease treatment in accordance with the present invention. Typically, the method tested herein was one in which the viscosity reached its plateau before the addition of the protease, although other embodiments are contemplated within the scope of the present invention.

[0063] Figure 7 shows the changes in viscosity of an oat fiber suspension treated with various proteases after storing the fiber suspension at 2°C for 100 min before protease treatment at 2°C. In the legend in Figure 7, 1=NONE709, 2=BRML652, 3=FLZM137, 4=TRY5595, 5=AAMY711, 6=ALKP270, 7=TRY1790, 8=NEUTN570, and 9=NEUTB352. Figure 7 shows illustrative test samples from a larger data set that are described in more detail in Example 10 and Table 14 below. Example 10 discloses the effect of a large number of proteases on the viscosity of an oat fiber suspension. Table 14 shows the changes in relative viscosity of oat fiber suspension treated with various enzymes at 2°C. A decrease in viscosity is the main factor contributing to the fiber suspension treatment and generally correlates with an increase in yield in accordance with the present invention.

[0064] Fig. 8 shows the changes in viscosity of the oat fiber suspension treated with NEUTB at different protease concentrations after storing the fiber suspension at 2°C for 100 min before the protease treatment at 2°C. In the notation in Fig. 8, 1=NEUTB0005, 2=NEUTB0025, 3=NEUTB005, 4=NEUTB01 and 5=NEUTB05. The data in Fig. 8 are discussed in more detail in Example 11 and Table 15, which disclose the relative changes in viscosity of the oat fiber suspension treated with neutral protease L (NEUTB) at different enzyme concentrations at 2°C.

[0065] Fig. 9 shows the changes in viscosity of the oat fiber suspension treated with microbial trypsin at different protease concentrations after storing the fiber suspension at 2°C for 100 min before the protease treatment at 2°C. In the notation in Fig. 9, 1=TRY1005, 2=TRY10025, 3=TRY1005, 4=TRY101, and 5=TRY105. The data in Fig. 9 are discussed in more detail in Example 12 and Table 16, which disclose the relative changes in viscosity of the oat fiber suspension treated with TRY1 at different enzyme concentrations at 2°C.

[0066] Taken together, the data show that, unexpectedly, the addition of certain proteases to the fiber suspension 150 results in a rapid and significant reduction in viscosity at very low temperatures and extreme pH values. The viscosity reduction due to NEUTB, which can be added at or below the standard application rate, is significant and unexpected given the low temperature of the fiber suspension 150 substrate. In some embodiments, the viscosity reduction after less than ten minutes of enzymatic reaction is sufficient to filter the retentate, producing a secondary plant milk with a unique nutritional profile. Without enzyme treatment, the diluted fiber suspension remains highly viscous and slimy and, from a practical standpoint, unsuitable for most applications, including plant milk production.

[0067] It has surprisingly been found that, according to the method of the present invention, a large part or a substantial part of the nutrients present in the fiber suspension 150 can be efficiently extracted at low temperatures under suboptimal or severely suboptimal conditions of protease activity, as defined herein; conditions under which the protease is expected to be minimally active or completely inactive. Incubation temperatures according to the present invention can range, in one embodiment and without limitation, from 0°C to 25°C, although lower temperatures may be preferred. Incubation times during low-temperature extraction can vary, in one embodiment and without limitation, from 1 minute to 1 hour, or more preferably from 2 minutes to 30 minutes.Protease treatment at pH below the expected minimum of protease activity, such as below pH 5.0, also unexpectedly resulted in the extraction of significant amounts of nutrients from the 150 fiber suspension, even when combined with very low temperatures.

[0068] In some embodiments, the rapid reduction in viscosity at 2°C caused by treatment with NEUTB or trypsin 154 in accordance with the present invention allows for rapid combined processing of multiple batches of fiber suspension 150 collected at different times during commercial processing. The short incubation time at low temperatures for treatment with protease 154 in accordance with the present invention prevents microbial growth while earlier batches of fiber suspension 150 are stored and ensures a rapid reduction in viscosity when combined batches are processed prior to high-temperature processing for long-term storage or aseptic products.

[0069] Proteases, like many enzymes, can catalyze more than one type of reaction. Some secondary activities occur under different conditions and may have a different operating temperature and pH range than the enzyme's primary activity. For example, many proteases are known to possess plastin-forming activity in addition to protease activity (Sun et al. 2021; Xu et al. 2014). Without being limited by theory, it is possible that Neutrase® and trypsin possess secondary activities that are responsible for the observed rapid decrease in viscosity at low temperatures and low pH.

[0070] Protease extraction followed by minimal to moderate protein digestion / hydrolysis. In some embodiments, protease treatment can be combined with other enzymes, such as amylase, to effectively and thoroughly hydrolyze and dissociate proteins from tightly bound other seed structural components, such as cell wall polysaccharide. Optionally, amylase or a mixture of amylases can be added to the fiber suspension 150.

[0071] In some embodiments, the fiber suspension 150 can be diluted with cold water 1-2X 152 before treatment with the protease 154. In some embodiments, to deactivate the enzyme, the fiber suspension 150 can be heated from 160 to 99°C, or in some embodiments to a temperature of 75°C to 99°C at a rate of 6°C per minute. In some embodiments, thermal inactivation can be carried out by direct or indirect steam treatment for rapid inactivation. The fiber suspension 150 can then be rapidly cooled to 82°C to obtain the treated fiber suspension 164. In some embodiments, heating 160 can be rapid, so that the enzyme is deactivated substantially without significant incubation time in the temperature range at which the protease is active. In some embodiments, this may be achieved by steam heating, which may include steam injection or direct and indirect steam heating.Alternative methods of rapid heating, including microwave radiation, may also be used, as known to one skilled in the art.

[0072] The processed fiber suspension 164 can then be sieved 166 through a No. 60-400 sieve to obtain processed secondary milk 170, which may also be referred to in the tables as secondary milk or 2nd milk, and clean fiber 168. Clean fiber 168 obtained as a result of this method may be substantially free of macronutrients such as proteins and fats, and may consist primarily of insoluble fibers. Clean fiber 168 is a by-product of the method of the present invention and may have value in the food industry and other applications.

[0073] The processed secondary milk 170 can then be combined with the processed primary milk 116 to obtain the combined milk 120 or the combined milk product 120. Alternatively, the processed primary milk 116 and the processed secondary milk 170 can be used separately.

[0074] The processed secondary milk 170 obtained from the fiber suspension 150 according to the method of the present invention contains a significant amount of protein, fat, ash, and carbohydrates found in the fiber suspension. Separation of the proteins, fiber, fat, and carbohydrates in the fiber suspension 150 leads to an increase in yield by allowing these components to disperse and dissolve in water, thereby forming the processed secondary milk 170. In addition, the reduction in viscosity caused by the protease can ensure an increase in the flow of nutrient material through the mesh during sifting 166, which also leads to an increase in the yield of nutrients in the processed secondary milk 170.

[0075] Processed secondary milk 170 can be combined with processed primary milk 116 or used separately. When combining processed primary milk 116 and processed secondary milk 170, the combined milk 120 has a higher yield, in some cases improved functionality, and additional nutrients, which may be present primarily in the fibrous portions of the grain and in the fibrous clusters of the nuts. The examples and tables below demonstrate that the protein hydrolysis process before milk extraction, as described by Conrad, significantly improved oat yield compared to the mechanical process alone.

[0076] The milk produced according to the present invention did not have a bitter taste, while the plant-based milk produced by the Conrad method had a bitter aftertaste. Furthermore, the plant-based milk produced by the Conrad method had reduced foaming performance and foam stability compared to the mechanical wet milling process and the method of the present invention.

[0077] The method of the present invention improved milk yield in all products tested, although the effect was greater for some materials. Without being limited by theory, the method of the invention appears to effectively separate most of the soluble proteins of small and medium molecular weight into the primary milk fraction and separate proteins tightly bound to cell wall components in the aleurone and subaleurone layers into the secondary milk. Thus, the present invention significantly improves milk yield compared to the prior art while minimizing the undesirable effects of treating all ground plant material with protease.

[0078] Furthermore, the present invention limits the formation of free amino acids and peptides, as well as low-mass protein molecules that create undesirable organoleptic characteristics in products. The present invention can prevent the interaction of primary, secondary, and tertiary reactants (i.e., browning) with other components in seeds. Furthermore, the method according to the present invention provides a by-product of pure fiber 168, which can be used in food products and other applications.

[0079] Additional advantages of the method according to the present invention include short processing times for extraction, which increases cost-effectiveness in industrial settings. Furthermore, low temperatures and low pH prevent microbial growth during processing.

[0080] In one embodiment, the present invention, especially with neutral proteases effective in the present invention, can be effective with substrates with low or high pH, ​​such as oxidized oat grain. During oat processing, oats that have been stored longer tend to oxidize and therefore have a lower pH, which can lead to a total pH decrease of 1 unit. Most of the grains are active, or some enzymes are still active in inactive grains, which leads to reactions that reduce the pH of the grain. In addition, pH adjustment can occur during processing for various reasons, and the effectiveness of the present invention at low and high pH can be beneficial in certain embodiments. In some embodiments, waste that can be processed according to the present invention, such as spent barley grain or other waste that has a higher or lower pH.

[0081] In some embodiments, the effective temperature range for the protease reaction according to the present invention may be between 0°C and the upper denaturation temperature of the proteases that are effective in the present invention. In some embodiments, the effective temperatures for the protease reaction according to the present invention may be between 0°C and the upper activity range of the proteases that are effective in the present invention.

[0082] In some embodiments, effective temperatures for the protease reaction according to the present invention may be suboptimal temperatures, wherein suboptimal temperatures are defined as values ​​below the suggested range specified in publications of protein suppliers or other publications, or that are expected to be used by those of ordinary skill in the art. In some embodiments, an effective temperature range for the protease reaction according to the present invention may be from 0°C to 80°C, or from 0°C to 70°C, or from 0°C to 60°C, or from 0°C to 50°C, or from 0°C to 40°C, or from 0°C to 35°C, or from 0°C to 30°C, or from 0°C to 25°C, or from 0°C to 20°C, or from 0°C to 15°C, or from 0°C to 12°C, or from 0°C to 10°C, or from 0°C to 9°C, or from 0°C to 8°C, or from 0°C to 7°C, or from 0°C to 6°C, or from 0°C to 5°C, or from 0°C up to 4°C, or from 0°C to 3°C, or from 0°C to 2°C, or from 0°C to 1°C.

[0083] In some embodiments, the effective temperature range for storing the materials used in accordance with the present invention, if the materials are not intentionally heated to inactivate enzymes or reduce microbial activity, may be from 0°C to 50°C, or from 0°C to 40°C, or from 0°C to 35°C, or from 0°C to 30°C, or from 0°C to 25°C, or from 0°C to 20°C, or from 0°C to 15°C, or from 0°C to 12°C, or from 0°C to 10°C, or from 0°C to 9°C, or from 0°C to 8°C, or from 0°C to 7°C, or from 0°C to 6°C, or from 0°C to 5°C, or from 0°C to 4°C, or from 0°C to 3°C, or from 0°C to 2°C, or from 0°C to 1°C.

[0084] In some embodiments, an effective pH range for the protease reaction according to the present invention may be from about 3.5 to 12, or from about 4 to 12, or from about 4.5 to 12, or from about 3.5 to 11, or from about 4 to 11, or from about 4.5 to 11, or from about 4.5 to 10, or from about 4.5 to 9, or from about 4.5 to 8, or from about 4.5 to 7, or from about 4.5 to 6.5, or from about 5 to 8, or from about 5 to 7, or from about 5 to 6, or from about 6 to 7, or from about 6 to 8.

[0085] In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 10 minutes, or from 2 minutes to 10 minutes, or from 5 minutes to 10 minutes. In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 20 minutes, or from 2 minutes to 20 minutes, or from 5 minutes to 20 minutes. In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 30 minutes, or from 2 minutes to 30 minutes, or from 5 minutes to 30 minutes. In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 45 minutes, or from 2 minutes to 45 minutes, or from 5 minutes to 45 minutes.In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 60 minutes, or from 2 minutes to 60 minutes, or from 5 minutes to 60 minutes. In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 90 minutes, or from 2 minutes to 90 minutes, or from 5 minutes to 90 minutes. In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 1 minute to 120 minutes, or from 2 minutes to 120 minutes, or from 5 minutes to 120 minutes. In some embodiments, the effective incubation period for the protease reaction according to the present invention may be from 10 seconds to 4 hours.

[0086] In some embodiments, effective conditions for the protease reaction of the present invention are conditions that result in limited hydrolysis of the protein or a low degree of hydrolysis (DH), as defined herein, which may also be referred to as the protein degradation ratio, as previously described herein.In some embodiments, a low DH sufficient for the method of the present invention is a DH that does not result in a noticeable, or significant, or negative, or substantially negative change in the taste of the final product, wherein the change in taste is caused by proteolysis; and wherein in some embodiments, the final product may be a secondary plant milk, or in some embodiments, may be a combination of a primary plant milk and a secondary plant milk, or a combination of secondary milk, or a dried or concentrated version of secondary milk and any other food product.

[0087] In some embodiments, an acceptable DH, as defined herein, for the purposes of the present invention may be less than 5%, or less than 1%, or less than 2%, or less than 3%, or less than 4%, or less than 6%, or less than 7%, or less than 8%, or less than 9%, or less than 10%, or less than 11%, or less than 12%, or less than 13%, or less than 14%, or less than 15%.

[0088] Proteases that may be effective in addition to the proteases disclosed in the examples include a neutral metalloprotease (class M4). In some embodiments, heat-labile neutral bacterial proteases known in the art can be used in accordance with the present invention. Heat-labile means that the enzyme is susceptible to irreversible deactivation at relatively moderate temperatures, as will be understood by one of skill in the art. An enzyme having a substrate cleavage specificity defined as P1 = a Leu, Val, or Phe residue, where P1 is a residue on the N-terminal side of the scissile bond, can be used. Suitable heat-labile bacterial neutral proteases include proteases obtained from Bacillus species, in particular Bacillus subtilis or Bacillus amyloliquefaciens.In a particular aspect, the method of the invention comprises the use of a neutral protease, which is sold by NovoZymes® under the trade name Neutrase®, in particular Neutrase 0.5 L, or an enzyme with similar properties. In a particular aspect, this enzyme may be NEUTB, sold by BIOCAT. In some embodiments, the metalloendoprotease (EC. 3.4.24) may be Neutrase® or Maxazyme NNP DS® (EC. 3.4.24.28; bacillolysin).

[0089] Neutrase® is a trademark of protease owned by Novozymes Biopharma US Inc. Neutrase® is a metalloprotease currently derived by Novozymes from Bacillus amyloliquefaciens (also known to be derived from Bacillus subtilis). Neutrase may have a CAS number of 9080-56-2. Neutrase is specific primarily for leucine and phenylalanine (Kunst, 2003). Neutral protease belongs to a class of proteases that act as catalysts in neutral, slightly acidic, or slightly alkaline environments. Its optimal pH is between 6.0 and 7.5, and it can catalyze the hydrolysis of peptide bonds in proteins, releasing amino acids or peptides.

[0090] The advantage of neutral proteases often lies in their high reaction rate and wide adaptability to reaction conditions. According to Novozymes, Neutrase® is a high-quality, broad-spectrum endoprotease for animal protein extraction. It provides gentle hydrolysis. It is often used alone in the hydrolysis process, but it can also be combined with an exoprotease for superior flavor. Available concentration (range) 0.8-1.5 AU-N / g. Hydrolytic action: Less aggressive. Formation of peptides or individual amino acids Peptides. Bitterness reduction: No. Formation of savory flavor: Yes. Working pH range*: 6-9. Working temperature range (°C)*: 30-65. Quality assessment: Food grade, (https: / / biosolutions.novozymes.com / en / animal-protein / products / neutrase).

[0091] NEUTB is provided by BIOCAT, 9117 Three Notch Road, Troy, VA 22974 (https: / / www.bio-cat.com / ). BIOCAT describes neutral protease L (NPL or NEUTB) as useful for hydrolyzing animal and plant proteins. BIOCAT further describes NPL as useful for reducing the viscosity of fish or chicken by-products on its product information page for NPL. BIOCAT produces hydrolysates with reduced bitterness compared to alkaline proteases and claims that NPL is food grade. According to BIOCAT's product information sheet, NPL has CAS number 76774-43-1 and EC number 3.4.24.28. According to the NIH website, the substance name for CAS No. 76774-43-1 is: Proteinase, Bacillus neutral, (https: / / chem.nlm.nih.gov / chemidplus / rn / 76774-43-1). EC No. 3.4.24.28 is listed on Expasy, the Swiss bioinformatics resource portal of the Swiss Institute of Bioinformatics, as Bacillolysin and, alternatively, Bacillus metalloendopeptidase, Bacillus subtilis neutral proteinase, and megatheriopeptidase. The catalyzed reaction is listed as similar, but not identical, to that of thermolysin. Variants of this enzyme have been found in Bacillus species, including B.subtilis, B.amyloliquefaciens, B.megaterium, B.mesentericus, B.cereus, and B.stearothermophilus. The enzyme belongs to the M4 peptidase family. Formerly EC 3.4.24.4. NEUTB may have an activity range of no more than 1600 AZO / g. Bacillus amyloliquefaciens is cited as the source of NEUTB in some publications. NEUTB is in liquid form.

[0092] Neutrase® (Novozymes®) and BIOCAT are metalloproteases, a subgroup of neutral proteases, derived from Bacillus amyloliquefaciens and members of the thermolysin M4 protease family. Metalloproteases depend on the presence of divalent metal cations and can be inactivated by dialysis or metal chelates. X-ray crystallography studies have shown that most metalloproteases form a metal-binding site in the enzyme structure during crystal formation. The metal cation is typically Zn2+, but other metal cations, such as Mg2+ and Cu2+, can also be present. The metal ion in the enzyme's active site can be chelated by a chelating agent such as EDTA, causing the enzyme to partially or completely lose its activity. This process is usually reversible, and enzyme activity can be restored by re-addition of metal ions.In some embodiments of the present invention, the proteases may be bacterial neutral metalloproteinases or fungal neutral metalloproteinases, depending on their sources.

[0093] Bacterial neutral proteases are the most commonly used neutral proteases on the market, especially those produced by Bacillus species, such as Bacillus subtilis and Bacillus licheniformis. The enzymatic activity of bacterial neutral proteases mainly depends on divalent cations such as Mg2+, Zn2+, and Ca2+. Bacterial proteases have strong hydrolysis ability, a fast reaction rate, and the hydrolyzed product has less bitterness, making them widely used in the food industry.

[0094] Sources of fungal neutral protease include Aspergillus oryzae, Rhizopus, and Mucor. The catalytic pH of fungal proteases is broad (typically from 4 to 11). Aspergillus oryzae can produce acidic, neutral, and alkaline proteases. Fungal proteases are produced primarily by solid-phase fermentation. Their protease activity depends primarily on divalent cations, which can be affected by metal chelates. In general, the reaction rate and stability of fungal proteases are relatively lower than those of bacterial proteases.

[0095] In some embodiments of the present invention, certain trypsin proteases have been shown to be effective. In particular and generally, these include bacterial and fungal trypsin. Aspergillus melleus and Bacillus subtilis can be sources of trypsin effective in the present invention. Bacterial and fungal trypsins belong to the S1 chymotrypsin family. Other sources of trypsin can also be effective, and therefore, any bacterial or fungal trypsin that is effective in the present invention is considered to be within the scope of the present invention.

[0096] In general, the proteases claimed in the present invention may have similar or equivalent effects to other proteases that are not listed herein but may be known or discoverable to those skilled in the art, and any of these proteases that have similar or equivalent effects for the purposes of the present invention are considered to be within the scope of the present invention.

[0097] In some embodiments, proteases that are effective according to the present invention may be combined with other enzymes. In some embodiments, these combinations may be between enzymes that are independently effective according to the present invention. In some embodiments, these combinations may include one enzyme that is a protease that is independently effective according to the present invention and an additional enzyme that may not be effective according to the present invention. The additional enzymes may include amylase, cellulase, hemicellulase, xylanase, lipase, phytase, or other enzymes.

[0098] In some embodiments of the present invention, the material to be processed may not be of plant origin. In some embodiments, the material to be processed may be wastewater. In some embodiments, the material to be processed may be meat. In some embodiments, the material may be a food material other than a food of plant origin. In some embodiments, the material may be pet food. In some embodiments, the material to be processed may be beta-glucan containing microbial organisms or fungi. Throughout the application, the use of the term "milk" shall include any liquid obtained in accordance with the method of the present invention, regardless of whether the product is edible.

[0099] In some embodiments, the method of the present invention may include heat treating the protease-treated material to reduce or eliminate microbial contamination. In some embodiments, the heat treatment may be aseptic processing. In some embodiments, the heat treatment may be ultra-high temperature (UHT) processing. In some embodiments, the heat treatment may be conducted at a temperature sufficient for pasteurization. In some embodiments, the heat treatment may be sufficient to produce a product with an extended shelf life (ESL).

[0100] In some embodiments, the heat treatment for deactivating the enzyme may be about 90°C, or about 85°C, or about 80°C, or about 75°C, or about 70°C; wherein, in some embodiments, the heat treatment for deactivating the enzymes will result in sufficient liquefaction of the treated material such that the treated material can be processed at a high temperature to reduce or kill microbes without fouling components of the processing equipment, including pipes or heat exchangers; wherein, in some embodiments, the protease to be deactivated is a neutral protease whose effectiveness is shown herein, including NEUTB; and wherein, in some embodiments, alpha-amylase or any non-protease enzymes are not required for sufficient liquefaction for further processing.

[0101] In one embodiment, the present invention can be regarded as a method for efficiently extracting beta-glucan and protein from cereal grains such as oats and barley, and potentially from other beta-glucan-containing organisms, while maximally protecting the native structure of the beta-glucan and protein molecules. Compared to known methods of wet milling or dry milling of oats or barley for the production of a nutritional drink or plant milk, which discard highly viscous fibrous material, the present invention can use this material to more than double the amount of beta-glucan yield from the grain, as shown in Table 11, and also almost double the protein yield from the grain, as shown in Table 8. In addition to beta-glucan and protein, the method of the invention also extracts other valuable nutrients from the fibrous portion of the grain, many of which are contained only in this material.Secondary milk has a low starch content, which can be useful as a low-carb drink.EXAMPLES.

[0102] The materials and methods used in the examples of the present invention are disclosed herein below.

[0103] Obtaining a fiber suspension

[0104] The fiber suspension is typically prepared as described herein for each of the examples below, where applicable. Typically, approximately 100 g, 200 g, 250 g, or 300 g of raw material, including grains, nuts, or seeds, is weighed and washed with approximately twice the amount of ice water (i.e., 400 ml per 200 g of grains), after which the water is drained through a sieve.

[0105] The washed raw materials were placed in the jug of a 64-ounce Vitamix® wet-blade blender, model VM0135 (Vitamix® Corp., Cleveland, OH, USA). Four times the amount of ice water (i.e., 765 g per 200 g of raw materials), the calculated amount of CaCl2, CaCO3, and / or alpha-amylase (DSM, Parsippany, NJ, USA) were added to the washed raw materials in the blender jug. The mixture was then blended at high speed (10 / 10 setting) using a Vitamix® TurboBlend 4500 (model VM0197, Vitamix® Corp., Cleveland, OH, USA) for 2 minutes.

[0106] The primary slurry was filtered through a 120-mesh US sieve using a 5.5 x 3.75-inch plastic scraper with a straight edge. Most of the milk was filtered by moving the scraper at a 30-40° angle across the screen surface in a circular motion. Gentle pressure was applied to the fiber with the scraper in a flat position to force the milk out of the retentate at the end, until the retentate solids content reached approximately 35%. For some experiments, the milk production process, including washing, mixing, and sieving, was repeated depending on the needs of different slurries. In some embodiments, the primary milk yield was calculated to be approximately 67% on a dry matter basis for oats.

[0107] Approximately 125 grams of fiber suspension (i.e., from 200 grams of oat grains) was added to 300 ml (1.5 times the original grain weight) of ice water. The suspension was then placed back into the Vitamix® blender bowl and blended on high speed (10 / 10) for 30 seconds using the Vitamix® TurboBlend 4500.

[0108] In some embodiments, for example, the diluted mixed fiber slurry contained approximately 10.8% total solids (i.e., oats). In some embodiments, 400 ml (2 times the original grain weight) of ice water was added. In some embodiments, 2X water was added to the diluted fiber slurry having approximately 8% total solids.

[0109] In some embodiments, where the effect of pH on the enzymatic activity and changes in viscosity of the fiber suspension were determined, the pH of the fiber suspension was adjusted by adding anhydrous citric acid or 50% KOH solution to the mixed suspension before 100 minutes of storage in a refrigerator (1.7°C).

[0110] The suspension was placed in a beaker, covered and left in a refrigerator (1.7°C) for 100 minutes in an undisturbed state until further analysis of texture, viscosity and other analyses.

[0111] Enzyme inactivation

[0112] In some cases, enzyme inactivation for primary milk was typically performed by heating in a water bath to 77°C for 15-20 minutes, followed by heating to boiling in a microwave oven, unless otherwise specified. Alternatively, in some cases, enzymes were inactivated by introducing high-pressure steam using a Nuova Simonelli Appia II V GR1 to 80°C for 1 minute, followed by heating to boiling in a microwave oven.

[0113] Filtration of processed fiber suspension

[0114] After treating the fiber suspension with protease, the treated fiber suspension was filtered through a mesh using the same methods previously described for filtering virgin milk.

[0115] Texture Analysis

[0116] Texture analysis was generally performed as described below for each of the examples, where applicable. The fiber suspension was stored in the refrigerator for 100 minutes (grain or nut suspension) or 30 minutes (chicken skin suspension) and then mixed with a hand blender (Oster, PN: 181439 Rev B) at a speed set to 1 / lower for 10 seconds before placing it in an acrylic reverse extrusion cup (25 mm (internal diameter) x 100 mm high, Texture Technologies Corp., South Hamilton, MA, USA).

[0117] One hundred fifty grams (150 g) of fiber mass was placed in an acrylic reverse extrusion cup. The height of the one hundred fifty grams (150 g) of fiber slurry in the acrylic reverse extrusion cup was approximately 72 mm.

[0118] The total solids content, pH before enzyme addition and viscosity were measured.

[0119] The extrusion cup containing the sample was placed in an ice water bath at 1.8°C for texture analysis. A pre-calculated amount of enzymes was then added to the suspension at the top right before testing. For the chicken skin experiment, a warm (49°C) or hot water bath (60°C) was used.

[0120] To ensure compression and mixing during viscosity change measurement, a 40 mm diameter disc reverse extrusion machine (Texture Technologies, Inc., South Hamilton, MA, USA) was used.

[0121] A TA.XTPlus C texture analyzer from Texture Technologies Co. (South Hamilton, MA, USA) running Expont Connect software version 8.0.7.0 was used to measure the compressive force of oat fiber suspension.

[0122] The maximum compression force of an oat slurry from 70 mm to 5 mm at a speed of 20 mm / s was measured using a 5 kg lead cell. Data collection continued for up to 200 cycles, and the peak compression force for each peak was measured and used to determine the rate and viscosity changes due to enzyme treatment.

[0123] From the raw data, only the peak compressive forces for each cycle were extracted and used for further analysis.

[0124] For the oat retentate suspension, it was observed that the viscosity of the suspension increased continuously until approximately 100 minutes (Fig. 5). Therefore, any texture change measurements of the grain / nut retentate suspension using a texture analyzer were performed after storing the suspension in a refrigerator (1.7°C) for 100 minutes. For testing each enzyme, a fresh suspension was prepared from the grains / nuts, stored for 100 minutes, and the corresponding test parameter was applied, and texture changes were measured using a texture analyzer.

[0125] For texture analysis, in some embodiments, the slurry is stored in a walk-in refrigerator for 100 minutes for grain and nut slurries, 30 minutes for chicken skin slurries, or overnight for protein isolate and concentrate slurries.

[0126] Viscosity measurement

[0127] Viscosity measurements were generally conducted as described in this document for each of the examples below, where applicable. Grain / nut suspensions, chicken skin suspensions, and milk bases, cooled to 1-2°C in an ice water bath or stored in a refrigerator, were transferred to beakers, placed in an ice water bath at 1.7°C, and left in the bath for 10 minutes to obtain samples and equilibrate the ice bath temperature. The ice bath temperature was controlled and maintained constant by adding water or ice.

[0128] The sample beaker was removed one by one from the ice sample bath and placed in another ice-water bath maintained at 1.7°C under the viscometer. The viscosity of the sample mixture was then measured using a Brookfield RVT Series viscometer (Brookfield Engineering Laboratories Inc., Middleborough, MA) equipped with a #3, 4, or 5 round disk probe while the sample tube was in the ice-water bath. The viscometer speed was 50 or 100 rpm, and the viscosity was converted to centipoise (cP) using the table provided by the viscometer manufacturer. Three readings were collected and averaged for viscosity.

[0129] Viscosity was measured at 1.7°C in an ice water bath to minimize differences between samples and to minimize viscosity variations, especially rate changes during heating of cooled samples to a higher temperature (i.e., room temperature, 21°C).

[0130] Organoleptic evaluation of milk and other products

[0131] Approximately 30 ml of milk or other products were assigned a three-digit random number and placed into 3-ounce Solo cups. A panel of experts rated the overall quality of the milk and products on a 9-point quality scale.

[0132] Lowest quality - highly unacceptable with many off-flavors and taste characteristics, such as odors, bitterness, sourness, saltiness, viscosity, throat irritation, darker or different in color, slimy, viscous in consistency, etc. In addition, it includes samples with low quality scores, lack of sweetness, lack of the intended flavor (i.e., oat flavor in oat milk). Average quality - Neither acceptable nor unacceptable. Highest quality - Very acceptable, no off-flavors, high intensity of the intended flavor, correct level of sweetness, mouthfeel, and good color.

[0133] Between samples, panelists rinsed the palate with distilled water, unsalted saltine crackers, and waited a minimum of 3 minutes until the palate was clear and free of any residual off-notes from the previous sample evaluation.

[0134] In some cases, organoleptic qualities were assessed on a 9-point quality scale. A score of 1 corresponds to the lowest quality product, with many off-notes and inferior qualities, while a score of 9 is assigned to the highest quality product, with no off-notes, a high intensity of the expected flavor, a desired level of sweetness and mouthfeel, and good color.

[0135] Analysis of protein isolate and protein concentrate

[0136] The protein isolates and concentrates used for the analysis were used without any modifications. Appropriate amounts of protein powders and cold ice water were weighed to obtain approximately 10% or 20% solids suspensions. The mixture of water and protein powder was blended at high speed (10 / 10 setting) for 2 minutes using a Vita-Mix TurboBlend 4500. The suspension was refrigerated (1.7°C) overnight (at least 16 hours) to fully hydrate the protein.

[0137] Degree of hydrolysis (DH) of protein

[0138] The degree of protein hydrolysis, or coefficient of protein degradation (CPD), was generally measured as described in this document for each of the examples below, where applicable. Total solids and protein content of the samples were measured using an Ohaus MB90 moisture analyzer (Parsippany, NJ) and by the Dumas method using a Dumas NDA 701 nitrogen analyzer (Velp Scientific, Inc., Bohemia, NY) with a conversion factor of 6.25.

[0139] The samples were diluted to a protein concentration of 4 mg / mL, then dissolved in an equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with or without 2-mercaptoethanol (2-ME), and heated in boiling water for 3 minutes.

[0140] After cooling the samples to room temperature, the solutions were centrifuged at 2000 g for 5 min to remove non-protein particles.

[0141] Precast gels purchased from Bio-Rad Lab. (Hercules, CA, USA) or SDS-PAGE gels (separating gel: 12% acrylamide; stacking gel: 5% acrylamide) prepared according to an established method were used. Electrophoresis was performed according to a developed method in a third-party laboratory that performed the SDS-PAGE analysis.

[0142] Molecular weight standards were purchased from Sigma-Aldrich Co. All chemical reagents and organic solvents were purchased from Sigma-Aldrich. Quantification of individual protein bands (pixels and %) was performed from SDS-PAGE images using digitizing software.

[0143] The degree of hydrolysis in oat milk was determined by the relative quantitative changes (% increase) in the amount of peptide with a molecular weight of less than 25 kDa in reducing SDS-PAGE gels containing 2-mercaptoethanol.

[0144] Calculation of substances

[0145] All material measurements were calculated on a dry basis (DSB) basis unless otherwise stated.

[0146] Foam quality

[0147] Foam quality was typically measured as described in this document for each of the examples below, where applicable. The final milk products with measured pH were diluted to 10% milk powder by adding distilled water and mixed.

[0148] One hundred grams (100 g) of each milk was placed in a Nespresso milk frother (Nespresso USA Inc., New York, NY) and frothed.

[0149] Warm foamed samples were placed in 400 ml measuring cups, and the foam volume and quality were observed and recorded.

[0150] According to the foam / liquid volume and foam quality, the foam quality was converted and rated from 1 to 5. (1) Poor foam quality The volume of the milk and foam mixture after foaming is 100-120ml, and the bubbles are large and collapse quickly. (2) Below average The volume of the milk and foam mixture after foaming is 120-150ml, and the bubbles are large and collapse quickly. (3) Average The volume of the milk and foam mixture after foaming is 125-175ml, a mixture of large microbubbles that collapse moderately. (4) Above average The volume of the milk and foam mixture after foaming is 150-200ml, mainly forming micro-foam, which collapses slowly. (5) Excellent The volume of the milk and foam mixture after foaming is> 200ml, mainly forming micro-foam, which collapses slowly.

[0151] Materials

[0152] The materials used in the present invention are listed below. Alkaline protease (Bacillus licheniformis), bromelain (Ananas comosus, pineapple), fungal protease A (Aspergillus niger), fungal protease A2 (Aspergillus niger), fungal protease HU (Aspergillus oryzae), neutral protease L™ (Bacillus amyloliquefaciens), Opti-Ziome NPL, otherwise called neutral protease (Bacillus subtilis), OPTI-Ziome Pro-ST, papain (Carica papaya (papaya), protease AM (Aspergillus melleus), microbial trypsin (Aspergillus melleus and Bacillus subtilis) and xylanase (Trichoderma longibrachiatum) was obtained from Bio-Cat (Troy, VA). Bacterial amylase was purchased from DSM (Parsippany, NJ).α-Chymotrypsin (bovine pancreas), carboxypeptidase A (bovine pancreas), proteinase K (Tritirachium album), thermolysin (Geobacillus stearothermophilus), trypsin type I (bovine pancreas), and trypsin type II-S (porcine pancreas) were purchased from MiliporeSigma (Burlington, MA, USA). Flavorzyme (Bacillus licheniformis and amyloliquefaciens) and neutrase (Bacillus amyloliquefaciens) were purchased from Novozymes (Franklinton, NC, USA). Calcium carbonate (CaCO3) was purchased from Specialty Minerals Inc. (Adams, MA). Anhydrous citric acid was purchased from Fisher Chemical (Fair Lawn, NJ). Calcium chloride (CaCl2) was purchased from Avantor Performance Material Inc. (Center Valley, PA). Potassium hydroxide (KOH) was obtained from Mallinckrodt Pharmaceuticals (Hampton, NJ).Chickpea protein isolate (Plantec, item SP24000) was obtained from Socius Ingredient LLC, Evanston, IL, USA. Pea protein (Puris 870MV) was obtained from World Food Processing LLC (Turtle Lake, WI, USA), and 80% isolate (YPVCP-80C) was obtained from Yantai T Full Biotech Co. (Zhaoyuan, Shandong, China).

[0153] Table 1 contains a list of enzymes used in the present invention, including abbreviations, suppliers and additional information. TABLE 1 Reduction Name (Source) Supplier Notes ALKP AAMY Alkaline protease α-amylase (Bacillus licheniformis) BIOCAT DSM pH 7-10, 25-70°C CHTR α-chymotrypsin (Bovine pancreas) MILIPORE SIGMA pH 7.5-8.5, 30-60°C (50°C opt.) BRML Bromelain (Ananas comosus, pineapple) BIOCAT 2.4 GDU / mg, pH 4-9.35-65°C CBPT Carboxypeptidase A (Bovine pancreas) MILIPORE SIGMA pH 7-8 FLZM Flavorzyme (Bacillus licheniformis and amyloliquefaciens) NOVOZYME pH 4-8, 30-65°C FGPTA Fungal protease A (Aspergillus niger) BIOCAT 800 HUT / mg, pH 3-6.5, 30-70°C FGPTA2 Fungal protease A2 (Aspergillus niger) BIOCAT 75 HUT / mg, pH 3-6.5, 30-70°C FGPTHU Fungal protease HU (Aspergillus oryzae) BIOCAT 400 HU / mg, pH 2-11, 30-70°C NEUTB / NPL Neutral Protease L™ (Bacillus amyloliquefaciens) BIOCAT NLT 1.6 AZO / mg, pH 5.5-9, 30-70°C NEUTN Neutrase (Bacillus amyloliquefaciens) NOVOZYMES pH 6-9, 30-65°C NEUTATKL IS MISSING Autoclaved Neutral Protease L™ No Added Enzymes (Bacillus amyloliquefaciens) BIOCAT NLT 1.6 AZO / mg, pH 5.5-9, 30-70°C NEUTBS Opti-Ziome NPL, also known as neutral protease (Bacillus subtilis) BIOCAT NTL 0.2 NU / mg, pH 5.0-11, pH 9 optimal, 30-70°C, 50°C optimal OZPST OPTI-ziome Pro-ST BIOCAT pH 3-9, 20-70°C PAPN Papain (Carica papaya (papaya) BIOCAT 800 TU / mg, pH 4-10, 25-70°C PTAMHUT Protease AM (Aspergillus melleus) BIOCAT 25 HUT / mg, pH 5.5-10, 30-55°C PRK Protease K (Tritirachium album) MILIPORE SIGMA pH 7.5-12, 20-65°C (50-60°C optimal) THERE Thermolysin (Geobacillus stearothermophilus) MILIPORE SIGMA pH 7-9, 65-85°C TRY1 Microbial trypsin (Aspergillus melleus and Bacillus subtilis) BIOCAT 20 HUT / mg, pH 5-8, 30-60°C TRY4 Trypsin type I (Bovine pancreas) MILIPORE SIGMA 10000 BAEE units / mg protein, T8003-100 mg, Lipolysed powder TRY5 Trypsin type II-S (Pig pancreas) MILIPORE SIGMA T7409-1G, lipolyzed powder, type-II-S, 1000-2000 U / mg dry solids TRY1ATKL Autoclaved microbial trypsin (Aspergillus melleus and Bacillus sublilis) BIOCAT 20 HUT / mg, pH 5-8, 30-60°C XYL Xylanase (Trichoderma longibrachiatum) BIOCAT 50 XU / mg Example 1

[0154] Referring to Example 1, specifically, 200 g of various plant materials were washed with 1× ice water, mixed manually, and sieved through a sieve, as shown in Tables 1–3. Washing was repeated two more times. The plant material was placed in a blender bowl (Vitamix®). 600~800 mL of ice water, 100–400 μL of alpha-amylase (AAMY, DSM), 60 mg of CaCl2, and 100–200 mg of CaCO3 were added. The mixture was stirred at high speed (10 / 10 setting) for 2 minutes to form a primary suspension.

[0155] The primary suspension was filtered through a 100 or 120 mesh screen to separate the primary milk from the retentate fiber suspension. The primary milk was covered and stored in the refrigerator. A portion of the fiber suspension was transferred to a blender bowl. Between 333 ml and 400 ml of cold water (2°C) and 66 mg of neutral protease L (NEUTB, BioCat) were added to the fiber suspension. The mixture was mixed at high speed (10 / 10) for 30 seconds and placed in the refrigerator (2°C) for 0.5-1 hour.

[0156] After cold storage of the protease and slurry mixture, 50-200 μL of alpha-amylase (AAMY, DSM) were added (in some embodiments, amylase is optional). The virgin milk and the processed fiber slurry were heated separately in a water bath to 76.7°C for 15-20 minutes (about 6°C per minute), and then heated to boiling in a microwave oven to inactivate the enzymes. The virgin milk was cooled to 71°C in a water bath and maintained warm in a water bath (60°C). In the case of oats, approximately 775 g of virgin milk was collected from 200 g of grain and 800 g of water, and the virgin milk content in the sale was 15% (the remaining 225 g were in the fiber slurry retentate). This calculation shows a yield of about 58%. Additional washing and grinding cycles (108 in Fig. 1) may change the results, however, additional washing and grinding cycles increase the cost due to increased time and energy and may be undesirable from a practical standpoint.The method of the present invention reduces the need for additional washing and grinding cycles and improves efficiency.

[0157] The fiber suspension was cooled to 82°C and filtered through a 100- or 120-mesh sieve (washing). An additional 333 or 400 ml of cold water was added to a portion of the washed fiber suspension, and the mixture was stirred for 30 seconds. The stirred mixture was filtered through a 100- or 120-mesh sieve to obtain secondary milk. For secondary oat milk from 200 g of grains, the amount was 640 g mechanically, and the dry matter content in the milk was 5%, respectively. In the case of secondary oat milk from 200 g of grains treated with neutral protease L (NEUTB) (invention), the quantity was 600 g, and the milk dry matter content was 8%, respectively (requires clarification / discussion). (Heating for deactivation caused moisture loss due to steam evaporation, resulting in 600 g compared to 640 g of the previous sample.)

[0158] The obtained secondary milk was mixed with primary milk. The combined milk at 60°C was homogenized at 2000 psi (1500 psi in the 1st stage, 500 psi in the 2nd stage) using a GEA Niro Sovavi™ homogenizer and placed in a refrigerator. The pH and total solids content of the homogenized milk were measured. The organoleptic and other functional properties of milk and finished products containing milk, including barista, creamer, and latte (Table 5), were evaluated as shown in Table 4. The remaining fiber fraction on the sieve was placed in a drying tray and dried at 93.3°C in an oven for approximately 16 hours until completely dry (moisture content <10%). TABLE 2 Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Oats (g) 200 200 200 200 200 200 Oat moisture content (%) 12,77 12,77 12,77 12,77 12,77 12,77 α-amylase (%) 0,10 0,05 0,10 0,05 0,10 0,05 NEUTB (%) 0 0 0,033 0,033 0,017 0,033 CaCl2 (%) 0,03 0,03 0,03 0,03 0,03 0,03 CaCO3 (%) 0,05 0,05 0,05 0,05 0,05 0,05 Total number of washes 3 3 3 3 3 3 Processing of fibrous suspension No No No No Yes Yes Incubation temperature (°C) n / a n / a 57 57 5-2 5-2 Incubation time (min) n / a n / a 30 30 30-60 30-60 Grid (No.) 120 100 120 100 120 100 Exit 86,51 84,45 94,06 92,91 91,97 94,14

[0159] Table 2 presents the oat milk production protocols and milk yield. The fiber suspension was prepared as described previously. Test samples 1 and 2 were obtained solely by mechanical means. Test samples 2 and 3 were supplemented with enzyme without separation. Test samples 5 and 6 were supplemented with enzyme and separated. Measurements were calculated based on the initial mass of the raw material. Yield was measured on a dry matter basis. TABLE 3 Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Chickpeas (g) 200 200 200 200 200 200 Chickpea moisture content (%) 10,5 10,5 10,5 10,5 10,5 10,5 alpha-amylase (%) 0,2 0,1 0,2 0,1 0,2 0,1 NEUTB (%) 0 0 0,033 0,033 0,017 0,017 CaCl2 (%) 0,03 0,03 0,03 0,03 0,03 0,03 CaCO3 (%) 0,10 0,05 0,10 0,05 0,10 0,05 Total number of washes 3 3 3 3 3 3 Processing of fibrous suspension No No No No Yes Yes Incubation temperature (°C) n / a n / a 57 57 5-2 5-2 Incubation time (min) n / a n / a 30 30 30-60 30-60 Grid (No.) 120 100 120 100 120 100 Exit 79,35 74,97 79,09 78,4 84,22 80,03

[0160] Table 3 presents the chickpea milk production protocols and milk yield. The fiber suspension was prepared as described previously. Test samples 1 and 2 were obtained mechanically. Test samples 2 and 3 were supplemented with enzyme without separation. Test samples 5 and 6 were supplemented with enzyme and separated. Measurements were calculated based on the initial mass of the raw material. Yield was measured on a dry matter basis. TABLE 4 Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Almonds (g) 200 200 200 200 200 200 Almond moisture content (%) 4,5 4,5 4,5 4,5 4,5 4,5 alpha-amylase (%) 0,05 0,05 0,05 0,05 0,05 0,05 NEUTB (%) 0 0 0,033 0,033 0,017 0,017 CaCl2 (%) 0,03 0,03 0,03 0,03 0,03 0,03 CaCO3 (%) 0,10 0,05 0,10 0,05 0,10 0,05 Total number of washes 3 3 3 3 3 3 Processing of fibrous suspension No No No No Yes Yes Incubation temperature (°C) n / a n / a 57 57 5-2 5-2 Incubation time (min) n / a n / a 30 30 30-60 30-60 Grid (No.) 120 100 120 100 120 100 Exit 83,07 83,7 84,7 86,68 88,49 88,33

[0161] Table 4 presents the almond milk production protocols and milk yield. The fiber suspension was prepared as described previously. Test samples 1 and 2 were obtained mechanically. Test samples 2 and 3 were supplemented with enzyme without separation. Test samples 5 and 6 were supplemented with enzyme and separated. Oat creamer was evaluated in hot coffee with a pH of 5.11. Barista base was evaluated in a frother. Almond latte prepared with barista base and coffee. Measurements were calculated based on the original weight of raw materials. Yield was measured on a dry matter basis. TABLE 5 Tests 1 and 2 (A) Tests 3 and 4 (B) Tests 5 and 6 (C) Oat milk Strong oat notes. Excellent mouthfeel compared to B and C. Slightly sweeter than B and C. The finest texture, the most neutral flavor and the least oat-based. Very neutral flavor, light patina and poor texture, and some metallic bitterness compared to A and B. Similar in taste to B. Oat cream Floats in 2 minutes. Floats the fastest, but not much faster than B and C. It floats into flakes in 2 minutes and at the second fastest speed. Floats in 2 minutes, but slower than A and B. A small but positive improvement over A and B. Barista Oat Milk White foam, the best microfoam. Produces large bubbles. Yellowish-brown (darker) foam color, worse foam density and bubble size. The foam is slightly yellowish-brown in color, the second-highest quality microfoam with large bubbles. It has good foam volume, but the lowest foam quality compared to A and B. Chickpea milk Strong earthy, starchy, and chickpea-like flavor. Heavy in texture and taste. The cleanest taste without a residue in the mouth. Slightly starchy. Has fruity and metallic notes. Strong earthy chickpea notes, not as strong as in mechanicals. Some culinary notes, sulfur, and bitterness. Almond milk Raw almond flavor and slimy texture. Diluted flavor. Finest in texture and darkest in color. Bitter aftertaste. Slightly darker in color than A. Similar in taste to B, but without the diluted taste. Unsweetened Almond Milk Formula Thick, grainy, and fatty. Strong tannins and no culinary notes. Fine texture and refreshing. Diluted taste. Sweeter, fattier, and waxier. Clean flavor. Notes of roasted almonds. A better-tasting product than A and B. Barista's Almond Milk Same volume as B and C. Good microfoam. Similar in volume, but the foam has larger bubbles and breaks down more quickly. This is the lowest quality foam compared to grades A and C. Looks like A. Almond latte Strong taste of raw almonds and peanuts. Good nutty almond flavour, but with fishy notes and bitterness. The most neutral in taste. Good foam quality. Light nutty and peanut notes. Chickpea milk Strong chickpea and earthy notes. Heavy, starchy flavor. Clean, neutral, and without a residue in the mouth. Slightly starchy, with fruity and metallic notes. Earthy, strong chickpea notes, but not as strong as A. Some culinary notes, hints of sulfur and bitterness. The most neutral in flavor.

[0162] Table 5 presents the organoleptic and functional properties of milk and milk-based products. The fiber suspension was prepared as described previously. Test samples 1 and 2 were obtained solely by mechanical means. Test samples 2 and 3 were supplemented with enzyme without separation. Test samples 5 and 6 were supplemented with enzyme and separated. Oat creamer was evaluated in hot coffee with a pH of 5.11. Barista base was evaluated in a foam frother. Almond latte prepared with barista base and coffee.

[0163] Example 1 showed that the extraction of nutrients by protease from the fiber suspension is significant at temperatures below 10°C and that in some cases the organoleptic properties can be improved by the method of the present invention. Example 2

[0164] As shown in Table 6, various groups of enzymes were tested with a fiber suspension for their ability to increase oat milk yield according to the methods of the present invention. Protease, amylase, and xylanase were tested at a suboptimal enzyme activity temperature of 10°C. Alpha-amylase (AAMY), neutral protease L (NEUTB), and xylanase (XYL) were compared with a control containing only alpha-amylase. Table 6 shows the oat milk recovery from fiber suspensions treated with various enzymes at a temperature below the optimal activity (10°C).

[0165] For the control, 200 g of oat grains were rinsed three times with ice water and decanted. The rinsed grains were mixed with 800 ml of ice water, 100 µl of alpha-amylase (DSM, AAMY), 60 mg CaCl2, and 100 mg CaCO3 in a blender cup (Vitamix®). The mixture was mixed at high speed (setting 10 / 10) for 2 minutes to obtain a primary suspension. The control sample was rinsed three times with water. The control sample largely reproduced the wet milling and mechanical extraction processes used in the test samples.

[0166] In Test Sample 1, an additional 60 mg of alpha-amylase, or 0.03% of the original grain weight, was added to the sample. In Test Sample 2, 66 mg of neutral protease L (BIOCAT), or 0.033% of the original grain weight, was added to the sample. In Test Sample 3, 66 mg of xylanase (XYL, BIO-CAT), or 0.033% of the original grain weight, was added to the sample.

[0167] The test suspensions were incubated at 10°C in a cold water bath for 2 hours with occasional stirring. After 2 hours of incubation, the suspensions were heated in a water bath to 79.5°C for 15-20 minutes, and then heated to boiling in a microwave oven. The heated fiber suspension was washed by filtering through a No. 120 US sieve while hot, at approximately 82°C, and washed again with 400 ml of water and stirred in a Vitamix® for 30 seconds. The retentate was added to the primary milk. A portion of the fiber in the retentate after washing was discarded. The total milk solids were measured and recorded to calculate the total milk yield. TABLE 6 Control AAMY NEUTB XYL Grain weight (g) 174,57 174,56 174,39 174,54 Total water content in milk (g) 1600 1600 1600 1600 Number of washes to obtain a suspension n / a 2 2 2 Amount of alpha amylase (mg) 100 120 60 60 Amount of NEUTB (mg) 0 0 66 0 Amount of xylanase in fiber suspension (mg) 0 0 0 66 Incubation temperature (°C) n / a 10 10 10 Incubation time (minutes) n / a 120 120 120 Milk solids content (%) 8,95 9,35 9,5 12,57 Milk yield % 84,23 87,6 95,07 80,49

[0168] The fiber suspension was prepared as described previously. The control sample was extracted using only mechanical extraction. Enzyme abbreviations are provided in a separate table.

[0169] Example 2 showed that the increase in yield resulting from the combined treatment of fiber suspension with neutral protease and amylase at 10°C is high, whereas treatment with amylase alone or amylase in combination with xylanase under the same conditions results in a relatively low increase in yield. Example 3

[0170] However, in Example 3, samples were tested only with neutral protease treatment, without amylase, and at various suboptimal activity temperatures. Incubation times also varied. As shown in Table 7, samples were incubated at approximately 4°C, 7°C, and 10°C. TABLE 7 Control Test 1 Test 2 Test 3 Test 4 Grain weight (g) 174,57 178,95 178,86 178,81 178,84 Total water content in milk (g) 1600 1600 1600 1600 1600 Number of washes to obtain a suspension n / a 2 2 2 2 Amount of alpha amylase (mg) 100 100 100 100 100 Neutral protease content (mg) 0 34 66 66 66 Incubation temperature (°C) n / a 7,2 4,4 10 10 Incubation time (minutes) n / a 120 60 60 10 Milk solids (g) 9,92±0,73 10,43 8,94 9,53 8,80 Milk yield (%) 83,33±0,09 89,96 87,37 89,72 88,22

[0171] The fiber suspension was prepared as described previously. In the control sample, only the mechanical extraction method was used. The mass % was measured on a dry matter basis. Test sample 1 was supplemented with 34 mg NEUTB (BIOCAT), which is 0.173% of the original grain weight. Test samples 2, 3, and 4 used 66 mg neutral protease L (BIOCAT), which is 0.033% of the original grain weight. As shown in Table 6, the suspensions of the test samples were incubated at different temperatures for different periods of time with occasional stirring.

[0172] Example 3 showed that adding NEUTB to the fiber suspension significantly increased the yield under all the different test conditions, including different temperatures, amounts of neutral protease L, and incubation time. As previously observed, the viscosity of the fiber suspension decreased rapidly and significantly within a few minutes of incubation at 4.4-10°C. When the fiber suspension was heated, the viscosity did not increase, and the secondary milk was easily separated from the fiber suspension by filtration. It was noted that the fiber suspension from Test Sample 3 (66 mg NEUTB, 10°C, and 60 minutes) was the driest and had the least slimy texture.

[0173] NEUTB is effective in reducing the viscosity of fiber suspensions across various enzyme concentrations, temperatures, and incubation times. The increase in yield at a very low incubation temperature (4.4°C) and short incubation time (10 minutes) was significantly higher than that of the control sample.

[0174] According to Example 3, the method of the present invention by treating with neutral protease L for 10 minutes at 10°C, as shown in test strip 4 in Table 7, increased the yield by about 7-8% of the total solids in the oat grain, as shown in Table 6. The method of the present invention for 1 hour at 10°C provided an increase in the yield by about 9-10% of the total solids. Example 4

[0175] As shown in Table 8, the yield of NEUTB-treated fiber suspension was tested at a pH below the enzyme activity and at a cold (10°C) temperature. To further investigate whether activities other than protease activity could participate in NEUTB in the effect of the present invention on yield, the method was carried out at a pH of approximately 4.96-5.3. The results of this test are shown in Table 8. Based on the published enzyme activity curves provided by enzyme supplier BIOCAT (shown in Fig. 3), NEUTB is expected to be inactive or minimally active at pH 4.5. The combination of low pH and low temperature, presented in Table 7, should significantly inactivate NEUTB. TABLE 8 Control Test 1 Test 2 Solids in fiber suspension (g) 35,75 35,98 37,82 Total amount of water added to the suspension (g) 586,01 589,82 619,9 Number of washes to obtain a suspension 2 2 2 Amount of alpha amylase (mg) 60 0 0 Amount of NEUTB (mg) 0 34 34 pH of the suspension 6,54 6,54 4,96 Incubation temperature (°C) 10 10 10 Incubation time (minutes) 75 75 75 Milk solids (g) 14,43 19,03 17,9 Milk yield (%) 40,36 52,88 49,81

[0176] The fiber suspension was prepared as described previously, α-amylase was added to the control fiber suspension, while neutral protease L (NEUTB) was added to the test samples.

[0177] The pH of test sample 2 after incubation was 5.3. NEUTB-containing suspensions (Test 1 and Test 2) showed a significant decrease in viscosity after a few minutes of addition. The viscosity decrease in the NEUTB sample without pH adjustment (Test 1) was faster than in the pH-adjusted sample (Test Sample 2). However, no decrease in viscosity was observed in the sample with the addition of alpha-amylase (control). In addition, NEUTB-treated samples (Test 1 and Test 2) showed separation and sedimentation of fiber during incubation (shown with milk separation from the top). In the NEUTB-treated fiber suspension, the suspension became less uniform, with a whiter upper layer and a darker lower layer. However, the alpha-amylase-treated fiber suspension maintained homogeneity and a uniform color.This effect correlates with the ease of filtration of the NEUTB-treated product, as little mechanical force is required to separate the product; gravity alone may be sufficient to filter the product. In some embodiments, particularly commercial embodiments, filtration may be accomplished using a continuous mechanical screen. When treated with NEUTB in accordance with the present invention, the screen may require less time and energy to filter the fiber suspension, or in some cases, a continuous mechanical screen may not be required. Example 4 shows that nutrient recovery is high even under pH and temperature conditions that are believed to prevent or strongly inhibit protease activity. The yield of the control amylase sample was 40.36% of the total solids from the fiber suspension.The yield of NEUTB treated samples was 52.88% and 49.81% of fiber suspension for test samples 1 and 2, respectively. Example 5.

[0178] Table 9 shows the recovery of oats from NEUTB-treated fiber suspension at a pH below the enzyme activity and at low temperature (10°C). To further investigate whether actions other than the protease activity in NEUTB could be involved in the observed increase in yield, the inventive process was carried out at a pH of approximately 4.5. At pH 4.5, as shown in Fig. 3, the neutral protease is expected to be inactive or minimally active. The combination of low pH and low temperature should theoretically inactivate the neutral protease. TABLE 9 Control Test 1 Test 2 Test 3 Solids in fiber suspension (g) 34,83 35,09 34,78 34,77 Total amount of water added to the suspension (g) 570,83 575,18 570,16 569,98 Number of washes to obtain a suspension 2 2 2 2 Amount of alpha amylase (mg) 60 0 0 0 Amount of NEUTB (mg) 0 34 34 34 pH of the suspension - initial value 6,64 6,64 4,62 10,24 pH of the suspension - end point 6,97 6,97 4,99 9,66 Incubation temperature (°C) 10 10 10 10 Incubation time (minutes) 70 60 50 65 Milk solids (g) 12,66 17,38 14,4 14,4 Milk yield (%) 36,36 49,54 41,4 50,58

[0179] The fiber suspension was prepared as described previously. In Table 8, the milk yield percentage was calculated as the percentage of total solids from the fiber suspension that was included in the secondary milk, rather than in the combined secondary and primary milk.

[0180] Suspensions with added NEUTB showed a significant decrease in viscosity within a few minutes after the addition of the protease: 2 minutes for Test 1, 3 minutes for Test 2, and 5 minutes for Test 3, based on visual observation during the process. Subsequent examples using a texture analyzer confirmed the observation that a significant decrease in viscosity of the oat fiber suspension treated with neutral proteases occurred within 5 minutes after adding the enzymes to the retentate at a low temperature of 2°C. The viscosity decrease in the neutral, unadjusted NEUTB sample was more rapid than in the pH-adjusted samples. No decrease in viscosity was observed in the alpha-amylase control sample. The viscosity of the sample with a basic pH showed a very slow decrease in viscosity, but the viscosity dropped rapidly near the end of the digestion.The sudden drop in viscosity of test sample 3 could be due to a decrease in pH below 10 during incubation.

[0181] The acidic pH adjusted samples from Table 7 and Table 8 showed a difference in yield increase, with the yield increase being 49.81% for the conditions in Table 7 compared to 41.4% for the conditions in Table 8. These differences may be due to slight changes in pH during digestion. For the experiment shown in Table 7, the pH of the low pH fiber suspension ranged from less than 5.0 (4.96) at the beginning of the incubation period to slightly above 5.0 (5.3; data not shown) at the end of the incubation period; whereas the pH of the Table 8 experiment remained below 5.0 (4.62-4.99) throughout the enzymatic digestion of the fiber suspension.

[0182] NEUTB is expected to be minimally active at 10°C, as shown in Fig. 2, and, as shown in Fig. 3, neutral protease is expected to be virtually inactive at pH < 5.0. Therefore, it can be assumed that significant atypical protease activity exists, causing extraction and a corresponding increase in nutrient yield. This atypical activity may involve the destruction of cellular structures by means other than the hydrolysis of large protein molecules into smaller molecules by protease activity.

[0183] Once the pH exceeds 5.0, as occurred during the low pH processing portion as shown in Table 7, proteolysis may become active or more active in the low pH fiber suspension samples, thereby creating a potential synergistic effect with the putative non-proteolytic activity. This synergistic effect may explain the observed increase in yield in addition to the increase in yield resulting from the putative non-proteolytic activity of NEUTB, as shown in Table 7. The non-proteolytic activity observed in NEUTB at low pH and low temperature may theoretically be due to the activity of a secondary enzyme, such as plastein activity, which is a known neutral protease activity. When combined with secondary activity, the proteolytic activity of the protease may synergistically increase the yield as a result of a potential synergistic effect between protease and non-protease activities.

[0184] The low temperature and low pH experimental data from Table 8 showed that at pH below 5.0 and at 10°C, the increase in yield was approximately 80% of the increase in yield when the method was carried out at the optimal pH. Thus, the difference between the conditions of low protease activity (10°C) and the supposedly negligible protease activity (10°C, pH < 5) was approximately 20%. This result suggests that most of the increase in yield may be due to non-proteolytic enzymatic activity.

[0185] Neutral protease is known to have plastein activity, which is highly active at 10°C (Xu et al. 2014), and Dermiki and Fitzgerald (2020) reported that plastein synthesis generally requires a pH in the range of 3.0–7.0. Without being limited by theory, it can be said that the plastein reaction is a possible explanation for the effective extraction at 10°C and pH ~ 4.8. Since plastein is known to aggregate protein molecules, plastein activity may attract proteins and cause them to separate from the fibrous material.

[0186] Other unknown or unidentified actions of NEUTB or Neutrase® may also be associated with the observed yield increase. For example, the substrate may be a significant factor in the observed effects, so the reaction may involve protein-fiber interactions, such as beta-glucan or other cell wall fiber molecules. Regardless of the mechanism, the level of increase in fiber suspension yield under suboptimal conditions is unexpected and surprising, given the conditions tested and the known activity of NEUTB under these conditions. Example 6

[0187] Example 6 shows the extraction of fiber suspension with protease at high and low temperatures. These temperature conditions correspond to the conditions under which the samples shown in the SDS-PAGE gel in Fig. 4 were processed, as shown in Table 10. The test lanes of the SDS-PAGE gel show the size of the proteins in the fiber suspension after protease treatment. The SDS-PAGE gels in Figs. 4 and 5 demonstrate the degree of protein hydrolysis from the fiber suspension and also provide some insight into the mechanism of action of protease extraction. TABLE 10 Control Test 1 Test 2 Test 3 Solids in fiber suspension (g) 87,59 79,95 81,64 85,54 Total amount of water added to the suspension (g) 297,49 575,18 570,16 569,98 Number of washes to obtain a suspension 3 3 3 3 Neutral protease content (mg) 0 66 66 66 Incubation temperature (°C) 10 57 10 57 Incubation time (minutes) 120 120 120 120 Milk solids (g) 0 2,03 1,73 2,02 Milk yield (%) 0 36,63 33,71 36,06 Degree of protein hydrolysis (%) n / a 8,3 3,6 31,3 In Example 6, the fiber suspension was prepared as described previously. As shown in Table 10, no enzyme was added to the fiber suspension in the control lane. Test Sample 1 contained the crude suspension with added protease digested at high temperature. Test Sample 2 contained the crude suspension with added protease digested at low temperature. Test Sample 3 contained the suspension prepared before the addition of the protease and digested at high temperature. The degree of hydrolysis was determined as described previously. The increase in yield was calculated based only on the total solids content of the secondary milk.

[0188] To determine whether the degree of hydrolysis of the protease-treated fiber suspension is related to the observed increase in yield, the increase in yield of the protease-treated suspension was measured at high temperature (57°C), low temperature (10°C), and high temperature (55°C), where the fiber suspension was pre-boiled. These conditions were then reproduced for SDS-PAGE analysis.

[0189] As shown in Table 10, oat fiber suspension samples were digested using NEUTB (BIO-CAT). The nutrient yields from the fiber suspension were measured. The increase in yield with protease hydrolysis at low temperature (10°C) was similar to the increase in yield at high temperature (57°C) and at high cooking temperatures (followed by boiling, then 57°C) when the sample was first boiled. The results show unexpectedly high protease extraction at low temperatures and low degrees of hydrolysis.

[0190] The number of washes is related to the yield for amylase treatment. Extraction after two washes will show an increase in yield when controlled for amylase treatment, as the washing / grinding process itself will extract some nutrients. After three washes, nothing more will be removed. The product of two washes will pass into the primary milk. The third wash will yield no results in terms of extraction. Therefore, for the method of the present invention, the fiber suspension separated for protease extraction is what remains after the second wash. The control lane shown in Table 9 represents the water extraction after the third wash.

[0191] SDS-PAGE gel electrophoresis was performed to show the effect of protease treatment on the size of proteins in the fiber suspension, as shown in Fig. 4. Lane #812 contains the prepared sample, showing the protein from the fiber suspension cooked in a microwave and treated with NEUTB. Lane #752 is a control sample, which shows the protein from the fiber suspension without protease treatment. Lane #243 shows the protein from the fiber suspension treated with protease at a higher (optimal) temperature, the optimal temperature for NEUTB, which is 57°C for 2 hours. Lane #277 shows the protein treated with protease at a low temperature, 10°C, for 1 hour.

[0192] The prepared control sample in lane #812 showed a high degree of protease hydrolysis. The control lane #752 showed intact fiber suspension proteins not treated with protease. Major bands were present at 35 kDa and 22 kDa, with minor bands present between these two. Lane #812, showing the prepared and protease-treated oat fiber suspension protein, showed a high degree of hydrolysis (DH), with a large band at 35 kDa completely hydrolyzed by protease, and increased intensity of bands at 14 kDa and 12 kDa, likely representing hydrolysis products of the 35 kDa band and increased hydrolysis products in the range from 0 to 12 kDa.

[0193] Higher reaction temperature conditions of 57°C for 2 hours, shown in lane 243, revealed significant hydrolysis of the 35 kDa band compared to the control. A slight increase in bands at 14 kDa and 12 kDa was also observed, likely representing hydrolysis products of the 35 kDa band. A decrease in the intensity of the 35 kDa band is expected with protease hydrolysis at optimal temperatures. Protease digestion at a higher temperature resulted in a slight increase in degradation products from 0 to 12 kDa.

[0194] Low temperature protease treatment is shown in lane #277. This sample showed a high level of increase in nutrient yield, close to that of processing under optimal protease conditions. However, unlike the high temperature treated fiber suspension (#243), the low temperature treated fiber suspension (#277) did not negatively affect the organoleptic properties of sample #243, was not exposed to conditions that could lead to microbial growth or protein denaturation, and showed no evidence of significant hydrolysis compared to the control lane #752. Thus, sample #277 unexpectedly showed a very low DH while significantly increasing yield, with the low DH likely contributing to its positive organoleptic and flavor qualities. Example 7

[0195] Table 11 shows the total amount of dietary fiber and beta-glucan in oat milk from fiber suspension treated with NEUTB at or below 10°C. Using the pooled samples shown in Table 6, the amount of beta-glucan extracted from the fiber suspension was determined. TABLE 11 Control Without protease Added protease Combined primary and secondary milk: Total dietary fiber (%) 2,67 5,06 β-glucan (%) 1,6 3,35 Fiber waste: Total dietary fiber (%) 6,77 4,94 β-glucan (%) 3,23 1,24 Total (fiber waste + milk): Total dietary fiber (%) 9,43 10,00 β-glucan (%) 4,83 4,59

[0196] In Example 7, several test samples of fiber suspension treated with neutral protease L (NEUTB) at 10°C or below for different incubation times were pooled to obtain sufficient material for beta-glucan content analysis. Beta-glucan content analysis was performed by Medallion Labs (Minneapolis, MN, USA). The control sample was subjected to mechanical treatment only, without the addition of enzyme. The test samples contained fiber suspension treated with neutral protease L (NEUTB) at 10°C or below for different incubation times. Percentage calculations were based on dry matter. Primary milk is considered to initially contain approximately 1% beta-glucan. 0.6% can be added by repeated washing of the fiber suspension.However, treatment of the fibre suspension with protease can result in a more than twofold increase in beta-glucan content, as shown in Table 11 for the combined data for primary and secondary milk.Example 8.

[0197] Example 8 refers to the approximate composition and yield of secondary oat milk from neutral protease L (NEUTB)-treated fiber suspension at 2°C. TABLE 12 Control NEUTB Grain weight (g) 86,7 86,7 Total water content in milk (g) 700 700 Number of washes to obtain a suspension 1 1 Amount of α-amylase (mg) 10 10 Neutral protease content (mg) 0 50 Incubation temperature (°C) n / a 2 Incubation time (minutes) n / a 120 Amount of solids in 2nd milk (g) 16,02 22,25 Total milk yield (%) 77 94 2nd milk: Ash (%) 1,19 1,51 Carbohydrates (%) 83,33 75,85 Fat (%) 8,60 7,85 Protein (%) 6,88 14,79 Total solids in 2nd milk (%) 5,82 7,65

[0198] The fiber suspension was prepared as described previously. The control sample did not include enzyme addition. The NEUTB sample contained fiber suspension treated with NEUTB at 2°C for 120 minutes. Enzyme inactivation for the test retentate was performed by heating the raw milk in a water bath to 77°C for 7 minutes, followed by heating to boiling in a microwave oven. Measurements were made on a dry matter basis. Total milk yield was measured as a combination of primary and secondary milk. The starch content of secondary milk is predicted to potentially reach 35% or lower than that of primary oat milk, which may be advantageous for low- or reduced-carbohydrate plant milks.

[0199] In a separate preliminary experiment designed to measure the increase in the yield of individual nutrients, when NEUTB was treated for 2 hours at 10°C, the distribution of the increase in overall yield was approximately 10% protein, 15% fat, 9% ash, with fiber measurements requiring further testing. The secondary milk had a good flavor, more similar to oatmeal than the primary milk, and a good consistency. It is likely that the large amount of beta-glucan present in the fiber suspension was extracted into the secondary milk and contributed to the richness of the secondary milk. The secondary milk obtained by the method of the present invention did not have a bitter taste associated with a high degree of protein hydrolysis. The addition of secondary milk to primary milk did not degrade the overall flavor or texture of the primary milk. Example 9

[0200] Example 9 discloses the yield, milk quality, and protein hydrolysis degree (DH) of the fiber suspension treated with various proteases. TABLE 13 CONT AAMY NEUTB TRY1 PAPN ALKP CaCl2 (%) 0,03 0,03 0,03 0,03 0,03 0,03 CaCO3 (%) 0,05 0,05 0,05 0,05 0,05 0,05 α-amylase (%) 0,05 0,05 0,05 0,05 0,05 0,05 2nd enzyme (%) 0 0,033 0,033 0,033 0,033 0,033 Incubation temperature (°C) n / a 2,8-5,4 2,8-5,4 2,8-5,4 2,8-5,4 2,8-5,4 Incubation time 0 30 30 30 30 30 (min) pH of milk 6,65±0,03 6,64±0,04 6,64±0,10 6,68±0,05 6,61±0,04 7,09±0,33 Exit (%) 84,02±0,11 85,74±0,88 88,77±0,86 87,21±0,42 85,82±1,51 86,38±0,40 Foam quality 4,8±0,3 4,3±0,8 4,6±0,6 4,6±0,4 3,9±0,5 4,8±0,2 Foam volume (ml) 220±13 214±25 219±27 218±18 195±20 219±18 Viscosity (cP) 44±14 37±5 37±8 41±12 52±1 57±7 Organoleptic qualities 5,3±0,4 5,1±0,7 6,3±0,3 6,0±0,7 6,1±0,9 5,4±0,8 Degree of hydrolysis (%) 0,0±0,0 0,5±0,5 2,0±1,0 2,0±0,0 8,5±1,5 6,5±1,5

[0201] The fiber suspension was prepared as described previously. The mass % was based on the original mass of the raw material. The yield was determined from the combined primary and secondary oat milk. The yield was measured on a dry matter basis. Enzyme inactivation for the primary milk was performed by heating in a water bath to 77°C for 15-20 minutes, followed by heating to boiling in a microwave oven. Enzyme inactivation in the fiber suspension for the secondary milk was performed using the steam method, as described previously. Foam quality, organoleptic qualities, and DH were determined as described previously.

[0202] The data in Table 13 are taken from the data in Figs. 5A and 5B, which show SDS-PAGE of oat fiber suspension samples digested with protease according to the present invention. The increase in yield compared to the control was highest for the fiber suspension treated with NEUTB at a low temperature according to the present invention, followed by the fiber suspension treated with trypsin. The proteases papain and alkaline protease showed a significantly smaller increase in yield. Amylase showed the smallest increase in yield. Other experimental data showed that the effect of NEUTB on viscosity and yield increase was similar to that of Neutrase® (data not shown).

[0203] Of the proteases tested, the DH was lowest for NEUTB and trypsin, where the DH calculated as described previously was approximately 2%. The DH for papain was approximately 4 times higher than that for NEUTB and trypsin, and the DH for alkaline protease was approximately 3 times higher than that for NEUTB and trypsin.

[0204] The results show that DH does not correlate with an increase in yield, and that NPL and trypsin produce a greater increase in yield at much lower levels of hydrolysis. This result was unexpected, since hydrolysis of proteins or other organic molecules is generally considered to result in a greater decrease in viscosity. Lower molecular weight typically correlates with lower viscosity solutions. Maximizing fiber suspension yield while minimizing proteolysis is critical to the present invention, as it preserves the functional properties of the protein and minimizes changes in organoleptic properties. The neutral proteases and trypsin described herein were the only proteases tested that met the requirements of the present invention in these respects. Example 10

[0205] Example 10 reveals the effect of a large amount of proteases on the viscosity of an oat fiber suspension. Reducing viscosity is a key factor in the processing of the fiber suspension. Table 14 shows the changes in the relative viscosity of oat fiber suspensions treated with various enzymes at 2°C. TABLE 14 Enzymes n Ratio of relative viscosity to initial viscosity 1 min 2 min 3 min 4 min 5 min 10 min NEUTBS 1 0,71 0,66 0,62 0,60 0,59 0,60 NEUTB 6 0,81 0,71 0,66 0,63 0,62 0,60 NEUTN 3 0,85 0,78 0,72 0,66 0,63 0,61 TRY1 6 0,87 0,82 0,77 0,72 0,70 0,67 FGPTA2 3 0,89 0,86 0,83 0,80 0,77 0,72 FGPTHU 3 0,89 0,86 0,82 0,80 0,80 0,76 ALKP 2 0,89 0,85 0,83 0,82 0,81 0,77 FGPTA 3 0,89 0,87 0,85 0,84 0,83 0,81 TRY1ATKL 1 0,88 0,86 0,85 0,84 0,83 0,84 OZPST 2 0,90 0,88 0,87 0,85 0,84 0,79 PTAMHUT 3 0,90 0,88 0,87 0,86 0,86 0,84 AAMY 3 0,93 0,91 0,88 0,87 0,86 0,86 NEUTATKL 1 0,90 0,89 0,88 0,87 0,87 0,86 PRK4 1 0,90 0,88 0,88 0,88 0,87 0,94 FLZM 2 0,91 0,90 0,89 0,88 0,88 0,89 CHTR 1 0,86 0,84 0,85 0,88 0,88 0,95 CBPT 1 0,88 0,85 0,85 0,85 0,89 0,94 PAPN 2 0,92 0,90 0,90 0,89 0,90 0,93 THERL 1 0,89 0,87 0,88 0,89 0,91 1,00 TRY4 1 0,92 0,92 0,91 0,92 0,91 0,96 TRY5 1 0,90 0,89 0,89 0,90 0,92 0,96 ABSENT 5 0,93 0,93 0,93 0,94 0,95 0,99 BRML 2 0,96 0,96 0,96 0,96 0,97 0,99

[0206] Fiber suspensions were prepared as described previously. Texture analysis was used to measure changes in fiber suspension viscosity after enzyme treatment. Texture analysis was performed as described previously. Texture analysis can be used to measure viscosity changes when a texture analyzer measures changes in the compressive force of a reaction over time. The decrease in compressive force over time, measured by the texture analyzer, correlates with the decrease in viscosity over time.

[0207] The initial viscosity is set to 1.0 after preparing the fiber suspension for texture analysis, as described previously. After adding the enzyme, the texture analyzer continuously measures the compressive force applied to the sample as the enzyme activity increases. The final measurement in Table 14 shows the degree of viscosity reduction at a given point in time, which in this document is 10 minutes.

[0208] Table 14 shows that the neutral proteases tested herein, NEUTB and Neutrase®, are the most effective in reducing the viscosity of oat fiber suspension at 2°C. Trypsin, to a lesser extent, is also effective in significantly reducing the viscosity of the fiber suspension. Fungal proteases FGPTA2 and FGPTHU reduce viscosity to a lesser extent. Fungal proteases are complex enzyme mixtures and may include the neutral proteases of the present invention and trypsin, as well as other enzymes.

[0209] Generally, all other proteases or enzymes tested had low viscosity reduction levels compared to NPL, Neutrase®, and trypsin. Lower viscosity reduction levels caused by enzymes other than neutral proteases and trypsin from Table 14 were unsatisfactory for the purposes of the present invention. Given that viscosity reduction generally correlates with an increase in fiber suspension yield, neutral proteases and trypsin were found to be effective for the purposes of the present invention, while other proteases were not substantially effective. Example 11

[0210] Example 11 describes the changes in relative viscosity of oat fiber suspension treated with neutral protease L (NEUTB) at different enzyme concentrations at 2°C. TABLE 15 Concentration (%) n Ratio of relative viscosity to initial viscosity 1 min 2 min 3 min 4 min 5 min 10 min 0,0005 1 0,93 0,88 0,84 0,82 0,78 0,71 0,0025 1 0,93 0,85 0,79 0,76 0,76 0,66 0,005 1 0,91 0,86 0,82 0,79 0,76 0,73 0,01 1 0,87 0,75 0,71 0,67 0,66 0,67 0,05 1 0,77 0,71 0,68 0,67 0,66 0,66

[0211] Fiber suspensions were prepared as described previously. Enzyme concentration was based on the mass of the raw material. Texture analysis to measure viscosity reduction was performed as described previously.

[0212] A significant reduction in viscosity was evident even at very low NPL concentrations. This indicates that very low levels of NPL or Neutrase® are sufficient to achieve increased yields of the fiber suspensions according to the present invention. Example 12

[0213] Example 12 shows the changes in relative viscosity of oat fiber suspension treated with microbial trypsin (TRY1) at different enzyme concentrations at 2°C. TABLE 16 Concentration (%) n Ratio of relative viscosity to initial viscosity 1 min 2 min 3 min 4 min 5 min 10 min 0,0005 1 0,96 0,94 0,92 0,93 0,92 0,91 0,0025 1 0,95 0,91 0,87 0,85 0,83 0,79 0,005 1 0,93 0,89 0,85 0,84 0,81 0,79 0,01 1 0,91 0,87 0,83 0,81 0,79 0,74 0,05 1 0,90 0,83 0,74 0,73 0,73 0,71

[0214] Fiber suspensions were prepared as described previously. Enzyme concentration was based on the mass of the raw material. Texture analysis to measure viscosity reduction was performed as described previously.

[0215] A significant reduction in viscosity was evident even at very low trypsin concentrations; however, compared to NPL, the viscosity reduction with trypsin appears to be more concentration-dependent. Overall, these results indicate that low trypsin levels are sufficient to achieve increased yields of fiber suspensions according to the present invention. Example 13

[0216] Example 13 shows the changes in relative viscosity of oat fiber suspension without adding enzyme at different pH values ​​at 2°C. TABLE 17 pH n Ratio of relative viscosity to initial viscosity 1 min 2 min 3 min 4 min 5 min 10 min 10,94 1 0,86 0,87 0,88 0,88 0,88 0,90 4,38 1 0,92 0,92 0,94 0,95 0,96 0,96 6,89 1 0,89 0,89 0,89 0,90 0,93 1,00

[0217] Fiber suspensions were prepared as described previously. No enzyme was added. pH was adjusted using anhydrous citric acid and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described previously.

[0218] The pH of the solution was adjusted before texture analysis. Neutral or unadjusted pH was also tested. After a slight initial drop in pH, likely due to mechanical disruption, the pH of all samples gradually increased to 1.0. At an acidic pH, the viscosity of the fiber suspension remained virtually unchanged after 10 minutes. At a basic pH, the viscosity of the fiber suspension decreased by approximately 10% after 10 minutes. Judging from these results, pH does not significantly affect viscosity at the levels tested. Example 14

[0219] Example 14 shows the effect of pH on the viscosity-reducing ability of NEUTB in oat fiber suspensions. Changes in the relative viscosity of oat fiber suspension treated with neutral protease L (NEUTB) were measured at different pH values ​​at 2°C. TABLE 18 pH n 1 min Ratio of relative viscosity to initial viscosity 2 min 3 min 4 min 5 min 10 min 9,36 1 0,72 0,55 0,46 0,40 0,40 0,37 6,88 1 0,70 0,61 0,53 0,52 0,51 0,49 4,36 1 0,83 0,76 0,72 0,68 0,63 0,54 10,91 1 0,80 0,73 0,69 0,65 0,63 0,60 10,84 1 0,81 0,73 0,70 0,68 0,66 0,61 4,35 1 0,87 0,81 0,77 0,75 0,70 0,63 11,25 1 0,82 0,80 0,77 0,73 0,72 0,68 11,38 1 0,83 0,79 0,77 0,74 0,73 0,69 4,04 1 0,87 0,83 0,82 0,81 0,79 0,74 3,76 1 0,88 0,85 0,83 0,80 0,80 0,81 3,24 1 0,90 0,89 0,88 0,86 0,86 0,87 2,92 1 0,91 0,89 0,88 0,87 0,86 0,87 12,13 1 0,91 0,90 0,90 0,90 0,91 0,94

[0220] Fiber suspensions were prepared as described previously. pH was adjusted using anhydrous citric acid and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described previously. Table 18 shows the pH of the fiber suspension after the addition of acid or base and before the addition of 0.05% enzyme. Example 15

[0221] Example 15 shows the effect of pH on the ability of microbial trypsin (TRY1) to reduce the viscosity of oat fiber suspensions. Changes in the relative viscosity of oat fiber suspension treated with microbial trypsin (TRY1) were measured at different pH values ​​at 2°C. TABLE 19 pHϕ n 1 min Ratio of relative viscosity to initial viscosity 2 min 3 min 4 min 5 min 10 min 6,89 1 0,77 0,69 0,61 0,56 0,54 0,52 8,93 1 0,80 0,70 0,65 0,63 0,62 0,60 3,97 1 0,86 0,80 0,76 0,74 0,71 0,67 4,38 1 0,85 0,82 0,80 0,80 0,78 0,71 4,42 1 0,88 0,85 0,81 0,79 0,76 0,72 3,45 1 0,88 0,85 0,81 0,81 0,79 0,76 10,43 1 0,88 0,84 0,85 0,83 0,82 0,78 3,25 1 0,91 0,90 0,89 0,87 0,86 0,83 11,35 1 0,86 0,85 0,80 0,82 0,83 0,83 2,89 1 0,88 0,85 0,86 0,86 0,86 0,85 11,68 1 0,87 0,87 0,87 0,88 0,87 0,94 11,03 1 0,88 0,90 0,89 0,90 0,92 0,98 11,94 1 0,88 0,92 0,93 0,94 0,96 1,01

[0222] Fiber suspensions were prepared as described previously. The pH was adjusted using anhydrous citric acid and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described previously. Table 18 shows the pH of the fiber suspension after the addition of acid or base and before the addition of enzyme. Example 16

[0223] Example 16 shows the changes in the relative viscosity of the fiber suspension with various substrates when treated with neutral protease L (NEUTB) or microbial trypsin (TRY1) at 2°C according to the present invention. TABLE 20 Substrates Ratio of relative viscosity to initial viscosity (Enzymes) n 1 min 2 min 3 min 4 min 5 min 10 min Barley (No) 1 0,93 0,92 0,91 0,91 0,91 0,90 (NEUTB) 1 0,83 0,76 0,72 0,69 0,67 0,67 (TRY1) 1 0,91 0,86 0,83 0,79 0,77 0,74 Black chia (No) 1 0,95 0,95 0,97 0,99 1,00 1,01 (NEUTB) 1 0,95 0,95 0,95 0,97 0,97 0,98 (TRY1) 1 0,93 0,90 0,89 0,89 0,88 0,91 Soybeans (No) 1 0,98 0,96 0,95 0,95 0,95 0,96 (NEUTB) 1 0,85 0,83 0,81 0,81 0,82 0,80 (TRY1) 1 0,84 0,82 0,81 0,81 0,82 0,81 Almond (No) 1 0,96 0,96 0,96 0,95 0,95 0,94 (NEUTB) 1 0,96 0,95 0,94 0,94 0,92 0,91 (TRY1) 1 0,93 0,94 0,93 0,92 0,92 0,89 Chickpeas (No) 1 0,88 0,86 0,84 0,84 0,83 0,83 (NEUTB) 1 0,82 0,82 0,81 0,81 0,81 0,82 (TRY1) 1 0,78 0,75 0,73 0,73 0,72 0,70 Chicken skin (No) 3 0,97 0,98 0,98 0,97 0,96 0,98 (NEUTB) 8 0,96 0,96 0,97 1,02 0,98 1,02 (TRY1) 3 0,93 0,93 0,95 0,98 0,98 1,05

[0224] Fiber suspensions were prepared as described previously. pH was adjusted using anhydrous citric acid and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described previously.

[0225] Chicken skin tests were conducted at 2°C, 49°C, and 60°C. For the chicken skin viscosity analysis, the chicken skin was tested as described below. For the chicken skin texture analysis, the skin was obtained from a fresh chicken thigh quarter by separating the skin from the muscle. The skin was washed approximately twice with ice water (based on weight) and cut into pieces approximately 5x5 mm in size using a sharp knife and a cutting board in a refrigerator (1.7°C).

[0226] Add 2 or 3 times the amount of ice and cold distilled water to the chopped skin, bringing the final solids content to approximately 10%. The mixture was then blended at high speed (10 / 10 setting) for 2 minutes using a Vita-Mix TurboBlend 4500. The high-concentration suspension contained approximately 15% solids for chicken. Ice water was added to the chopped skin twice and mixed. For low-solid concentrations, ice water was added three times, and the resulting suspension for texture analysis had approximately 10% solids.

[0227] Tests on chicken skin were conducted in part because the BIOCAT product information sheet for NEUTB suggests the use of Neutrase, among other things, to reduce the viscosity of fish and chicken by-products. The product information sheet also provides information on the optimal conditions of activity for the use of NEUTB: 55°C and pH 6.5. The optimal temperature indicated by BIOCAT is much higher than the temperature used in the present invention, and therefore the low, suboptimal temperatures used in the present invention were tested on chicken skin, one of the substrates suggested in the BIOCAT product information sheet. As shown in Table 20, at a temperature within the scope of the present invention (2°C), no reduction in viscosity due to NEUTB (NEUTB) was observed.

[0228] To measure the viscosity of chicken skin, the chicken skin suspension was stored undisturbed in the refrigerator for 30 minutes. The same parameters used for measuring the texture changes of grains and nuts were used to measure the changes in the viscosity of the chicken skin. In addition to texture analysis at 2°C, the chicken skin suspension was heated to 49°C and 60°C before adding enzymes and analyzing the texture. During the texture analysis, the temperature of the chicken skin suspension was maintained at the same level as the initial temperature by placing the texture analysis cup in a cold ice water bath, warm water, or a hot water bath throughout the texture analysis.

[0229] Regarding the general data presented in Table 20, the yield data in Tables 2-4 and the viscosity reduction in Table 20 showed that there is a close relationship (correlation) between the increase in milk yield and the viscosity reduction in the texture analysis. For the oat fiber suspension, the viscosity reduction was high, and thus the increase in oat milk yield as a result of the method of the present invention was high; whereas, the viscosity reduction for almonds and chickpeas was not as high as that of oats, and similarly, the increase in milk yield was low. Thus, the viscosity reduction in the texture analyzer is useful for predicting the increase in the yield of plant milk. Based on the data in Table 20, it is evident that the viscosity reduction caused by the protease treatment according to the present invention is synergistic with the presence of beta-glucan in the substrate material.Although the present invention has been primarily described as a low temperature protease processing method, in some embodiments the method may also find application at higher temperatures to extract nutrients from milled cereal grains containing beta-glucan.

[0230] The beta-glucan-containing substrates tested in the present invention, oats and barley, showed a much greater reduction in viscosity, even when the initial viscosity of beta-glucan-free substrates, such as soybeans, was similar to that of oats and barley. As shown in Table 20, for barley, the relative viscosity reduction of the control after 10 minutes (0.90) compared to NEUTB (0.67) and trypsin-treated (0.74). Example 17

[0231] Example 17 shows the changes in viscosity, measured in centipoise (cP), for an unrefined oat fiber suspension at 2°C over 22 minutes. TABLE 21 Processing NEUTB TRY1 Total solids (%) 10,61 10,88 pH To 6,72 6,66 After 6,48 6,49 Viscosity (cP) To 513 445 After 29 59

[0232] Fiber suspensions were prepared as described previously. Viscosity was measured with a viscometer as described previously. The term "before" refers to the addition of enzymes, and "after" refers to the completion of enzyme treatment.

[0233] The pH remained virtually unchanged before and after enzyme treatment. NPL and trypsin showed similar viscosity reduction, although NPL demonstrated a greater viscosity reduction than trypsin. Example 18

[0234] Example 18 shows the viscosity and other properties of secondary oat milk from fiber suspension when treated with NPL and trypsin in combination with alpha-amylase at 2°C for 2 hours with slower (non-steam) enzyme deactivation. TABLE 22 Amount of enzymes (%) NEUTB (0.05) TRY1 (0.05) Solid suspension of retentate (%) 7,97 7,97 Amount of α-amylase (%) 0,05 0,05 Total solids (%) 2nd milk (%) 6,27 6,49 Exit (%) 88 87 pH 6,67 6,69 Viscosity (cP) 29 24 β-glucan (%) 10,16 9,37 Organoleptic qualities 6,5 7,5 Ease of sifting 1,5 2,0

[0235] Fiber suspensions were prepared as described previously. Viscosity was measured with a viscometer as described previously. Organoleptic properties were evaluated as described previously. To inactivate enzymes, as described previously, samples were heated in a hot water bath to 77°C for 7 minutes and then heated to boiling in a microwave oven for less than 2 minutes. Sample concentrations were based on the original mass of the raw material. Sifting was assessed on a 5-point scale: (1) very easy to sift, (3) neither easy nor difficult to sift, and (5) very difficult or impossible to sift. For samples from Examples 18, 20, and 21, milk from the sample combination was pooled and oven-dried, and β-glucan was determined at Medallion Laboratories. Example 19

[0236] Example 19 relates to the properties of secondary oat milk from fiber suspension treated with various enzymes with alpha-amylase at 2°C for 2 hours with slow (steam-free) heat deactivation of the enzymes. TABLE 23 Processing (% of enzymes) NEUTB (0.05) TRY1 (0.05) Fibrous suspension 10,97 10,97 Solids in suspension (%) Quantity 0,01 0,01 α-amylase (%) Total solids (%) 6,74 7,01 2nd milk (%) Exit (%) 81 79 pH 6,58 6,58 Protein (%) 13,85 14,09 Organoleptic qualities 7 6,5 Ease of sifting 1,7 2,5

[0237] Fiber suspensions were prepared as described previously. Viscosity was measured with a viscometer as described previously. Organoleptic properties were evaluated as described previously. To inactivate enzymes, as described previously, samples were heated in a hot water bath at 77°C for 7 minutes and then heated to boiling in a microwave oven for less than 2 minutes. Sample concentrations were based on the original mass of the raw material. Sifting was assessed on a 5-point scale: (1) very easy to sift, (3) neither easy nor difficult to sift, and (5) very difficult or impossible to sift. Example 20

[0238] Example 20 shows the viscosity and other properties of secondary oat-milk suspensions with various enzymes without α-amylase at 2°C for 2 hours with slow (non-steam) heat deactivation of the enzymes. TABLE 24 Amount of enzymes (%) NEUTB (0.05) TRY1 (0.05) Retentate 8,51 8,51 Solids in suspension (%) Quantity 0,00 0,00 α-amylase (%) Total solids (%) 6,45 6,63 2nd milk (%) Exit (%) 80 72 pH 6,29 6,34 Viscosity (cP) 47 386 Organoleptic qualities 7 6,5 Ease of sifting 2,0 4,5

[0239] Fiber suspensions were prepared as described previously. Viscosity was measured with a viscometer as described previously. Samples were heated to 80°C for 0.5 min by directly introducing steam into the suspension using a Nuova Simonelli Appia II V GR1 and then heated to boiling in a microwave oven for less than 1 minute, as described previously. Measurements were based on the original mass of the raw material. Sieving was assessed on a 5-point scale: (1) very easy to sift, (3) neither easy nor difficult to sift, and (5) very difficult or impossible to sift, as previously described. Example 21

[0240] Example 21 shows the effect of rapid (steam-treated) enzyme deactivation on the viscosity and other properties of secondary oat milk with fiber suspension treated with NPL and trypsin without alpha-amylase at 2°C for 2 hours. TABLE 25 Amount of enzymes (%) NEUTB (0.05) TRY1 (0.05) Retentate 8,43 8,43 Solids in suspension (%) Quantity 0,00 0,00 α-amylase (%) Total solids (%) 5,50 5,51 2nd milk (%) Exit (%) 78 71 pH 6,68 6,66 Viscosity (cP) 52 175 Organoleptic qualities 8 5,5 Ease of sifting 1,5 4,5

[0241] Fiber suspensions were prepared as described previously. Viscosity was measured with a viscometer, as described previously. Samples were heated to 80°C for 0.5 min by directly introducing steam into the suspension using a Nuova Simonelli Appia II V GR1, and then heated to boiling in a microwave oven for less than 1 minute, as described previously. Measurements were based on the original mass of the raw material. Sieving was assessed on a 5-point scale: (1) very easy to sift, (3) neither easy nor difficult to sift, and (5) very difficult or impossible to sift, as described previously. Example 22

[0242] Example 22 shows the initial viscosity, pH, and solids content of untreated, diluted fiber suspensions of various materials for texture analysis at 2°C. Steam injection provided a slightly better product compared to slower thermal deactivation of the enzyme. Table 25 shows that steam deactivation (or rapid deactivation) in combination with NEUTB in the absence of alpha-amylase results in a product with superior organoleptic properties and is easier to sieve. Viscosity remained low for NEUTB, but not for the trypsin proteases effective in the present invention. Although microbial trypsin has been shown to be effective, although not as effective as metalloproteases, in reducing viscosity and yield in accordance with the present invention, NEUTB and Neutrase® were more effective in some respects, as shown in Table 25. TABLE 26 Materials n Parameters Solids (%) pH Viscosity (cP) Oats 23 10,34±0,58 6,67±0,05 513±84 Barley 3 14,42±0,26 5,67±0,06 527±80 Black chia 3 13,02±0,30 6,91±0,12 106±16 Soybeans 3 12,40±0,31 6,68±0,07 414±79 Chickpeas 3 11,87±0,16 6,43±0,05 181±23 Almond 3 10,35±0,10 6,50±0,03 39±4 Chicken skin 3 15,94±0,53 7,25±0,00 469±116

[0243] Fiber suspensions were prepared as described previously. Measurements are shown as mean ± standard deviation.

[0244] The data in Table 26 show the initial concentrations of the untreated fiber suspensions. These data can be used as a general reference to other data presented in the present invention. Example 23

[0245] Example 23 shows the changes in relative viscosity of a 10% chickpea protein isolate suspension treated with different enzymes at 2°C or 50°C. TABLE 27 Enzymes n Temperature (°C) Initial viscosity (0 min) (cP) Ratio of relative viscosity to initial viscosity 1 (min) 5 (min) 10 (min) 20 (min) ABSENT 1 2 27 0,94 0,95 0,94 0,94 NEUTB 1 2 26 0,94 0,94 0,98 0,98 NEUTB 1 50 10 0,93 0,84 0,78 0,72 TRY1 1 2 25 0,94 0,97 0,98 0,98

[0246] Protein suspensions were prepared as described previously. Temperature is the incubation temperature during texture analysis. Initial viscosity is before enzyme addition and texture analysis.

[0247] The results show that at 2°C and a reaction time of 20 minutes, the neutral proteases and trypsin of the present invention, which are effective in reducing the viscosity of oats, barley, and other plant materials, had no effect on reducing the viscosity of a 10% chickpea protein isolate suspension. At 50°C, NEUTB reduced the viscosity of the chickpea material by approximately 25%. Example 24

[0248] Example 24 describes the changes in relative viscosity of a 21% pea protein isolate suspension treated with different enzymes at 2°C or 50°C. TABLE 28 Ratio of relative viscosity to initial viscosity Enzymes n Temperature (°C) Viscosity (cP) 1 (min) 5 (min) 10 (min) 20 (min) ABSENT 1 2 567 0,96 0,94 0,94 0,91 NEUTB 1 2 567 0,94 0,92 0,93 0,96 NEUTB 1 50 207 0,94 0,92 0,93 0,92 TRY1 1 2 567 0,94 0,93 0,98 0,97

[0249] Protein suspensions were prepared as described previously. Temperature is the incubation temperature during texture analysis. Initial viscosity is before enzyme addition and texture analysis.

[0250] The results show that at 2°C, the neutral proteases and trypsin of the present invention, which are effective in reducing the viscosity of oats, barley, and other plant material, had no effect on reducing the viscosity of the chickpea protein isolate suspension. At 50°C, NEUTB reduced the viscosity of the chickpea material by approximately 8% after 20 minutes of incubation. OTHER EMBODIMENTS

[0251] It should be understood that although the present invention has been set forth in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for producing a herbal drink, comprising: wet milling in an aqueous medium of plant raw material to obtain a suspension of raw material; sifting the raw material suspension to obtain primary milk and fibrous suspension; crushing of fibrous suspension; treating the fiber suspension with a protease selected from the group consisting of a metalloendoprotease and a trypsin selected from the group consisting of a bacterial trypsin and a fungal trypsin; treating the fiber suspension with said protease at a temperature below a specified operating temperature for said protease to obtain a treated fiber suspension; and sifting the processed fiber suspension to obtain secondary milk and clean fiber.

2. The method according to claim 1, wherein the plant raw material is at least one of oat grain and barley grain.

3. The method according to claim 1, wherein said temperature is below 10°C and the incubation period of treatment with protease is less than 30 minutes.

4. The method of claim 1, wherein the fiber suspension is treated with a protease at a pH below the established working pH range for said protease to obtain a treated fiber suspension.

5. The method of claim 1, wherein the viscosity of the treated fiber suspension is at least 35% lower than the viscosity of the fiber suspension when the incubation period of the protease treatment is less than 10 minutes.

6. The method according to claim 1, wherein the relative increase in the amount of peptides having a molecular weight of less than 25 kDa is less than 5%.

7. The method according to claim 1, wherein the relative increase in the amount of peptides having a molecular weight of less than 25 kDa is less than 2%.

8. The method according to claim 1, wherein the degree of protein hydrolysis is insignificant.

9. The method according to claim 1, further comprising combining the secondary milk with the primary milk to obtain combined milk.

10. The method according to claim 1, wherein the concentration of beta-glucan in the secondary milk is at least twice the concentration of beta-glucan in the primary milk, calculated on a dry matter basis.

11. The method of claim 9, wherein the combined milk contains at least half of all the beta-glucans contained in the plant raw material, calculated on a dry matter basis.

12. The method of claim 1, wherein the protease is selected from the group consisting of bacillolysin, a metalloendoprotease derived from Bacillus subtilis, and a metalloendoprotease derived from Bacillus amyloliquefaciens.

13. The method of claim 1, wherein significant growth of microorganisms is prevented during the production of plant-based milk from plant-based raw material.

14. The method according to claim 1, wherein the incubation period for treating the fibrous suspension with protease is less than 20 minutes.

15. The method of claim 1, further comprising treating the secondary milk at a high temperature sufficient to extend shelf life or aseptic packaging, without adding an additional enzyme for liquefaction, and wherein the protease is a metalloendoprotease.

16. The method of claim 1, wherein the protease is a metalloendoprotease, and wherein the fiber suspension is rapidly heated by direct steaming or indirect steaming to heat-inactivate the metalloendoprotease and prevent substantial protein hydrolysis during heat inactivation, resulting in secondary milk having at least one superior organoleptic property and increased ease of sifting compared to trypsin-treated secondary milk and treated secondary milk obtained by a slower heat inactivation method.

17. A method for producing a herbal drink, comprising: wet milling in an aqueous medium of unprocessed cereal grain containing beta-glucan to obtain a suspension of raw material; sifting the raw material suspension to obtain primary milk and fibrous suspension; crushing of fibrous suspension; treating the fiber suspension with a protease selected from the group consisting of a metalloendoprotease and a trypsin selected from the group consisting of a bacterial trypsin and a fungal trypsin; treating the fiber suspension with said protease at a temperature below 5°C for an incubation period of less than 30 minutes to obtain a treated fiber suspension; and sifting the treated fiber suspension to obtain secondary milk and clean fiber.

18. The method of claim 17, further comprising treating the secondary milk at a high temperature sufficient to extend shelf life or aseptic packaging, without adding an additional liquefaction enzyme, and wherein the protease is a metalloendoprotease.

19. A method for reducing the viscosity of a fibrous suspension, comprising: wet milling in an aqueous medium of raw material containing beta-glucan to obtain a suspension of raw material; sifting the raw material suspension to obtain the primary liquid and fibrous suspension; crushing of fibrous suspension; treating the fiber suspension with a protease selected from the group consisting of a metalloendoprotease and a trypsin selected from the group consisting of a bacterial trypsin and a fungal trypsin to obtain a treated fiber suspension; and incubating at a temperature below the specified operating temperature for the specified protease for an incubation period of less than 60 minutes; and wherein the viscosity of the treated fibrous suspension is at least 30% lower than the viscosity of the fibrous suspension.

20. A method for producing a herbal beverage, comprising: wet milling in an aqueous environment plant-based raw materials material to obtain a suspension of raw material; sifting the raw material suspension to obtain primary milk and fibrous suspension; crushing of fibrous suspension; treatment of fibrous suspension with metalloendoprotease; treating the fiber suspension with said metalloendoprotease at a temperature below the specified operating temperature for said metalloendoprotease to obtain a treated fiber suspension; and sifting the processed fiber suspension to obtain secondary milk and clean fiber.

21. A method for producing a herbal beverage, comprising: wet milling in an aqueous medium of unprocessed cereal grain containing beta-glucan to obtain a suspension of raw material; sifting the raw material suspension to obtain primary milk and fibrous suspension; crushing of fibrous suspension; treatment of fibrous suspension with metalloendoprotease; treating the fiber suspension with said metalloendoprotease at a temperature below 5°C for an incubation period of less than 30 minutes to obtain a treated fiber suspension; and sifting the processed fiber suspension to obtain secondary milk and clean fiber.

22. A method for reducing the viscosity of a fibrous suspension, comprising: wet milling in an aqueous environment raw material containing beta-glucan , with the production of a suspension of raw material; sifting the raw material suspension to obtain the primary liquid and fibrous suspension; crushing of fibrous suspension; treating the fiber suspension with a metalloendoprotease to obtain a treated fiber suspension; and incubating at a temperature below the specified operating temperature for the specified metalloendoprotease for an incubation period of less than 60 minutes; and wherein the viscosity of the treated fibrous suspension is at least 30% lower than the viscosity of the fibrous suspension.

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