Treatment to increase yield of plant-derived products

Abstract

A process utilizing bacterial or fungal metalloproteases and trypsin that solves problems associated with conventional protease extraction techniques by significantly reducing incubation times and significantly reducing temperature and protein degradation during protease extraction. This disclosure relates to a protease treatment that increases the yield of plants or other materials by extracting nutrients from fibrous waste portions of the broken down plant material while preserving the nutritional and functional qualities of the extracted raw materials for use in food products. The process preserves the quality of the extracted raw materials, including beta-glucans and proteins, by utilizing low temperatures and minimal protease activity and minimal digestion times for extraction.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 151,321, filed February 19, 2021. The disclosure of that priority application is incorporated by reference in its entirety into this application.

[0002] The present disclosure relates to processes for increasing plant nutrient yields in the production of plant-based foods and beverages, including plant-based milk. [Background technology]

[0003] In the production of plant-based milks derived from grains, nuts or seeds, some of the plant material may be discarded as waste. This waste may consist of a viscous, insoluble fibrous by-product or slurry produced during the processing of plant-based milks that is difficult or impossible to pass through mesh filters, even after dilution. The fibrous slurry is primarily composed of fibrous cell wall material such as bran and seed coats, which often contain important nutrients. This material may contain β-glucans, proteins, and bioactive phenolics and antioxidants, which are more abundant in the cell walls. It is therefore highly desirable to utilize the fibrous slurry rather than discard it.

[0004] Currently, the primary use of bran or bran-like materials is for human and animal consumption as a low nutritional value ingredient. Its relatively infrequent use as a food ingredient is related to the sensory attributes and texture of bran and bran extracts, as well as the inefficiency of nutrient extraction methods. For example, bran nutrient extracts can have a bitter taste associated with the presence of degradation products of certain proteins or lipids that become rancid upon oxidation, and can have incompatibility with certain food matrices.

[0005] Utilization of this cereal-derived fibrous material often requires further processing of the material to break down fiber interactions and reduce viscosity for improved texture. The interactions can be broken by treatments using enzymes that degrade and separate cell wall material. For example, in wheat processing, enzymes including cellulases, hemicellulases, lipases, proteases, amylases and xylanases are used to facilitate separation of the cereal material (WO2008132238A1). Cellulases, hemicellulases and xylanases are known to act directly on the main components of the cell wall to facilitate their degradation. Amylases, lipases and proteases act on starches, fats and proteins that are not the main components of the cell wall, but their enzymatic degradation can destroy some of the interactions between cell wall components.

[0006] In the brewing industry, xylanases are used to break down cell wall material in grains used in brewing to facilitate processing and increase yields (Novozymes®, 2013). Xylanases break down xylan, the main component of grain cell wall material. Neutrase®, a protease, is sometimes used in combination with xylanases in the brewing process; this increases the amount of free amino nitrogen (FAN) released during protein hydrolysis, making FAN available for yeast growth (Novozymes®, 2013). It is also believed that Neutrase® breaks down proteins that are part of the matrix, which may enhance cell wall stability (Novozymes®, 2013). Although xylanases, cellulases, and hemicellulases are effective in reducing the viscosity of fibrous grain waste, they can also have undesirable side effects on the final food product and in further processing, including the production of sugars as by-products. Lipases, amylases and proteases can also have undesirable effects on the end product, although these effects may differ from those caused by xylanases, cellulases, and hemicellulases.

[0007] Proteases have not traditionally been used alone to reduce the viscosity of cereal materials. Novozymes®, a major supplier of commercial enzymes, lists xylanases, cellulases, hemicellulases and β-glucanases, as well as α-amylases in its brewing publications for viscosity reduction, while Neutrase® is listed for fermentation enhancement through protein digestion (Novozymes® Brewing Manual, p. 40). Novozymes® markets Neutrase for oat processing with its “Neutrase® for Oats” product, stating, “Neutrase® is a high-quality broad-spectrum endoprotease that provides gentle hydrolysis. It can be used to improve protein solubility” (Novozymes®, 2021). Novozymes® describes the working temperature range of Neutrase® for oats as 30-65°C and the working pH range as 6-9 (Novozymes®, 2021). BIOCAT, an enzyme supplier, discloses in its product information sheet that Neutral Protease L (a version of Neutrase®) “reduces the viscosity of by-products from fish or chicken” (BIOCAT, 2019). BIOCAT discloses that the temperature range is 30°C-70°C, with an optimum temperature of 55°C. BIOCAT discloses that the pH range is 5.5-9.0, with an optimum pH of 6.5. BIOCAT discloses that the typical hydrolysis working rate varies depending on the application, but is typically in the range of 0.1%-1.0% (BIOCAT, 2019).

[0008] Although the use of proteases to reduce the viscosity of cereal materials is uncommon, protease extraction, in which nutrients are isolated and purified, is a well-known method for increasing nutrient yields from fibrous plant materials, including cereals. Protease extraction typically involves endoproteases that cleave peptide bonds within proteins. However, peptide bond cleavage during protease extraction can reduce the functionality of proteins and other nutrients. For example, native or near-native proteins may have better organoleptic properties and foaming capacity, as well as other properties.

[0009] The effect of proteolysis on protein functionality strongly depends on the hydrolysis conditions, including pH, temperature, hydrolysis duration, enzyme selection, and enzyme and substrate concentrations (Wouters et al., 2016).Several studies have shown that proteolysis leads to a negative impact on gel strength compared to intact proteins (Lamsal et al., 2007; Fan et al., 2005; Pinterits and Arntfield, 2007).

[0010] In terms of taste and aroma, protein hydrolysates affect the quality of the finished product. In the case of soy proteins, beany or grassy taste of the hydrolysates is an issue (Rackis et al., 1979; Wansink and Chan, 2001; Wansink and Park, 2002; Damodaran and Arora, 2013). Furthermore, protein hydrolysis often induces bitter tastes (Guigoz and Solms, 1976; Maehashi and Huang, 2009).

[0011] Protease treatment is typically carried out at temperatures of approximately 30°C to 65°C where enzyme activity is optimal or near optimal, but this may vary depending on the enzyme, enzyme source, and application. As disclosed above, Novozymes® indicates that the working temperature range for Neutrase® for Oats™ is 30-65°C, with an operating pH range of 6-9 (Novozymes®, 2021), with an optimum temperature for activity of approximately 42°C. According to the Novozymes® manual, the optimum pH for Neutrase® treatment is approximately 6, but activity drops sharply to 0 at approximately pH 4.3.

[0012] Regarding the temperatures used for Neutrase® digestion of grains, U.S. Patent No. 4,377,602 to Conrad discloses a process for preparing hydrolysates from broken whole grains using proteases. Conrad's process produced a product containing protein and sugars from a grain slurry by converting water-insoluble proteins to water-soluble products. According to Conrad, after one hour at 50°C, all of the protein was converted to water-soluble products. These conditions resulted in a product with a relatively high degree of hydrolysis and protein denaturation. This resulted in a low viscosity milk with poor texture and mouthfeel.

[0013] Protease extraction of proteins and other nutrients from fibrous materials has been effective but has limitations. Protease treatment generally hydrolyzes proteins to some extent, so that intact proteins are broken down into smaller fragments. This can affect the functionality of the protein, imparting a bitter taste to the protein and affecting its emulsifying properties, digestibility and viscosity. It has further been described that protease extraction from bran and other insoluble materials is carried out at relatively high temperatures, typically in the range of approximately 30°C to 65°C, and is generally carried out for long periods of time. Like most enzymes, proteases also have an optimum range of time and temperature at which they are effective. For proteases used in nutrient extraction from bran, the times and temperatures are typically in the range of 30°C to 65°C and 1 to 24 hours.

[0014] Janse's US Patent Application No. 20150257411 also discloses the use of Neutrase®-like proteases to extract protein from fibrous rice bran. Janse increased protein yield from rice bran while limiting the degree of hydrolysis of the protein, aiming to maintain a molecular weight of the hydrolysate generally above 500 kDa. Janse describes incubation of the protease with rice bran at 45°C and 65°C for approximately 1-4 hours, or more preferably, optimal metalloendoprotease extraction at pH 7.0 and 50°C, with incubation temperatures between 48°C and 55°C. Janse claimed a relatively low degree of hydrolysis (DH) of 10% to 16% for the claimed process.

[0015] Similarly, US Patent No. 8,575,310 to Hettiarachchy describes limited hydrolysis protease extraction from rice bran, with reaction conditions optimized at pH 8.0, 50°C for 1 hour. Janse and Hettiarachchy disclosed relatively low DH, typically around 10%-25%. US Patent No. 5,716,801 to Nielsen describes the use of proteases to produce protein hydrolysates from plant-derived proteins that are acceptable in terms of taste and organoleptic properties. Nielsen describes a protease treatment at 55°C for 18 hours, with a pH of 8.5 for the Alcalase treatment and an optimum of pH 7.0 for the Neutrase treatment, disclosing a DH of 15-35%. After 18 hours, the proteolysis was stopped by inactivating the protease by lowering the pH to 4.2 using 30% HCl.

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

[0017] Hanmoungjai et al. (2001) in their paper titled "Enzymatic Process for Extracting Oil and Protein from Rice Bran" disclose an enzymatic extraction method for oil and protein using a commercial protease (Alcalase). Hanmoungjai describes extraction conditions of 1-3 hours and 40-60°C, respectively. In their paper titled "Effect of hydrolyzing enzymes on wheat bran cell wall integrity and protein solubility" (2016), Arte et al. disclose a process for treating wheat bran with proteases for protein extraction. Arte describes an optimized protease treatment at 35°C for 3 hours. Santo Domingo et al. (2015) disclose a protease treatment method for insoluble plant fiber waste to extract fiber. Santo Domingo describes optimal protease conditions as 40°C for 5 hours.

[0018] Abdulkarim et al. (2006) in their paper titled "Use of Enzymes to enhance oil recovery during aqueous extraction of Moringa Oleifera seed oil" disclose a method for protease extraction of oil from Moringa seeds. Abdulkarim describes optimal protease extraction using Neutrase under conditions of pH 6.8, 45°C and 2 hours incubation time.

[0019] In a review on protease extraction of plant nutrients (2014), Yussof et al. disclosed the optimal conventional reaction conditions.

[0020] According to Rui et al. (2009), the optimum temperature range for enzymatic hydrolysis is between 40 and 55 °C, so most researchers use AEE (aqueous enzyme extraction) temperatures that fall within this range. In practice, the lowest possible temperature that gives adequate activity is often preferred (Passos et al., 2009). In the case of olive fruit, a lower temperature of 30 °C has been found to be favorable, especially for the preservation of 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 linseed oil extraction for similar reasons. Sharma et al. (2002) reported a significant effect of temperature on oil yield; the maximum groundnut oil yield was obtained at 40 °C, but the yield was significantly reduced when the temperature was reduced to 37 °C.

[0021] Yussof et al. (2014) further pointed out that incubation time is another limiting factor in the enzymatic extraction of nutrients from plant material; longer incubation times can have a negative impact on the quality of nutrients extracted from plant material.

[0022] 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 extending the incubation time. Passos et al. also reported that a 120-h treatment with an enzyme mixture of cellulase, protease, xylanase, and pectinase resulted in a 3.8% higher yield compared to a 24-h incubation time (2009). However, this duration (i.e., 120 h) is much longer than what is actually acceptable (Passos et al., 2009) and can result in a decrease in oil quality (Jiang et al., 2010), increased energy utilization, and the production of undesirable products (Abdulkarim et al., 2006).

[0023] A review by Mwaurah et al. (2020) compares known techniques for extracting oils from plant materials, including seeds. With regard to enzymatic extraction, Mwaurah states that "research has revealed that enzyme:substrate ratios between 1% and 8%, temperatures between 40 and 55°C, and pH between 4 and 8 are typical for enzymatic extraction of oils from different oil seeds" (Mwaurah et al., 2020).

[0024] According to Mwaurah, oil extraction from grains relies on proteolytic activity, which is sensitive to temperature and pH. Proteases are temperature sensitive, so Mwaurah explains that "temperature is one of the main factors in any oil extraction technique."

[0025] Protease extraction has also been used to extract β-glucan from cereals such as oats and barley, as well as other sources of β-glucan. β-glucans are found in cereal grains, including oats and barley, as well as in bacteria, fungi, yeasts, algae, and lichens. β-glucan has applications in several fields, particularly in the field of functional foods. β-glucan has been shown to have medical benefits, particularly with regard to immunity and cholesterol reduction.

[0026] β-glucan is an important structural component of cell walls in cereal grains, but is generally difficult to extract from these plant products. Traditional methods for β-glucan extraction include the use of acid or alkaline solutions, which often result in degradation of β-glucan polymers, thereby reducing their biological activity and health benefits. "To avoid alkaline / acid methods that are believed to degrade β-glucans, some researchers have introduced enzymatic extraction as an alternative to treatment with strong chemical solvents" (Avramia and Amariei, 2021). However, when using protease treatment to extract β-glucan from cell wall material, long treatment times and high temperatures are generally used; for example, cell wall treatment at pH 10.5 and 45°C for 5 hours followed by successive washing of the sediment with acetone or ethanol (Avramia and Amariei, 2021). Although these conditions induce strong proteolysis, these conditions would be acceptable if β-glucan extraction is the only goal, as proteolysis is not an issue. In applications where extraction of native β-glucan and native proteins is desired, such as plant-derived milks, traditional methods of β-glucan extraction using proteases are not desirable.

[0027] The references cited above indicate that nutrient extraction from plant material using proteases under optimal or conventional conditions can have detrimental effects on the quality of the extracted nutrients. Protease treatment under optimal or conventional conditions can cause proteolysis, which can reduce the functionality of the protein. Furthermore, protease treatment under conventional conditions utilizes temperatures that promote rapid microbial growth. Furthermore, the incubation conditions for conventional protease reactions promote lipase activity, which can cause oxidation of fats and oils, negatively impacting flavor. Furthermore, heating or high DH under standard protease reaction conditions can alter protein structure, which affects the functional properties of the protein. In summary, lower temperatures and shorter incubation times are desirable for protease extraction of nutrients from plant material, especially for applications such as plant-based milk. Summary of the Invention

[0028] The present disclosure solves problems associated with conventional protease extraction techniques by significantly reducing temperature, incubation time, and protein degradation during protease extraction. The present disclosure relates to a protease treatment that increases yields from plants or other materials by extracting nutrients from fibrous waste portions of the broken down plant material while preserving the nutritional and functional qualities of the extracted raw material intended for use as a food product. The process achieves quality preservation of the extracted raw material, including β-glucan and protein, by using low temperatures, minimal protease activity, and minimal digestion times during extraction. In some embodiments, the process of the present disclosure utilizes aqueous wet milling in combination with the production of plant- or microbial-derived milk or liquid. In some embodiments of the present disclosure, total nutrient yields from raw grains can be increased by approximately 5-10% or more, and for certain desirable nutrients, including β-glucan in oats, yields can be increased by up to approximately 80% or more, thereby providing a yield of approximately 80% or more of total β-glucan from the grain in the final product.

[0029] In one embodiment, oat grains are aqueous wet milled at low temperature and filtered to produce a first plant-based milk. After filtration, a fibrous slurry or by-product is separated from the primary milk. The fibrous slurry has a viscosity and texture that prevents the material from passing through a mesh filter even after dilution. After filtration (sometimes referred to herein as sifting), the fibrous slurry, which is approximately 40% total solids, may be diluted to approximately 5-15% total solids for a brief milling period. In one embodiment, the diluted fibrous by-product or fibrous slurry is maintained at a low temperature, slightly above 0°C, throughout the process. The fibrous slurry may then be transferred into a tank and maintained at a low temperature. The diluted fibrous slurry may then be treated with a protease, preferably Neutrase® or equivalent, which reacts at a low temperature of approximately 0-5°C, or with microbial trypsin. Low temperature reactions, with negligible or undetectable proteolytic activity, preserve the native structure of the protein, thus maintaining the functionality of the native protein.

[0030] Surprisingly, the addition of Neutrase® to the fibrous slurry results in a rapid and significant reduction in viscosity. The viscosity reduction with Neutrase® added at or below the standard use rate is substantial and unexpected given the low temperature of the substrate fibrous slurry. In some embodiments, after less than 10 minutes of enzyme reaction time, the viscosity reduction is sufficient to allow for by-product filtration to produce a commercially viable secondary plant-based milk. Without enzyme treatment, the diluted fibrous slurry remains highly viscous and slimy, and as a practical matter, unprocessable for most applications, including the production of plant-based milk.

[0031] Secondary milk is produced by Neutrase treatment followed by filtration. In some embodiments, the secondary milk comprises approximately 10% of the total solids of the grain feedstock. Given the low cost of the disclosed process, a 10% increase in yield is significant and commercially reasonable.

[0032] Before the secondary milk can be packaged, it must first be heat treated to inactivate the proteases or other enzymes used in the process in addition to the proteases. Heat treatment typically involves rapid heating to a temperature at which the enzymes denature, which in the present case is approximately 75°C to 90°C. The rapid heating of the enzyme inactivation prevents significant protease activity and proteolysis during the inactivation step and also limits thermal denaturation of proteins as well as microbial growth. In some applications, the enzyme inactivation heat treatment may be followed by a second heat treatment to prevent microbial growth in the final product after packaging.

[0033] During the heat inactivation process, the viscosity of the grain product generally increases due to gelatinization of starch or other interactions. Surprisingly, in the present disclosure, no significant viscosity increase occurred due to heating the Neutrase® treated fibrous slurry for enzyme inactivation. When a product containing starch granules is heated to a temperature that causes starch molecular bond breakage, gelatinization occurs, which causes water absorption and viscosity increase. Although the starch content in the oat fibrous slurry of the present disclosure is relatively low compared to the primary oat milk, it was expected that gelatinization would cause a significant viscosity increase, thereby resulting in this unexpected result.

[0034] Contrary to expectations, protease inactivation does not result in a significant increase in viscosity, which has important implications for further processing of the secondary milk according to the present disclosure. In general, amylase is added to the plant-based milk in liquefaction. Typically, amylase is required to break down starch during gelatinization, thereby reducing the viscosity and liquefying the product. Liquefaction allows the plant-based milk to be subjected to high temperature heat treatment without clogging the pipes of the processing machine.

[0035] The relatively low viscosity of the heat-inactivated secondary milk after Neutrase® treatment and enzyme inactivation allows for complete processing of the secondary milk without the use of amylase or other enzymes for liquefaction. Eliminating or reducing the need for amylase in the product has significant advantages in terms of cost and consumer demand. As with many other enzyme treatments used to reduce viscosity, amylase treatment can result in the production of sugars, which are undesirable in some products. Amylase treatment can also have a negative effect on flavor. Furthermore, with increasing consumer demand for clean label products, removal of ingredients such as enzymes can improve consumer perception of products, especially plant-based products such as oat milk.

[0036] In some embodiments, the total milk yield from grains such as oats is increased by approximately 9-10% and from nuts such as almonds by approximately 5%. The process increases the protein, fat, fiber, carbohydrate and ash yields from the grain or nut. For grains such as oats, the fibrous slurry is particularly rich in β-glucan, a nutrient known to have health benefits. In some embodiments, the process of the present disclosure may increase the yield of β-glucan by up to 80% or more, while generally preserving the native structure of β-glucan. Retention of the native structure of β-glucan is important to maintain the full functionality and health benefits of this molecule.

[0037] The low temperature treatment of the present disclosure also prevents microbial growth, particularly during protease treatment of the fibrous slurry. The low temperature minimizes or completely prevents proteolysis during protease treatment. Although the chemical mechanism that causes the rapid viscosity drop of the fibrous slurry is not clear, the degree of hydrolysis after protease treatment is surprisingly virtually zero or very close to zero. The advantages of the present process, including increased yield from plant material, inhibition of microbial growth, minimal protein degradation and structural changes to nutrients, and no need for amylase or other enzymes during high temperature treatment, are a significant improvement over existing technology. [Brief description of the drawings]

[0038] [Figure 1] 1 is a flow chart illustrating one embodiment of a process according to the present disclosure. [Diagram 2] 1 is a chart showing relative activity of Neutral Protease L™ with respect to temperature, according to one embodiment of the present disclosure. [Diagram 3] 1 is a chart showing relative activity of Neutral Protease L™ with respect to pH, according to one embodiment of the present disclosure. [Figure 4] 1 shows a reducing SDS-PAGE gel showing protein size and degree of hydrolysis according to one embodiment of the present disclosure. [Figure 5A] 1 shows an SDS-PAGE gel under non-reducing conditions showing protein size and degree of hydrolysis according to one embodiment of the present disclosure. [Figure 5B] 1 shows a reducing SDS-PAGE gel showing protein size and degree of hydrolysis according to one embodiment of the present disclosure. [Figure 6] 1 shows a graph of viscosity increase of an oat fibrous slurry at low temperature according to one embodiment of the present disclosure. [Figure 7] 1 is a graph of the viscosity change of an oat fibrous slurry at low temperature after enzyme treatment according to one embodiment of the present disclosure. [Figure 8]1 is a graph showing the viscosity change of oat fibrous slurry at low temperature after Neutral Protease L™ treatment at different enzyme concentrations according to one embodiment of the present disclosure. [Figure 9] 1 is a graph showing the viscosity change of an oat fibrous slurry at low temperature after trypsin treatment with different enzyme concentrations according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Various embodiments of the present disclosure are described in detail below. However, such detailed description is for the purpose of facilitating understanding of the present disclosure and for describing exemplary embodiments for carrying out the present disclosure. Such detailed description should not be considered as being limited to the specific embodiments described in the present disclosure, because other variations and embodiments are possible within the scope of the present disclosure. The contents of all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. As one skilled in the art would expect, there may be some variation in results in different experiments.

[0040] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the 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 by reference in their entirety. In the event of any conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. All references to percentages (%) are by weight unless otherwise specified. One or more embodiments of the invention are described in detail in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0041] Moreover, although various details are set forth in order to provide a thorough understanding of the present disclosure, it will be apparent to one skilled in the art that these specific details are not required to practice the present disclosure. In other instances, details of well-known methods, data types, protocols, procedures, processes, etc. are not described in full detail.

[0042] The present disclosure relates to a protease treatment for extracting nutrients from fibrous waste fractions or fibrous slurries of crushed plant material to increase yields from plant material while preserving the nutritional and functional qualities of the extracted raw material for use as a food product. The process preserves the quality of the extracted raw material by utilizing low temperatures, minimal protease activity and minimal digestion time for extraction. In some embodiments, the process of the present disclosure is used in combination with an aqueous wet milling process to produce plant-based milk. In some embodiments of the process of the present disclosure, the total nutrient yield from the wet milled grains can be increased by approximately 8-10%.

[0043] The process produces a secondary milk product that may be added to the primary milk product, thereby increasing the total milk yield by approximately 9-10% for grains such as oats and approximately 5% for nuts such as almonds in some embodiments. It is believed that these figures could be higher in an industrial setting where commercial use of the process may be achieved using industrial filtration and crushing systems. The process increases the yield of protein, fat, fiber, carbohydrates and ash from the grains or nuts.

[0044] In one embodiment of the process 10, as shown in FIG. 1, aqueous wet-milled oat grains are filtered at low temperature to produce a plant-based primary milk. This process involves size reduction of the grains, nuts or seeds by wet-milling in cold water to produce a primary slurry, which is then sieved through a mesh. A fibrous slurry 150 remains on the filter as a by-product. The fibrous slurry may also be referred to as a fibrous by-product or fibrous fraction. Throughout the process steps of the present disclosure, the fibrous slurry 150 may be maintained at a low temperature, slightly above 0° C. The fibrous slurry 150 is then transferred into a tank and maintained at a low temperature. The diluted fibrous slurry 150 or by-product may then be treated with a protease, preferably NEUTB; the protease is added to the diluted fibrous slurry for reaction at a low temperature of approximately 0° C. to 5° C. The low temperature reaction with negligible or undetectable proteolytic activity preserves the native structure of the proteins, thereby preserving the functionality of the native proteins.

[0045] As shown in FIG. 1, raw material 100, which may be grains, nuts or seeds, is added to cold water 102, which may be at 7° C., and crushed or ground 104 to reduce the size of raw material 100. In some embodiments, the size may be reduced to less than 1 mm at 7° C. Crushing 104 produces raw material slurry 106. After filtration, fibrous slurry 150 is separated from the primary milk. The fibrous slurry has a viscosity and texture that does not allow the material to pass through a mesh filter. Raw material slurry 106 may be sieved 108 at 10° C. through a mesh of #60-400, or more preferably a mesh of #80-160, or more preferably a mesh of #100-140, or more preferably approximately US #120, to separate a primary milk 110 fraction. The primary milk 110 fraction is comprised primarily of the white starchy soft endosperm component of the fibrous slurry 150 fraction.

[0046] The sieving step typically separates the primary milk (filtrate) from the thick, coarse, insoluble fraction of the primary slurry (by-product). In this disclosure, sieving is sometimes used as a synonym for filtration. The primary milk is primarily composed of white, starchy, soft endosperm components. The thick by-product is likely primarily composed of fibrous protein aggregates and structural seed components derived from the aleurone and subaleurone, or bran, and tough, clear endosperm portions.

[0047] The fibrous slurry, which may be approximately 40% total solids, may then be diluted with cold water to approximately 5-15% total solids and briefly stirred or milled prior to enzymatic treatment. The crushing or grinding of the feedstock 104 to the fibrous slurry 150 and the sieving of the feedstock 108 are typically performed at temperatures below the protein denaturation temperature. From the sieved feedstock 108, a primary milk 110 and a fibrous slurry 150 are produced.

[0048] In some embodiments, primary milk 110 may be produced and processed by known methods, including those described in U.S. Patent No. 7,678,403 to Mitchell. As shown in FIG. 1, primary milk 110 may be heated 112 at a rate of 6° C. / min up to 99° C. In a next step, primary milk 110 may be rapidly cooled 114 to 71° C. Cooling 114 produces processed primary milk 116.

[0049] In addition to the primary milk 110, sieving the raw slurry 108 as described above produces a fibrous slurry 150 by-product. In some embodiments, the fibrous slurry 150, which is primarily comprised of bran material, is subjected to enzyme-assisted extraction to extract nutrients from the fibrous slurry 150. Prior to protease treatment, the fibrous slurry 150 may be diluted. Protease extraction 154 according to the present disclosure includes treating the fibrous slurry 150 by adding a protease 154, which in some embodiments may be a bacterial or fungal neutral metalloendoprotease or abbreviated "neutral protease" (neutral protease is used herein as a synonym for Neutrase® or Neutral Protease L™).

[0050] For protease extraction, cold water 152, typically around 0-25° C., may be added to the fibrous slurry 150, followed by the neutral protease 154. The protease-containing fibrous slurry 150 may then be stirred 156 at low speed. It is important that the protease extraction 154 be carried out under suboptimal conditions for the protease, i.e., at a temperature or pH lower than the generally established working temperature or pH range (preferably 0° C.-15° C., or more preferably 0° C.-5° C.). Additionally, in some embodiments, protease extraction 154 from the fibrous slurry 150 may be carried out for a short period of time, in some embodiments around 10 minutes at 10° C.

[0051] As mentioned above, in normal cases, protease extraction of nutrients is typically carried out under optimal or near optimal protease activity conditions. However, optimal protease activity conditions are not optimal with respect to keeping the nutrients and plant milk products in an ideal state. Higher temperatures and longer incubation times can cause the nutrients to degrade, thereby causing their quality to deteriorate.

[0052] FIG. 2 shows the effect of temperature on the activity of BIOCAT's Neutral Protease L (NEUTB). As shown in FIG. 2, NEUTB is expected to be minimally active at 10° C. Accompanying this graph, BIOCAT states that the temperature range is 30° C. to 70° C., with an optimum temperature of 55° C. FIG. 3, also published by BIOCAT, shows that NEUTB is expected to be substantially inactive below pH 5.0. Accompanying this graph, BIOCAT states that the pH range is 5.5 to 9.0, with an optimum pH of 6.5. Novozymes® has published similar data regarding the activity of Neutrase®. Thus, based on the data presented herein, and without being limited by theory, it can be hypothesized that there will be significant atypical protease activity that will achieve extraction under strictly suboptimal conditions, with a corresponding increase in nutrient yield. This atypical activity may include disrupting cellular structures by means other than hydrolysis of large protein molecules into smaller molecules by protease activity.

[0053] Figure 4 shows the effect of protease extraction according to the present disclosure on the molecular structure of oat proteins in a fibrous slurry at high and low temperatures. These temperature conditions correspond to the conditions under which the samples shown in the SDS-PAGE gel of Figure 4 were processed; the processing conditions are shown in Table 10 and further described in detail in Example 6.

[0054] Figures 5A and 5B further illustrate the effect of the protease extraction of the present disclosure on the molecular structure of oat proteins in fibrous slurries under various conditions. The data in Table 13 are taken from the data in Figures 5A and 5B; Figures 5A and 5B show SDS-PAGE of samples of protease-digested oat fibrous slurries according to the present disclosure. Test samples and controls are shown that were treated with various proteases or α-amylase and subjected to SDS-PAGE under reducing or non-reducing conditions. Lane 162 is treated with ALKP; lane 264 is treated with NEUTB; lane 391 is treated with TRY1; lane 527 is treated with PAPN; lane 650 is a non-enzyme control, and lane 903 is treated with AAMY. The data in Figures 5A and 5B are described in more detail in Example 9, and the data is shown in Table 13. The degree of hydrolysis (DH) was calculated as described above and SDS-PAGE was generally performed as previously described herein.

[0055] Figure 6 shows the viscosity change over time of an oat fibrous slurry stored at 2°C. After wet-crushing or wet-milling and initial mesh filtration, the by-product fibrous slurry becomes more viscous over time during storage. The data shown in Figure 6 is for an oat fibrous slurry held at 2°C, which is the preferred temperature to avoid microbial growth and maintain nutrient structure before and during treatment with the proteases of the present disclosure. In general, the processes described herein that were tested were those in which the viscosity reached its plateau before the addition of the protease, although other embodiments are also considered to be within the scope of the present disclosure.

[0056] FIG. 7 shows the viscosity change of oat fibrous slurries treated with various proteases after holding the fibrous slurry at 2° C. for 100 minutes before protease treatment at 2° C. Legend for FIG. 7: 1=NONE709, 2=BRML652, 3=FLZM137, 4=TRY5595, 5=AAMY711, 6=ALKP270, 7=TRY1790, 8=NEUTN570, and 9=NEUTB352. FIG. 7 shows representative test samples from a larger data set that is described in more detail in Example 10 and Table 14 below. Example 10 discloses the effect of various proteases on the viscosity of oat fibrous slurries. Table 14 shows the relative viscosity change of oat fibrous slurries treated with various enzymes at 2° C. Viscosity reduction is a key factor that facilitates processing of fibrous slurries and generally correlates with yield increase in the present disclosure.

[0057] Figure 8 shows the viscosity change of oat fibrous slurries treated with different protease concentrations of NEUTB after the fibrous slurries were held at 2°C for 100 minutes prior to protease treatment at 2°C. Legend for Figure 8: 1=NEUTB0005, 2=NEUTB0025, 3=NEUTB005, 4=NEUTB01, and 5=NEUTB05. The data in Figure 8, which is explained in more detail in Example 11 and Table 15, discloses the relative viscosity change of oat fibrous slurries treated with different enzyme concentrations of Neutral Protease L (NEUTB) at 2°C.

[0058] Figure 9 shows the viscosity change of oat fibrous slurries treated with different protease concentrations of microbial trypsin after the fibrous slurries were held at 2°C for 100 minutes prior to protease treatment at 2°C. Legend for Figure 9: 1=TRY1005, 2=TRY10025, 3=TRY1005, 4=TRY101, and 5=TRY105. The data in Figure 9, which is explained in more detail in Example 12 and Table 16, discloses the relative viscosity change of oat fibrous slurries treated with different enzyme concentrations of TRY1 at 2°C.

[0059] In summary, the above data unexpectedly demonstrates that the addition of specific proteases to the fibrous slurry 150 results in rapid and significant viscosity reduction at very low temperatures and extreme pH. The viscosity reduction by NEUTB, which may be added at or below the standard use rate, is significant and unexpected given the low temperature of the substrate fibrous slurry 150. In some embodiments, the viscosity reduction after less than 10 minutes of enzyme reaction time is sufficient to allow for filtration of the by-product to produce a secondary plant-based milk with a unique nutritional profile. Without enzyme treatment, the diluted fibrous slurry remains highly viscous and slimy, and as a practical matter, unprocessable for most applications, including the production of plant-based milks.

[0060] Surprisingly, according to the process of the present disclosure, most or a significant portion of the nutrients contained in the fibrous slurry 150 can be efficiently extracted at low temperatures under suboptimal or strictly suboptimal protease activity conditions as defined herein; where the protease is expected to be minimally active or completely inactive. The incubation temperature of the present disclosure may range from, but is not limited to, 0° C. to 25° C. in one embodiment, although lower temperatures may be preferred. The incubation time for the low temperature extraction may range from, but is not limited to, 1 minute to 1 hour, or more preferably, 2 minutes to 30 minutes in one embodiment. Protease treatment at a pH lower than the minimum pH expected for protease activity, such as below pH 5.0, also unexpectedly extracted significant amounts of nutrients from the fibrous slurry 150, even when combined with very low temperatures.

[0061] In some embodiments, the rapid viscosity reduction at 2° C. resulting from NEUTB or trypsin treatment 154 according to the present disclosure allows for rapid processing of multiple combined batches of fibrous slurry 150 taken at different times during commercial processing. In the protease treatment 154 of the present disclosure, the low incubation time at low temperature preserves the initial batches of fibrous slurry 150 and allows for rapid viscosity reduction while also preventing microbial growth when processing combined batches prior to high temperature processing for extended shelf life or sterile product.

[0062] Like many enzymes, proteases can catalyze more than one reaction. There are several secondary activities under different conditions, which have different working temperature and pH ranges from the main enzyme activity. For example, many proteases are known to have plastein-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 have secondary activities that are responsible for the rapid viscosity reduction observed at low temperature and low pH.

[0063] Protease extraction involves minimal to moderate protein digestion / hydrolysis. In some embodiments, protease treatment may be combined with other enzymes, such as amylase, to hydrolyze and effectively and completely dissociate proteins tightly bound to other structural seed components, such as cell wall polysaccharides. Optionally, an amylase or mixture of amylases may be added to the fibrous slurry 150.

[0064] In some embodiments, the fibrous slurry 150 may be diluted with 1-2 volumes of cold water 152 prior to protease treatment 154. In some embodiments, the fibrous slurry 150 may be heated 160 to 99° C. to inactivate the enzymes, or in some embodiments, may be heated at a rate of 6° C. / min to 75° C.-99° C. In some embodiments, the heat inactivation may be by direct or indirect steam treatment for rapid inactivation. The fibrous slurry 150 may then be rapidly cooled to 82° C. to produce treated fibrous slurry 164. In some embodiments, the heating 160 may be rapid heating to substantially inactivate the enzymes without prolonged incubation in the temperature range in which the proteases are active. In some embodiments, this may be accomplished by steam heating, including steam injection or direct and indirect steam heating. Other rapid heating methods, including microwave, as known to those skilled in the art, may also be utilized.

[0065] The treated fibrous slurry 164 may then be sieved 166 through a #60-400 screen to produce processed secondary milk 170, which may also be referred to in the tables as secondary milk or second milk, and clean fiber 168. The clean fiber 168 produced in this process may be substantially free of macronutrients such as protein and fat, and may consist primarily of insoluble fiber. Clean fiber 168 is a by-product of the process of the present disclosure and may be valuable in food and other applications.

[0066] The processed secondary milk 170 may then be combined with the processed primary milk 116 to produce a combined milk 120 or a combined milk product 120. Alternatively, the processed primary milk 116 and the processed secondary milk 170 may be used individually.

[0067] The processed secondary milk 170 obtained from the fibrous slurry 150 by the process of the present disclosure contains significant amounts of the protein, fat, ash, and carbohydrates contained in the fibrous slurry. Separation of the protein, fiber, fat, and carbohydrates in the fibrous slurry 150 allows for increased yields by allowing these components to be dispersed and solubilized in water, thereby producing the processed secondary milk 170. Additionally, the reduced viscosity caused by the proteases allows for increased passage of nutritional materials through the mesh in the sieve 166, thereby increasing the nutrient yield of the processed secondary milk 170.

[0068] The processed secondary milk 170 can be combined with the processed primary milk 116 or can be used separately. When the processed primary milk 116 is combined with the processed secondary milk 170, the combined milk 120 can have higher yields, may have increased functionality, and contains additional nutrients that may be primarily found in the fibrous portions of grains and fibrous masses of nuts. The examples and tables below show that a pre-milking protein hydrolysis process such as that described by Conrad significantly improves yield in oats compared to mechanical processes alone.

[0069] The plant-based milk produced according to the Conrad process had a bitter aftertaste, whereas the milk produced according to the present disclosure did not have a bitter taste. Additionally, the plant-based milk produced by the Conrad process exhibited reduced functionality in terms of foaming capacity and foam stability compared to the mechanical process with wet milling and the process of the present disclosure.

[0070] The process of the present disclosure improved milk yield in all products tested, but the effect was stronger in certain materials. Without being limited by theory, the process effectively separates the mostly soluble low and medium molecular weight proteins into the primary milk fraction, and the proteins tightly bound to cell wall components in the aleurone and subaleurone layers into the secondary milk. Thus, the present disclosure significantly improves milk yield compared to the prior art, while minimizing the undesirable effects of treating all ground plant materials with proteases.

[0071] Additionally, the present disclosure limits the production of free amino acids and peptides and low molecular weight protein molecules that exhibit undesirable organoleptic properties in the product. The present disclosure may prevent primary, secondary and tertiary reactants that react with other components of the seed (i.e., browning). Additionally, the process of the present disclosure produces a by-product of clean fiber 168 that can be used for food and other applications.

[0072] Further advantages of the disclosed process include the short processing time of the extraction, which makes it more profitable in an industrial environment. Additionally, the low temperature and low pH prevent microbial growth during processing.

[0073] In one embodiment, the present disclosure, using neutral proteases that are particularly effective in the present disclosure, can be effective in low pH or high pH substrates, such as oxidized oat grain. In oat processing, oats that have been stored for longer tend to be oxidized and therefore have a lower pH, and the pH can even drop by a full 1. Most grains are viable, or certain enzymes are still active in inactivated grains, causing reactions that lower the pH of the grain. In addition, pH adjustments may be made during processing for various reasons, and the effectiveness of the present disclosure at low and high pH can be useful in certain embodiments. In some embodiments, waste products that can be processed by the present disclosure, such as spent barley grain, or other waste products with higher or lower pH.

[0074] In some embodiments, the effective temperature range for a protease reaction of the present disclosure can range from 0° C. to the upper limit of the denaturation temperature of a protease effective in the present disclosure. In some embodiments, the effective temperature range for a protease reaction of the present disclosure can range from 0° C. to the upper limit of the activity range of a protease effective in the present disclosure.

[0075] In some embodiments, the effective temperature of a protease reaction of the present disclosure can be a suboptimal temperature, where "suboptimal" is defined to mean lower than a recommended range provided in a publication by the protein supplier or other publication, or as would be expected to be used by one of skill in the art. In some embodiments, the effective temperature range of a protease reaction of the present disclosure can be 0°C to 80°C, or 0°C to 70°C, or 0°C to 60°C, or 0°C to 50°C, or 0°C to 40°C, or 0°C to 35°C, or 0°C to 30°C, or 0°C to 25°C, or 0°C to 20°C, or 0°C to 15°C, or 0°C to 12°C, or 0°C to 10°C, or 0°C to 9°C, or 0°C to 8°C, or 0°C to 7°C, or 0°C to 6°C, or 0°C to 5°C, or 0°C to 4°C, or 0°C to 3°C, or 0°C to 2°C, or 0°C to 1°C.

[0076] In some embodiments, when materials used in accordance with the present disclosure are not intentionally heated for the purposes of enzyme inactivation or microbial reduction, the effective temperature range for maintaining these materials can be 0°C to 50°C, or 0°C to 40°C, or 0°C to 35°C, or 0°C to 30°C, or 0°C to 25°C, or 0°C to 20°C, or 0°C to 15°C, or 0°C to 12°C, or 0°C to 10°C, or 0°C to 9°C, or 0°C to 8°C, or 0°C to 7°C, or 0°C to 6°C, or 0°C to 5°C, or 0°C to 4°C, or 0°C to 3°C, or 0°C to 2°C, or 0°C to 1°C.

[0077] In some embodiments, the effective pH range of the protease reactions of the present disclosure can be approximately 3.5-12, or approximately 4-12, or approximately 4.5-12, or approximately 3.5-11, or approximately 4-11, or approximately 4.5-11, or approximately 4.5-10, or approximately 4.5-9, or approximately 4.5-8, or approximately 4.5-7, or approximately 4.5-6.5, or approximately 5-8, or approximately 5-7, or approximately 5-6, or approximately 6-7, or approximately 6-8.

[0078] In some embodiments, the effective incubation time of the protease reaction of the present disclosure can be 1 minute to 10 minutes, or 2 minutes to 10 minutes, or 5 minutes to 10 minutes. In some embodiments, the effective incubation time of the protease reaction of the present disclosure can be 1 minute to 20 minutes, or 2 minutes to 20 minutes, or 5 minutes to 20 minutes. In some embodiments, the effective incubation time of the protease reaction of the present disclosure can be 1 minute to 30 minutes, or 2 minutes to 30 minutes, or 5 minutes to 30 minutes. In some embodiments, the effective incubation time of the protease reaction of the present disclosure can be 1 minute to 45 minutes, or 2 minutes to 45 minutes, or 5 minutes to 45 minutes. In some embodiments, the effective incubation time of the protease reaction of the present disclosure can be 1 minute to 60 minutes, or 2 minutes to 60 minutes, or 5 minutes to 60 minutes. In some embodiments, the effective incubation time for the protease reaction of the present disclosure can 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 time for the protease reaction of the present disclosure can 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 time for the protease reaction of the present disclosure can be from 10 seconds to 4 hours.

[0079] In some embodiments, the effective conditions for the protease reaction of the present disclosure are conditions that provide limited protein hydrolysis, or a low degree of hydrolysis (DH) as defined in the present disclosure; as set forth herein above, the degree of hydrolysis may also be referred to as the proteolysis coefficient. In some embodiments, a sufficiently low DH for the process of the present disclosure is a DH that does not provide a significant or significant or negative or substantially negative change in the taste of the end product; where the change in taste is due to proteolysis; and where, in some embodiments, the end product may be a plant-derived secondary milk, or in some embodiments, a combination of a plant-derived primary milk and a plant-derived secondary milk, or a combination of secondary milk, or a dried or concentrated type of secondary milk, and any other food product.

[0080] In some embodiments, acceptable DH as defined herein, for purposes of this disclosure, can be less than 5%, less than 1%, less than 2%, or less than 3%, or less than 4%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, or less than 15%.

[0081] In addition to those disclosed in the examples, effective proteases include neutral metalloproteases (class M4). In some embodiments, heat-labile neutral bacterial proteases known in the art may be used in accordance with the present disclosure. As will be appreciated by those skilled in the art, heat-labile means that the enzyme is susceptible to irreversible inactivation at relatively moderate temperatures. Enzymes with substrate cleavage specificity defined as P1 = Leu, Val or Phe residues may be used, where P1 is the residue N-terminal to the scissile bond. Suitable heat-labile bacterial neutral proteases include those from multiple species of Bacillus, particularly Bacillus subtilis or Bacillus amyloliquefaciens. In a particular aspect, the method of the present invention includes the use of a neutral protease marketed by NovoZymes® under the trademark Neutrase®, particularly Neutrase 0.5 L, or an enzyme with similar properties. In one particular aspect, the enzyme may be NEUTB marketed by BIOCAT. In some embodiments, the metalloendoprotease (EC.3.4.24) may be Neutrase® or Maxazyme NNP DS® (EC.3.4.24.28; bacillolysin).

[0082] Neutrase® is a trademark owned by Novozymes Biopharma US for proteases. Neutrase® is a metalloprotease from Novozymes currently derived from Bacillus amyloliquefaciens (and also known to be derived from Bacillus subtilis). The CAS number for Neutrase is 9080-56-2. Neutrase has specificity primarily for leucine and phenylalanine (Kunst, 2003). Neutrase refers to a class of proteases that act as catalysts in neutral, weakly acidic, or weakly alkaline environments. Its optimum pH is 6.0-7.5 and it can catalyze the hydrolysis of protein peptide bonds, releasing amino acids or peptides.

[0083] Neutral proteases often have the advantage of fast reaction rate and broad compatibility with reaction conditions. According to Novozymes, Neutrase® for animal protein extraction is a high quality broad spectrum endoprotease. This enzyme provides gentle hydrolysis. It is often used alone in the hydrolysis process, but can also be combined with exoproteases for superior flavor benefits. Usable strength (range) is 0.8-1.5 AU-N / g. Hydrolysis action: Not very strong. Production of peptides or single amino acids: Peptides. Bitterness removal: No. Full-bodied flavor production: Yes. Working pH range*: 6-9. Working temperature range (℃)*: 30-65. Quality grade: Food quality. (https: / / biosolutions.novozymes.com / en / animal-protein / products / neutrase).

[0084] NEUTB is provided by BIOCAT, 9117 Three Notch Road Troy, VA 22974 (https: / / www.bio-cat.com / ). BIOCAT states that Neutral Protease L (NPL or NEUTB) is useful for both animal and plant protein hydrolysis. BIOCAT further states on its product information page for NPL that NPL is useful for reducing the viscosity of fish or chicken by-products. BIOCAT states that it produces hydrolysates with reduced bitterness compared to alkaline proteases and that NPL is food grade. According to the product information sheet, the CAS number for BIOCAT NPL is 76774-43-1 and the EC number is 3.4.24.28. According to the NIH website, the name of the substance with CAS number 76774-43-1 is listed as Proteinase, Bacillus Neutral (https: / / chem.nlm.nih.gov / chemidplus / rn / 76774-43-1). The EC number 3.4.24.28 is listed in Expasy (the Swiss Bioinfomatics Resource Portal at the Swiss Institute of Bioinformatics) as Bacillolysin or alternatively as Bacillus metalloendopeptidase, Bacillus subtilis neutral protease and Megateriopeptidase. The reaction catalyzed is described as similar but not identical to that of thermolysin. Variants of this enzyme have been found to exist in the following Bacillus species: B. subtilis, B. amyloliquefaciens, B. megaterium, B. mesentericus, B. cereus, and B. stearothermophilus. The enzyme belongs to the peptidase family M4. Formerly, it was EC 3.4.24.4. The activity range of NEUTB is NLT 1,600 AZO / g. In several publications, the source of NEUTB is stated to be Bacillus amyloliquefaciens. The form of NEUTB is liquid.

[0085] Neutrase® (Novozymes®) and BIOCAT are metalloproteases of the neutral protease subgroup derived from Bacillus amyloliquefaciens and are members of the M4 thermolysin family of proteases. Metalloproteases depend on the presence of divalent metal cations and can be inactivated by dialysis or metal chelators. X-ray crystallographic studies have shown that most metalloproteases form metal binding sites in the enzyme structure upon crystal formation. The metal cation is usually Zn2+, but can also be other metal cations such as Mg2+ and Cu2+. The metal ion in the active site of the enzyme can be chelated by a chelator such as EDTA, which partially or completely inactivates the enzyme. This process is usually reversible, and enzyme activity can be restored by re-addition of the metal ion. In some embodiments of the present disclosure, the protease is a bacterial or fungal neutral metalloprotease consistent with its source.

[0086] Bacterial neutral proteases are the most commonly used neutral proteases on the market, especially those produced by Bacillus such as Bacillus subtilis and Bacillus licheniformis. The enzymatic activity of bacterial neutral proteases is mostly dependent on divalent cations such as Mg2+, Zn2+, and Ca2+. Bacterial proteases have strong hydrolysis ability, fast reaction rate, and their hydrolysis products have little bitterness, so they are widely used in the food industry.

[0087] Sources of fungal neutral proteases include Aspergillus oryzae, Rhizopus, and Mucor. Fungal proteases have a wide catalytic pH range (usually 4-11). Aspergillus oryzae can produce acidic, neutral, and alkaline proteases. The production of fungal proteases is mainly by solid-state fermentation. The activity of the protease is mainly dependent on divalent cations, which can be affected by metal chelation. In general, the reaction rate and stability of fungal proteases are relatively low compared to bacterial proteases.

[0088] In some embodiments of the present disclosure, certain trypsin proteases have been shown to be effective. In particular, and generally, these include bacterial and fungal trypsins. Aspergillus melleus and Bacillus subtilis can be effective trypsin sources of the present disclosure. Bacterial and fungal trypsins belong to the chymotrypsin family S1. Other trypsin sources are also considered to be effective, so any bacterial or fungal trypsin that is effective in the present disclosure is considered to be within the scope of the present disclosure.

[0089] In general, the proteases claimed in this disclosure may have similar or equivalent effects as other proteases not enumerated in this disclosure, but which would be known or recognizable to one of ordinary skill in the art, and any such proteases having similar or equivalent effects are also considered to be within the scope of this disclosure for purposes of this disclosure.

[0090] In some embodiments, the effective proteases of the present disclosure may be combined with other enzymes. In some embodiments, these combinations may be combinations of enzymes of the present disclosure that are independently effective. In some embodiments, these combinations may include one enzyme that is an independently effective protease of the present disclosure and one auxiliary enzyme of the present disclosure that is not effective. The auxiliary enzyme may include amylase, cellulase, hemicellulase, xylanase, lipase, phytase, or other enzymes.

[0091] In some embodiments of the present disclosure, the material being treated may not be of plant origin. In some embodiments, the material being treated may be wastewater. In some embodiments, the material being treated may be meat. In some embodiments, the material may be a food material other than a plant-based food. In some embodiments, the material may be a pet food. In some embodiments, the material being treated may be a β-glucan-containing microorganism or fungus. Use of the term "milk" as used throughout this application is intended to include any liquid produced according to the processes of the present disclosure, regardless of whether the product is edible.

[0092] In some embodiments, the process of the present disclosure may include heat treatment of the protease treated material to reduce or eliminate microbial contamination. In some embodiments, the heat treatment may be aseptic. In some embodiments, the heat treatment may be ultra-high temperature treatment (UHT). In some embodiments, the heat treatment may be at a temperature sufficient for pasteurization. In some embodiments, the heat treatment may be consistent with the production of an extended shelf life (ESL) product.

[0093] In some embodiments, the enzyme inactivation heat treatment may be at approximately 90°C, or approximately 85°C, or approximately 80°C, or approximately 75°C, or approximately 70°C; wherein in some embodiments, the enzyme inactivation heat treatment may be at a high temperature to liquefy the material to be processed for the purpose of reducing or removing microorganisms without clogging components of the processing equipment, including pipes or heat exchangers; wherein in some embodiments, the protease to be inactivated is a neutral protease, including NEUTB, which has been shown to be effective in the present disclosure; and wherein in some embodiments, α-amylase and other non-protease enzymes are not required for sufficient liquefaction for further processing.

[0094] In one embodiment, the present disclosure can be viewed as a process for effectively extracting β-glucan and protein from cereal grains such as oats and barley, and potentially from other β-glucan-containing organisms, while maximizing the preservation of the native structure of the β-glucan and protein molecules. Compared to known methods of wet-grinding or dry-milling oats or barley for the purpose of producing nutritional beverages or plant-based milks, in which a thick fibrous material is discarded, the present disclosure can utilize this material to nearly double the protein yield from the grain, as shown in Table 8, while also more than double the β-glucan yield from the grain, as shown in Table 11. The process extracts β-glucan and protein from the fibrous portion of the grain, as well as other important nutrients, many of which are found only in this material. Secondary milk is low in starch, which can be an advantage for low sugar beverages.

[0095] Working Example Materials and methods used in the examples of the present disclosure are disclosed herein below.

[0096] Fibrous Slurry Preparation Fibrous slurries are generally prepared as described herein for each of the examples below, where applicable. Typically, approximately 100g, 200g, 250g or 300g of grain, nut or seed-containing material is weighed and washed with approximately twice the amount of ice-cold water (i.e., 400mL / 200g grain) and drained through a strainer.

[0097] The washed ingredients were placed in a 64 oz Vitamix® Blender Cup Model VM0135 (Vitamix®, Cleveland, OH, USA) equipped with a wet blade. To this washed ingredient, 4 volumes of ice cold water (i.e., 765 g / 200 g ingredient), calculated amounts of CaCl2, CaCO3, and / or α-amylase (DSM, Parsippany, NJ, USA) were added to the blender cup. The mixture was then blended for 2 minutes on high speed (10 / 10 setting) using a Vitamix® TurboBlend 4500 (Model VM0197, Vitamix®, Cleveland, OH, USA).

[0098] The primary slurry was filtered through a US#120 mesh screen using a 5.5"x3.75" straight edge plastic bowl scraper. The majority of the milk was filtered by moving the scraper in a circular motion at a 30°-40° angle over the screen surface, and the scraper was then tilted down to apply gentle flat pressure to the fibers to squeeze the milk out of the by-product until the final solids content of the by-product was approximately 35%. In some experiments, the milking process, including washing, blending, and sieving, was repeated as necessary for each of the different slurries. In some embodiments, the primary milk yield for oats was calculated to be approximately 67% based on dry matter.

[0099] To approximately 125 grams of fibrous slurry (i.e., from 200 grams of oat grain), 300 mL (1.5 times the amount of initial grain weight) of ice cold water was added. The slurry mix was placed back into the Vitamix® blender cup and blended with a Vitamix® TurboBlend 4500 at high speed (10 / 10) for 30 seconds.

[0100] In some embodiments, for example, the diluted blended fibrous slurry contained approximately 10.8% total solids (i.e., oats). In some embodiments, 400 mL (2x the amount relative to the initial grain weight) of ice cold water was added. In some embodiments, 2x the amount of water was added to the diluted fibrous slurry having approximately 8% total solids.

[0101] In some embodiments to determine the effect of pH on enzyme activity and viscosity change of the fibrous slurry, the pH of the fibrous slurry was adjusted by adding anhydrous citric acid or 50% KOH solution to the combined slurry before keeping it in the refrigerator (1.7°C) for 100 minutes.

[0102] The slurry was placed in a beaker, covered, and placed in a refrigerator (1.7° C.) for 100 minutes before further texture, viscosity, and other analyses were performed.

[0103] enzyme inactivation In some cases, enzyme inactivation of the primary milk was generally 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 stated. Alternatively, in some cases, enzymes were inactivated by injecting 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.

[0104] Filtration of treated fibrous slurry After protease treatment of the fibrous slurry, the treated fibrous slurry was filtered through a mesh in a manner similar to that described above for filtering primary milk.

[0105] Texture Analysis Texture analysis was generally performed as described herein for each example below, where applicable. The fibrous slurries were stored in a walk-in refrigerator for 100 minutes (grain or nut slurries) or 30 minutes (chicken skin slurries) and then blended for 10 seconds using a handheld blender (Oster, PN:181439 Rev B) at the 1 / low speed setting before being placed into acrylic back-extrusion cups (25 mm (id) x 100 mm high, Texture Technologies, South Hamilton, MA, USA).

[0106] One hundred fifty grams (150 g) of the fibrous slurry was placed in an acrylic back extrusion cup. The height of the one hundred fifty grams (150 g) of the fibrous slurry in the acrylic back extrusion cup was approximately 72 mm.

[0107] Total solids, pH before enzyme addition, and viscosity were measured.

[0108] For texture analysis, the extrusion cup containing the sample was placed in an ice water bath at 1.8° C. A pre-calculated amount of enzyme was then added to the top right of the slurry before the start of the test. In the case of the chicken skin experiment, a warm water bath (49° C.) or a hot water bath (60° C.) was used.

[0109] A back-extrusion setup with a 40 mm disk (Texture Technologies, South Hamilton, MA, USA) was used to provide compression and agitation during viscosity change measurements.

[0110] A TA. XTPlus C Texture Analyser operated with Exponent Connect version 8.0.7.0. software from Texture Technologies, Inc. (South Hamilton, MA, USA) was used to measure the compressive force of the oat fiber slurries.

[0111] A 5 kg lead cell was used to measure the maximum force required to compress an oat slurry from 70 mm to 5 mm at a rate of 20 mm / s. Data was collected continuously for up to 200 cycles, and the peak compression force at each peak was measured to provide insight into the rate and change in viscosity due to enzyme treatment.

[0112] From the raw data, only the peak compressive force for each cycle was extracted and used for further analysis.

[0113] For the oat by-product slurries, it was observed that the viscosity of the slurry increased continuously for approximately up to 100 minutes (Figure 5). Therefore, texture change measurements of the grain / nut by-product slurries were performed using a texture analyzer after storing the slurries for 100 minutes in a walk-in refrigerator (1.7°C). For each enzyme tested, fresh slurries were prepared from grains / nuts and stored for 100 minutes; texture changes were measured using the texture analyzer using the appropriate test parameters.

[0114] For texture analysis, in some embodiments, the stored slurries were stored in a walk-in refrigerator for 100 minutes for grain and nut slurries and 30 minutes for chicken skin slurries, while overnight storage was performed for protein isolate and concentrate slurries.

[0115] Viscosity measurement Viscosity measurements were generally performed as described herein for each of the Examples below, where applicable. The grain / nut by-product slurry, chicken skin slurry, and milk-like base, cooled to 1-2°C in an ice-water bath or walk-in refrigerator, were transferred to a beaker, which was placed in an ice-water bath at 1.7°C and allowed to sit in the bath for 10 minutes to allow the samples to equilibrate to the ice bath temperature. The ice bath temperature was monitored and maintained constant by adding water or ice.

[0116] The sample beakers were removed one at a time from the sample ice-water bath and placed in another ice-water bath maintained at 1.7°C under the viscometer. The viscosity of the sample mixes was then measured with a Brookfield RVT series viscometer (Brookfield Engineering Laboratories, Middleboro, MA) equipped with a #3, #4 or #5 disk-type probe while the sample tubes were immersed in the ice-water bath. The viscometer speed was either 50 or 100 rpm, and the viscosity was converted to centipoise (cPs) based on tables provided by the viscometer manufacturer. Data was collected by repeating the data readings in triplicate and averaging to obtain viscosity values.

[0117] Viscosity measurements were performed at 1.7°C in an ice-water bath to minimize sample-to-sample variability and to minimize viscosity changes, especially rate changes, during the initial warming-up of the refrigerated samples to a higher temperature (i.e., room temperature, 21°C).

[0118] Sensory evaluation of milk and other products Approximately 30 mL of milk or other product was assigned a three-digit random number and placed in a 3 oz. Solo cup. Using a nine-point quality scale, expert panel members assessed and rated the overall quality of the milk and product.

[0119] Minimum quality: very unacceptable in many off-odor and taste aspects, such as rancid, bitter, sour, salty, astringent, sore, dark or off-color, sticky, greasy texture, etc. Additionally, this includes samples with low or no sweetness, lack of intended flavor (i.e. oat odor in oat milk). Medium quality: neither acceptable nor unacceptable. Best quality: very acceptable with no off-odors, high level of intended flavor, right level of sweetness, mouthfeel, good color.

[0120] Between samples, panel members rinsed their palate with distilled water and unsalted soletan crackers and waited a minimum of 3 minutes to allow the palate to be clean and free of any off-flavors remaining from the previous sample evaluation.

[0121] In some cases, the sensory attributes were evaluated using a nine-point quality scale, where a score of 1 indicates the lowest quality product with many off-flavors and poor flavor, while a score of 9 is given to the highest quality product with no off-flavors, a high degree of the intended flavor, the desired level of sweetness and mouthfeel, and good color.

[0122] Analysis of Protein Isolates and Concentrates Protein isolates and protein concentrates used in the analysis were used without modification. Appropriate amounts of protein powder and ice cold water were weighed to create an approximate 10% or 20% solids slurry. The water and protein powder mix was blended / mixed for 2 minutes using a Vita-Mix TurboBlend 4500 on high speed (setting 10 / 10). The slurry was placed in a walk-in refrigerator (1.7°C) overnight (minimum 16 hours) to allow the protein to fully hydrate.

[0123] Protein hydrolysis degree (DH) The degree of protein hydrolysis or proteolysis coefficient (CPD) was generally measured, where applicable, as described herein for each of the Examples below. Total solids and protein content of samples were measured using an Ohaus MB90 Moisture Analyzer (Parsippany, NJ) and by the Dumas method using an NDA 701 Dumas Nitrogen Analyzer (Velp Scientific, Bohemia, NY) with a conversion factor of 6.25.

[0124] Samples were diluted to a protein concentration of 4 mg / mL, 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 min.

[0125] After the samples were cooled to room temperature, the solutions were centrifuged at 2000 xg for 5 minutes to remove non-protein particles.

[0126] Precast gels were purchased from Bio-Rad (Hercules, CA, USA) and SDS-PAGE gels (separating gel: 12% acrylamide; stacking gel: 5% acrylamide) were prepared according to established methods. Electrophoresis was performed using procedures developed by a third-party laboratory that performed the SDS-PAGE analysis.

[0127] Molecular weight standards were purchased from Sigma-Aldrich. All chemical reagents and organic solvents were purchased from Sigma-Aldrich. Individual protein bands were quantified (pixels and %) from SDS-PAGE images using digitizing analysis software.

[0128] The degree of hydrolysis of oat milk was determined based on the quantitative relative abundance change (% increase) of peptides with molecular weights below 25 kDa on SDS-PAGE gels under reducing conditions containing 2-mercaptoethanol.

[0129] Substance calculations All material measurements were calculated on a dry matter basis (DSB) unless otherwise stated.

[0130] foam quality Foam quality was generally measured as described herein for each example below, where applicable. The pH measured final milk was diluted and blended to 10% milk solids by adding distilled water.

[0131] One hundred grams (100 g) of each milk was placed into a Nespresso milk frother (Nespresso USA, Inc., New York, NY) and foamed.

[0132] After pouring the warm foam sample into a 400 mL graduated beaker, the foam volume and quality were observed and recorded.

[0133] The foam quality was rated from 1 to 5 based on the foam / liquid volume and foam quality. (1) Low quality foam: The volume of the milk / foam mix after foaming is 100-120mL, and the air bubble size is large and disappears quickly. (2) Below average: The volume of the milk / foam mix after foaming is 120-150 mL, and the air bubble size is large and disappears quickly. (3) Average: The volume of the milk / foam mix after foaming is 125-175 mL, with the mixture of large fine bubbles, disappearing at a moderate rate. (4) Better than average: The volume of the milk / foam mix after foaming is 150-200mL, and most of the bubbles are fine bubbles that disappear slowly. (5) Excellent: The volume of the milk / foam mix after foaming is more than 200 mL, and most of the bubbles are fine bubbles that disappear slowly.

[0134] material The materials used in this disclosure are listed here below.

[0135] 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 aka 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 MilliporeSigma (Burlington, MA, USA). Flavourzyme (Bacillus licheniformis and amyloliquefaciens) and Neutrase (Bacillus amyloliquefaciens) were obtained from Novozymes (Franklinton, NC, USA). Calcium carbonate (CaCO3) was obtained from Specialty Calcium chloride (CaCl2) was purchased from Avantor Performance Materials (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., Ltd. (Zhaoyuan, Shandong, China).

[0136] Table 1 contains a list of the enzymes used in this disclosure, including abbreviations, suppliers and further information.

[0137] [Table 1] TIFF2024522023000002.tif240159TIFF2024522023000003.tif81159

[0138] Example 1 For Example 1, as shown in Tables 1-3, specifically, 200 g of each plant material was washed with 1 volume of ice-cold water, mixed manually, and drained through a strainer. Washing was repeated two more times. The plant material was placed in a blender cup (Vitamix®). 600-800 mL of ice-cold water, 100-400 microliters of α-amylase (AAMY, DSM), 60 mg of CaCl2, and 100-200 mg of CaCO3 were added. The mixture was blended for 2 minutes on high speed (10 / 10 setting) to produce a primary slurry.

[0139] The primary slurry was filtered through a #100 or #120 mesh screen to separate the primary milk from the fibrous slurry by-product. The primary milk was covered and stored in the refrigerator. The fibrous slurry portion was transferred to a blender cup. To the fibrous slurry, 333 mL to 400 mL of cold water (2°C) and 66 mg of Neutral Protease L (NEUTB, BioCat) were added. The mixture was blended on high speed (10 / 10) for 30 seconds and placed in the refrigerator (2°C) for 0.5 to 1 hour.

[0140] After refrigeration of the protease and slurry mix, 50-200 microliters of α-amylase (AAMY, DSM) was added (in some embodiments, amylase is optional). The primary milk and treated fibrous slurry were placed separately in a water bath and heated to 76.7°C for 15-20 minutes (approximately 6°C / min), then further heated to boiling in a microwave oven to inactivate the enzymes. The primary milk was cooled to 71°C in the water bath and kept warm in a water bath (60°C). For oats, approximately 775 grams of primary milk was recovered from 200 g of grain and 800 g of water, with a solids content of 15% (the remaining 225 g was included in the fibrous slurry by-product). Based on this calculation, the yield was approximately 58%. Although additional washing and crushing cycles (108 in FIG. 1) may alter the results, in practice they are undesirable because of the increased time and energy required to perform the additional washing and crushing cycles, which increases costs. The process of the present disclosure reduces the need for additional washing and crushing cycles and improves efficiency.

[0141] The fibrous slurry was cooled to 82°C and filtered through a #100 or #120 mesh screen (washed). An additional 333mL or 400mL of cold water was added to the washed fibrous slurry portion and the mixture was blended for 30 seconds. The blended mixture was filtered through a #100 or #120 mesh screen to prepare the secondary milk. For the secondary oat milk, the mechanical process yielded 640 grams from 200g grains with 5% solids in the milk. For the secondary oat milk, the neutral protease L (NEUTB) treatment process (invention) yielded 600 grams from 200g grains with 8% solids in the milk. (Need to confirm / consult). (The inactivation heating resulted in 600g of water loss from steam evaporation compared to the previous sample of 640g).

[0142] The resulting secondary milk was mixed with the primary milk. The combined milk at 60°C was homogenized using a GEA Niro Sovavi® homogenizer at 2000 PSI (1500 psi on the first stage and 500 psi on the second stage) and then placed in a refrigerator. The pH and total solids content of the homogenized milk were measured. The organoleptic and other functional properties of the milk and milk-containing end products, including baristas, creamers and lattes (Table 5), were evaluated as shown in Table 4. The fiber fraction retained on the mesh screen was placed in a drying dish and dried in an oven at 93.3°C for approximately 16 hours until dry (moisture content less than 10%).

[0143] [Table 2]

[0144] Table 2 discloses the oat milk extraction processing protocol and milk yield. The fibrous slurry was prepared as described above. Test samples 1 and 2 were prepared using only mechanical process. Test samples 2 and 3 were added with enzymes and no separation was performed. Test samples 5 and 6 were added with enzymes and separation was performed. The measurements were calculated based on the initial raw material weight. The yield was measured based on dry matter.

[0145] [Table 3]

[0146] Table 3 discloses the chickpea soy milk processing protocol and milk yield. Fibrous slurry was prepared as described above. Test samples 1 and 2 were prepared using mechanical process. Test samples 2 and 3 were added with enzymes and no separation was performed. Test samples 5 and 6 were added with enzymes and separation was performed. The measurements were calculated based on the initial raw material weight. Yield was measured based on dry matter.

[0147] [Table 4]

[0148] Table 4 discloses the almond milk extraction processing protocol and milk yield. Fibrous slurry was prepared as described above. Test samples 1 and 2 were prepared using mechanical process. Test samples 2 and 3 had enzymes added and no separation. Test samples 5 and 6 had enzymes added and separation. Oat coffee creamer evaluated in hot coffee at pH 5.11. Barista base stock was evaluated in foaming agent. Almond latte made with barista base and coffee. Measurements were calculated based on initial ingredient weight. Yield was measured on dry matter basis.

[0149] [Table 5] TIFF2024522023000008.tif137159

[0150] Table 5 discloses the sensory and functional properties of milks and products prepared from expressed milk-like substrates. Fibrous slurries were prepared as described above. Samples 1 and 2 were prepared using only mechanical processes. Samples 2 and 3 had enzymes added and no separation was performed. Samples 5 and 6 had enzymes added and separation was performed. Oat coffee creamer evaluated in hot coffee at pH 5.11. Barista base stocks were evaluated in foaming agents. Almond latte made with barista base and coffee.

[0151] Example 1 shows that nutrient recovery from fibrous slurries by proteases is significant below 10° C., and in some cases the disclosed process can improve sensory properties.

[0152] Example 2 Different groups of enzymes were tested using fibrous slurries for their ability to increase oat milk yield using the disclosed method, as shown in Table 6. Proteases, amylases and xylanases were tested at the suboptimal enzyme activity temperature of 10°C. Alpha-amylase (AAMY), neutral protease L (NEUTB) and xylanase (XYL) were compared to an alpha-amylase only control. Table 6 shows the recovery of oat milk from fibrous slurries treated with various enzymes at temperatures below their optimum activity temperatures (10°C).

[0153] For the control, 200 g of oat grains were washed three times with ice-cold water and drained. The washed grains were placed in a blender cup (Vitamix®) with 800 mL of ice-cold water and 100 μl of α-amylase (DSM, AAMY), 60 mg of CaCl2 or 100 mg of CaCO3 were added. The mixture was blended for 2 minutes on high speed (10 / 10 setting) to prepare a primary slurry. The control was washed three times with water. This control sample substantially replicated the wet milling and mechanical extraction process used in the test samples.

[0154] In test sample 1, α-amylase was additionally added to the sample at 60 mg or 0.03% of the initial grain weight. In test sample 2, neutral protease L (BIOCAT) was added to the sample at 66 mg or 0.033% of the initial grain weight. In test sample 3, xylanase (XYL, BIO-CAT) was added to the sample at 66 mg or 0.033% of the initial grain weight.

[0155] The test slurries were incubated in a 10°C cold water bath for 2 hours with occasional stirring. After the 2 hour incubation, the slurries were heated to 79.5°C in a water bath for 15-20 minutes and then heated to boiling in a microwave oven. The heated fiber slurry was washed by filtering through a US#120 mesh screen while hot, approximately 82°C. It was washed again with 400 mL of water and blended with a Vitamix® for 30 seconds. The secondary milk was added to the primary milk. The by-product fiber portion was discarded after washing. The total solids in the milk were measured and recorded, and the total milk yield was calculated.

[0156] [Table 6]

[0157] Fibrous slurries were prepared as described above. In the control samples, only mechanical processes were used for extraction. Enzyme abbreviations are listed in a separate table.

[0158] Example 2 shows that combined treatment of fibrous slurry with neutral protease and amylase at 10°C results in a high yield increase, whereas treatment with amylase alone or in combination with xylanase under the same conditions results in a relatively low yield increase.

[0159] Example 3 On the other hand, in Example 3, samples were tested only with neutral protease treatment in the absence of amylase at different suboptimal activity temperatures. Incubation times were also varied. Samples were incubated at approximately 4°C, 7°C and 10°C as shown in Table 7.

[0160] [Table 7]

[0161] Fibrous slurries were prepared as described above. In the control sample, only mechanical processes were used for extraction. Weight percentages were determined based on dry matter. 34 mg NEUTB (BIOCAT) was added to test sample 1, corresponding to 0.173% of the initial grain weight. In test samples 2, 3 and 4, 66 mg Neutral Protease L (BIOCAT) was used, corresponding to 0.033% of the initial grain weight. The test sample slurries were incubated at different temperatures for different times with occasional stirring as shown in Table 6.

[0162] Example 3 shows that the addition of NEUTB to the fibrous slurry significantly increased yield under all test conditions including different temperature changes, different amounts of neutral protease L, and different incubation times. As noted above, at temperatures between 4.4 and 10°C, the viscosity of the fibrous slurry rapidly and significantly decreased within a few minutes of incubation. Heating the fibrous slurry did not increase viscosity, and the secondary milk was easily separated from the fibrous slurry by filtration. Test sample 3 (66 mg NEUTB, 10°C, and 60 min) showed the driest fiber slurry and the least slimy texture.

[0163] At different enzyme concentrations, different temperatures and different incubation times, NEUTB was effective in reducing the viscosity of the fibrous slurry. The yield increase at very low incubation temperature (4.4°C) and short incubation time (10 min) was significantly higher than that of the control sample.

[0164] According to Example 3, as shown in Table 7, row 4, the disclosed process using neutral protease L treatment at 10° C. for 10 minutes increased the yield of oat grain by approximately 7-8% of the total solids, as shown in Table 6. The disclosed process at 10° C. for 1 hour results in an increase in yield of approximately 9-10% of the total solids.

[0165] Example 4 The yield of fibrous slurry treated with NEUTB was evaluated at a pH lower than the enzyme activity pH and at a low temperature (10° C.), as shown in Table 8. To further investigate whether activities other than the protease activity of NEUTB may be responsible for the effect of the present disclosure on yield, the process was carried out at a pH of approximately 4.96 to 5.3. The results of this testing are shown in Table 8. At pH 4.5, NEUTB is expected to be inactive or minimally active, given the enzyme activity curve provided and published by the enzyme supplier, BIOCAT (shown in FIG. 3). At the combination of low pH 9 and low temperature shown in Table 7, NEUTB is essentially inactive.

[0166] [Table 8]

[0167] Fibrous slurries were prepared as described above. The control fibrous slurries contained the addition of α-amylase, while the test samples contained the addition of neutral protease L (NEUTB).

[0168] At the end of the incubation, the pH of Test Sample 2 was 5.3. The slurries containing NEUTB (Tests 1 and 2) showed a significant viscosity drop a few minutes after addition. The viscosity drop in the pH unadjusted NEUTB sample (Test 1) was more rapid than that in the pH adjusted sample (Test Sample 2). However, the sample with added α-amylase (control) did not show any viscosity drop. In addition, the NEUTB treated samples (Tests 1 and 2) showed fiber separation and settling during the incubation (milk separation was observed at the top). The NEUTB treated fibrous slurries became less uniform with a whiter upper layer and a darker lower layer. On the other hand, the α-amylase treated fibrous slurries maintained their uniformity and color. This effect correlates with the ease of filtering the NEUTB treated product, since less mechanical force is required to separate the product; gravity alone is sufficient to filter the product. In some embodiments, particularly in commercial embodiments, filtration may be performed by a mechanical sieve.When using the NEUTB process of the present disclosure, the time and energy required to filter the fibrous slurry using a sieve may be reduced; or, in some cases, the need for a mechanical sieve may be eliminated.

[0169] Example 4 shows that nutrient extraction is high even under conditions of pH and temperature that would prevent or strongly inhibit protease activity. The yield in the amylase control sample was 40.36% of the total solids of the fibrous slurry. The yields in the NEUTB treated samples were 52.88% and 49.81% of the fibrous slurry for test samples 1 and 2, respectively.

[0170] Example 5 Table 9 shows the recovery of milky oats from NEUTB treated fibrous slurry at pH below the enzyme activity pH and at low temperature (10° C.). To further explore whether activities other than protease activity in NEUTB may be responsible for the observed yield increase, the process was run at a pH of approximately 4.5. At pH 4.5, the neutral protease is expected to be inactive or minimally active, as can be seen in FIG. 3. The combination of low pH and low temperature should theoretically inactivate the neutral protease.

[0171] [Table 9]

[0172] The fibrous slurry was prepared as described above. In Table 8, the milk yield (%) was calculated as the % of total solids of the fibrous slurry that was incorporated into the secondary milk, not the secondary and primary milk combined.

[0173] The NEUTB-added slurries showed a significant viscosity drop 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. This observation was confirmed in the examples below using a texture analyzer; at a low temperature of 2°C, a significant viscosity drop in the neutral protease-treated oat fibrous slurry occurred within 5 minutes after the enzyme was added to the by-product. The viscosity drop in the neutral unadjusted NEUTB samples was more rapid than the pH-adjusted samples. The α-amylase control samples showed no viscosity drop. The basic pH samples showed a very slow viscosity drop, but a rapid viscosity drop near the end of digestion. The rapid viscosity drop in Test Sample 3 may be related to the pH change below 10 during incubation.

[0174] There was a difference in yield increase between the samples adjusted to acidic pH in Tables 7 and 8; the condition in Table 7 had a 49.81% increase in yield compared to the condition in Table 8, which had a 41.4% increase in yield. This difference may be related to slight pH changes during digestion. For the experiments shown in Table 7, the pH of the low pH fibrous slurry ranged from below 5.0 (4.96) at the start of incubation to a pH just above 5.0 (5.3; data not shown) at the end of incubation; on the other hand, the pH for the experiments in Table 8 remained below 5.0 (4.62-4.99) throughout the course of enzymatic digestion of the fibrous slurry.

[0175] As can be seen from Figure 2, NEUTB is expected to be minimally active at 10°C; and as can be seen from Figure 3, neutral proteases are expected to be essentially inactive below pH 5.0. It can therefore be hypothesized that there is significant atypical protease activity that causes extraction and a corresponding increase in nutrient yield. This atypical activity may involve disruption of cellular structure by means other than hydrolysis of large protein molecules into smaller molecules by protease activity.

[0176] At pH levels above 5.0, proteolysis may be active or more active in low pH fibrous slurry samples, as indeed occurred in some of the low pH treatments shown in Table 7, which may result in potential synergistic effects with putative non-proteolytic activities. This synergistic effect may explain the observed yield increases in addition to the yield increases due to the putative non-proteolytic activity of NEUTB as shown in Table 7. The non-proteolytic activity of NEUTB observed at low pH and low temperature may theoretically be related to a second enzymatic activity, such as plastein activity, a known activity in neutral proteases. In combination with the second activity, the proteolytic activity of this protease may synergistically increase yield as a result of the potential synergistic effects of protease and non-protease activities.

[0177] The low temperature and low pH experimental data in Table 8 indicates that the yield increase at a pH below 5.0 and 10° C. was approximately 80% of the yield increase when the process was performed at the optimum pH. Thus, the difference between conditions between low protease activity (10° C.) and estimated negligible protease activity (10° C., pH below 5) was approximately 20%. This result suggests that most of the yield increase may be related to non-proteolytic enzyme activity.

[0178] Neutral proteases are known to have highly active plastein activity at 10 °C (Xu et al., 2014); Dermiki and Fitzgerald also reported that plastein synthesis generally requires a pH in the range of 3.0-7.0 (2020). Without being limited to theory, the plastein reaction is one of the possible explanations for the efficient extraction at about pH 4.8 at 10 °C. Plastein is known to aggregate protein molecules, so plastein activity may attract proteins and cause dissociation from fibrous materials.

[0179] It is also possible that other unknown or unidentified activities of NEUTB or Neutrase® may be responsible for the observed yield increase. For example, the reaction may involve protein / fiber interactions, such as β-glucan or other fibrous molecules of the cell wall, as the substrate may be an important factor in the observed effect. Regardless of the mechanism, the level of increase in yield obtained from a fibrous slurry under suboptimal conditions is unexpected and surprising, given the conditions tested and the known activity of NEUTB under these conditions.

[0180] Example 6 Example 6 shows protease extraction of fibrous slurry at high and low temperatures. These temperature conditions correspond to the conditions for treating the samples shown in the SDS-PAGE gel of Figure 4 in the manner described in Table 10. The test lane of the SDS-PAGE gel shows the protein size of the fibrous slurry proteins after protease treatment. The SDS-PAGE gels of Figures 4 and 5 provide some understanding of the degree of protein hydrolysis of the fibrous slurry as well as the mechanism of action of protease extraction.

[0181] [Table 10]

[0182] For Example 6, a fibrous slurry was prepared as described above. As shown in Table 10, the control lanes did not receive any enzyme addition to the fibrous slurry. Test sample 1 includes unheated slurry with digestive protease added at high temperature. Test sample 2 includes unheated slurry with digestive protease added at low temperature. Test sample 3 includes slurry heated prior to protease addition with digestive protease added at high temperature. The degree of hydrolysis was determined as described above. Yield increase was calculated based on total solids of the secondary milk only.

[0183] To determine whether the degree of hydrolysis of the protease-treated fibrous slurry was related to the observed yield increase, the level of yield increase was measured for the protease-treated slurries at high temperature (57° C.), low temperature (10° C.), and at high temperature (55° C.) after previously boiling the fibrous slurry. Replicates of these conditions were then prepared for SDS-PAGE analysis.

[0184] For Table 10, oat fibrous slurry samples were digested with NEUTB (BIO-CAT). Nutrient yields from the fibrous slurry were measured. The yield increase from protease digestion at low temperature (10°C) was similar to that from digestion at high temperature (57°C) and from high temperature heating where the samples were first boiled (57°C after boiling). The results unexpectedly indicate that high protease extraction occurs at low temperature, but the degree of hydrolysis is low.

[0185] The number of washes is related to the yield of the amylase treatment. Extraction from the second wash will show an increase in yield over the amylase treatment control; this is because the washing / breaking process alone extracts some nutrients. After the third wash, there will be nothing else taken out by the washing alone; what comes from the second wash becomes part of the primary milk. The third wash produces nothing at all for extraction. Thus, for the process of this disclosure, the fibrous slurry that is separated for protease extraction is the one remaining after the second wash. The control column in Table 9 shows the extraction with the third wash water.

[0186] SDS-PAGE gel electrophoresis was performed to show the effect of protease treatment on the size of proteins in the fibrous slurry, as shown in Figure 4. Lane #812 contains the heated sample and shows the proteins of the fibrous slurry treated with NEUTB after boiling in a microwave oven. Lane #752 is the control sample and shows the proteins of the fibrous slurry not treated with protease. Lane #243 shows the proteins of the fibrous slurry treated with protease at a higher (optimal) temperature (57°C for 2 hours, optimal for NEUTB). Lane #277 shows the proteins treated with protease at a lower temperature of 10°C for 1 hour.

[0187] The heated sample control in lane #812 showed a high degree of protease hydrolysis. The control lane #752 showed intact protein from the protease untreated fibrous slurry. Major bands were present at 35 kDa and 22 kDa with minor bands between these two. Lane #812, showing protein from the heated and protease treated fibrous oat slurry, showed a high degree of hydrolysis (DH); a large band at 35 kDa that was completely hydrolyzed by the protease; increased intensity in the 14 kDa and 12 kDa bands that likely represent hydrolysis products of the 35 kDa band; and an increase in hydrolysis products from 0 to 12 kDa.

[0188] The high reaction temperature condition shown in lane #243 at 57°C for 2 hours showed significant hydrolysis of the 35 kDa band compared to the control. There was also some increase in the 14 kDa and 12 kDa bands, which are likely hydrolysis products of the 35 kDa band. The decrease in intensity of the 35 kDa band is expected to be due to protease hydrolysis at the optimal temperature. There was some increase in the 0-12 kDa digests with protease at higher temperatures.

[0189] Low temperature protease treatment is shown in lane #277. This sample showed high levels of nutrient yield increase close to that of treatment under optimal protease conditions. However, in contrast to the high temperature treated fibrous slurry (#243), the low temperature treated fibrous slurry (#277) did not adversely affect the sensory properties of the #243 sample; it was not exposed to conditions that may cause microbial growth or protein denaturation; and it showed no significant signs of hydrolysis compared to the control lane #752. In summary, the #277 sample surprisingly showed a very low DH while increasing yield to a significant extent, which likely contributes to its good sensory and taste properties.

[0190] Example 7 Table 11 shows the amount of total dietary fiber and β-glucan in oat milk obtained from the fibrous slurry NEUTB treated at or below 10° C. The amount of β-glucan recovered from the fibrous slurry was measured using the combined samples disclosed in Table 6.

[0191] [Table 11]

[0192] In Example 7, multiple test samples of fibrous slurries treated with Neutral Protease L (NEUTB) at 10°C or less for different incubation times were combined to obtain sufficient material for β-glucan content analysis. β-glucan content analysis was performed at Medallion Labs (Minneapolis, MN, USA). Control samples were subjected to mechanical process only without enzyme addition. Test samples included fibrous slurries treated with Neutral Protease L (NEUTB) at 10°C or less for different incubation times. Percentage calculations were based on dry matter. Primary milk is expected to contain approximately 1% β-glucan at the beginning. An additional 0.6% can be added by washing the fibrous slurry multiple times. However, protease treatment of the fibrous slurry can increase β-glucan by more than two-fold, as shown in Table 11 for the combined data of primary and secondary milk.

[0193] Example 8

[0194] Example 8 relates to the general composition and yield of secondary oat milk obtained from a fibrous slurry treated with neutral protease L (NEUTB) at 2°C.

[0195] [Table 12]

[0196] The fibrous slurry was prepared as described above. No enzyme was added to the control sample. The NEUTB sample contained fibrous slurry treated with NEUTB for 120 min at 2°C. To obtain the tested by-products, the raw milk was heated to 77°C in a water bath for 7 min and then boiled in a microwave oven to inactivate the enzymes. Measurements were performed on dry matter. The total milk yield was determined for the combination of primary and secondary milk. The secondary milk potentially contains up to 35% starch and has a lower starch content than the primary oat milk, which is expected to be advantageous for obtaining a plant-based low-sugar or plant-based sugar-restricted milk.

[0197] In separate pilot experiments designed to measure yield increases for individual nutrients, with NEUTB treatment at 10°C for 2 hours, the increase in total yield was approximately 10% protein, 15% fat, and 9% ash when broken down individually, but fiber measurements require further testing. The secondary milk had a good taste, described as more oat-like than the primary milk, and also had a good texture. The high amount of β-glucan contained in the fibrous slurry was likely extracted into the secondary milk and contributed to the full body of the secondary milk. The secondary milk produced by the process of the present disclosure did not have a high degree of protein hydrolysis associated with bitterness. The addition of the secondary milk to the primary milk did not detract from the overall taste or texture of the primary milk.

[0198] Example 9 Example 9 discloses the yield, milk quality and degree of protein hydrolysis (DH) of fibrous slurries treated with different proteases.

[0199] [Table 13]

[0200] The fibrous slurry was prepared as described above. Weight percentages were based on initial raw material weight. Yields were determined for the combined primary and secondary oat milk. Yields were measured on a dry matter basis. Enzyme inactivation for the primary milk was performed by heating to 77°C in a water bath for 15-20 minutes followed by heating to boiling in a microwave oven. Enzyme inactivation of the fibrous slurry to produce the secondary milk was performed using the steam method as described above. Foam quality, sensory attributes and DH were determined as described above.

[0201] The data in Table 13 is based on the data in Figures 5A and 5B showing SDS-PAGE of samples of protease digested oat fibrous slurry according to the present disclosure. The yield increase compared to the control was highest in the fibrous slurry treated with NEUTB at low temperature according to the present disclosure and further treated with trypsin. The proteases papain and alkaline protease showed substantially lower yield increases. Amylase showed the lowest yield increase. Other experimental data suggested that the effect of NEUTB on viscosity and yield increase was similar to that of Neutrase® (data not shown).

[0202] Among the proteases tested, NEUTB and trypsin had the lowest DH, but in this case the DH calculated by the above method was approximately 2%. The DH of papain was approximately 4-fold higher than that of NEUTB and trypsin; and the DH of alkaline protease was approximately 3-fold higher than that of NEUTB and trypsin.

[0203] The results suggest that DH does not correlate with yield increase, but NPL and trypsin provide greater yield increases at a much lower degree of hydrolysis. This result was unexpected, since it is generally believed that hydrolysis of proteins or other organic molecules results in a significant decrease in viscosity. Lower molecular weight generally correlates to a less viscous solution. Maximizing the yield increase obtained from the fibrous slurry while minimizing protein degradation is a key focus of this disclosure, since it maintains the functional properties of the protein and minimizes sensory property changes. Of all the proteases tested, the neutral protease and trypsin disclosed herein were the only two proteases that met the requirements of this disclosure in these respects.

[0204] Example 10 Example 10 discloses the effect of various proteases on the viscosity of oat fibrous slurry. Viscosity reduction is a key factor in enabling the processing of fibrous slurries. Table 14 shows the relative viscosity change of oat fibrous slurries treated with various enzymes at 2°C.

[0205] [Table 14]

[0206] The fibrous slurry was prepared as disclosed above. Texture analysis was used to measure the viscosity change of the fibrous slurry after enzyme treatment. Texture analysis was performed as described above. Texture analysis can be used to measure the viscosity change, where the compressive force change over time during the reaction is measured with a texture analyzer. The decrease in compressive force over time as measured with the texture analyzer correlates to the decrease in viscosity over time.

[0207] As previously disclosed, the initial viscosity after preparation of the fibrous slurry for texture analysis was set to 1.0. After enzyme addition, the texture analyzer continuously measures the compressive force exerted on the sample by the presence of enzyme activity. The last measurement in Table 14 shows the amount of viscosity reduction at a specific time point, which in this case is 10 minutes.

[0208] Table 14 shows that the neutral proteases tested herein, NEUTB and Neutrase®, are the most effective at reducing oat fibrous slurry viscosity at 2° C. Trypsin is somewhat less effective, but is also effective at substantially reducing the viscosity of the fibrous slurry. The fungal proteases FGPTA2 and FGPTHU reduce viscosity to a lesser extent. Fungal proteases are a complex mixture of enzymes, but may include the neutral proteases and trypsin of the present disclosure along with other enzymes.

[0209] In general, all other proteases or enzymes tested provided less viscosity reduction compared to NPL, Neutrase®, and trypsin. The lower levels of viscosity reduction provided by enzymes other than neutral proteases and trypsin shown in Table 14 were insufficient for purposes of this disclosure. Given that viscosity reduction generally correlates with increased yield from a fibrous slurry, neutral proteases and trypsin were determined to be effective for purposes of this disclosure, while other proteases were substantially less effective.

[0210] Example 11 Example 11 discloses the relative viscosity change of oat fibrous slurries treated with Neutral Protease L (NEUTB) at different enzyme concentrations at 2°C.

[0211] [Table 15]

[0212] Fibrous slurries were prepared as disclosed above. Enzyme concentrations were based on the initial raw material weight. Texture analysis was performed as described above to measure viscosity reduction.

[0213] Substantial viscosity reduction was evident even at very low NPL concentrations, suggesting that very low levels of NPL or Neutrase® are sufficient to achieve the increased yields obtained from the fibrous slurries of the present disclosure.

[0214] Example 12 Example 12 discloses the relative viscosity change of oat fibrous slurries treated with different enzyme concentrations of microbial trypsin (TRY1) at 2°C.

[0215] [Table 16]

[0216] Fibrous slurries were prepared as disclosed above. Enzyme concentrations were based on the initial raw material weight. Texture analysis was performed as described above to measure viscosity reduction.

[0217] Substantial viscosity reduction was evident even at very low concentrations of trypsin, but the viscosity reduction with trypsin appeared to be more concentration dependent when compared to NPL. In general, these results suggest that low levels of trypsin are sufficient to achieve increased yields from the fibrous slurries of the present disclosure.

[0218] Example 13 Example 13 shows the relative viscosity change of oat fibrous slurries without added enzymes at various pH values ​​at 2°C.

[0219] [Table 17]

[0220] A fibrous slurry was prepared as disclosed above. No enzyme addition was performed. The pH was adjusted using citric anhydride and 50% KOH solution. Texture analysis was performed as described above to measure viscosity reduction.

[0221] The pH of the solutions was adjusted prior to texture analysis. Neutral or unadjusted pH was also tested. After a small initial drop in pH likely due to mechanical disruption, all samples gradually increased toward 1.0. At acidic pH, the viscosity of the fibrous slurry was essentially unchanged after 10 minutes. At basic pH, the viscosity of the fibrous slurry decreased by approximately 10% after 10 minutes. Based on these results, pH does not significantly affect viscosity at the levels tested.

[0222] Example 14 Example 14 shows the effect of pH on the ability of NEUTB to reduce the viscosity of oat fibrous slurries. The relative viscosity change of oat fibrous slurries treated with Neutral Protease L (NEUTB) was measured at various pH values ​​at 2°C.

[0223] [Table 18]

[0224] The fibrous slurry was prepared as disclosed above. The pH was adjusted using citric anhydride and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described above. Table 18 shows the pH of the fibrous slurry after addition of acid or base but before addition of 0.05% enzyme.

[0225] Example 15 Example 15 shows the effect of pH on the ability of microbial trypsin (TRY1) to reduce the viscosity of an oat fibrous slurry. The change in viscosity of an oat fibrous slurry treated with microbial trypsin (TRY1) was measured at various pH values ​​at 2°C.

[0226] [Table 19]

[0227] The fibrous slurry was prepared as disclosed above. The pH was adjusted using citric anhydride and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described above. Table 18 shows the pH of the fibrous slurry after addition of acid or base but before enzyme addition.

[0228] Example 16 Example 16 shows the relative viscosity change of fibrous slurries containing various substrates when treated with neutral protease L (NEUTB) or microbial trypsin (TRY1) at 2° C. in accordance with the present disclosure.

[0229] [Table 20] TIFF2024522023000024.tif46159

[0230] The fibrous slurry was prepared as disclosed above. The pH was adjusted using citric anhydride and 50% KOH solution. Texture analysis to measure viscosity reduction was performed as described above.

[0231] Chicken skin testing was performed at 2°C, 49°C and 60°C. For chicken skin viscosity analysis, chicken skin was tested as described below. For chicken skin texture analysis, skin was obtained from fresh chicken thigh quarters by peeling the skin from the muscle. The skin was washed with approximately two volumes of ice water (by weight) and sliced ​​and chopped into approximately 5x5mm pieces using a sharp knife and cutting board in a walk-in refrigerator (1.7°C).

[0232] The shredded chicken skin was mixed with either 2 or 3 times the amount of chicken skin plus ice + cold distilled water to a final solids content of approximately 10%. The mixture was then mixed for 2 minutes at high speed (10 / 10 setting) using a Vita-Mix TurboBlend 4500. For the chicken that was mixed with the shredded chicken skin plus 2 times the amount of ice water, the high concentration slurry had approximately 15% solids. For the low solids concentration, 3 times the amount of ice water was added; the slurries for texture analysis had approximately 10% solids, respectively.

[0233] Chicken skin testing was performed in part because the BIOCAT product information sheet for NEUTB suggests that Neutrase may be used to reduce the viscosity of fish and chicken by-products, among other things. The product information sheet also provides information about the optimum activity conditions for using NEUTB, described as 55° C. and pH 6.5. This optimum temperature as described by BIOCAT is much higher than the temperatures used in this disclosure, and therefore the lower suboptimal temperatures used in this disclosure were tested using chicken skin, one of the substrates listed in the BIOCAT product information sheet. As shown in Table 20, no viscosity reduction was observed with NEUTB (NEUTB) at temperatures within the range of this disclosure (2° C.).

[0234] For the chicken skin viscosity measurements, the chicken skin slurry was allowed to sit in a walk-in refrigerator for 30 minutes and then the viscosity change of the chicken skin was measured using the same parameters as the texture change measurements for grains and nuts. In addition to the texture analysis at 2° C., the chicken skin slurries were warmed to 49° C. and 60° C. prior to enzyme addition and texture analysis. The chicken skin slurry temperature during the texture change analysis was maintained the same as the initial temperature by placing the texture analysis cup in an ice-cold water bath, warm water, or hot water bath during the course of the texture analysis.

[0235] For the overall data shown in Table 20, the yield data in Tables 2-4 and the viscosity reduction in Table 20 indicate that there is a close correlation between increased milk yield and reduced viscosity in texture analysis. Oat fibrous slurry had a larger viscosity reduction and therefore a larger increase in oat milk yield with the disclosed process; whereas, the viscosity reduction in almond and chickpea was not as large as oat and similarly the increase in milk yield was also smaller. Thus, the viscosity reduction in the texture analyzer is useful for predicting increased yield of plant-based milk. Based on the data in Table 20, it is believed that the viscosity reduction from the disclosed protease treatment was synergistic with the presence of β-glucan in the matrix material. Although the present disclosure is described primarily as a low temperature protease treatment process, the process also has application in some embodiments to extract nutrients from β-glucan-containing milled cereal grains at higher temperatures.

[0236] The β-glucan-containing substrates tested in this disclosure are oats and barley, which showed a much greater viscosity reduction, even when the starting viscosity of a non-β-glucan-containing substrate, such as soybean, is similar to that of oats and barley. As shown in Table 20, for barley, the relative viscosity reduction at 10 minutes for the control (0.90) is compared to treatment with NEUTB (0.67) and trypsin (0.74).

[0237] Example 17 Example 17 shows the change in viscosity measured in centipoise (cPs) for an uncooked oat fibrous slurry at 2° C. for 22 minutes.

[0238] [Table 21]

[0239] The fibrous slurry was prepared as disclosed above. The viscosity was measured by a viscometer as described above. In this specification, "Pre" refers to before the enzyme addition and "Post" refers to after the enzyme treatment is completed.

[0240] The pH was essentially unchanged before and after enzyme treatment. NPL and trypsin showed similar viscosity reductions, but the viscosity reduction by NPL was greater than that by trypsin.

[0241] Example 18 Example 18 shows the viscosity and other properties of secondary oat milk obtained from a fibrous slurry when treated with NPL and trypsin together with α-amylase for 2 hours at 2°C and slower (non-steam) enzyme inactivation was performed.

[0242] [Table 22]

[0243] Fibrous slurries were prepared as disclosed above. Viscosity was measured by viscometer as described above. Sensory properties were evaluated as described above. Samples were heated to 77°C in a water bath for 7 minutes and then heated to boiling in a microwave oven for less than 2 minutes as described above for enzyme inactivation. Sample concentrations were based on initial ingredient weight. Sieving was evaluated using a 5-point scale: (1) very easy to sieve, (3) neither difficult nor easy to sieve, and (5) very difficult to unsievable. For the samples of Examples 18, 20, and 21, the milks of the combined samples were combined, oven dried, and β-glucan was measured by Medallion labs.

[0244] Example 19 Example 19 relates to the properties of secondary oat milk obtained from a fibrous slurry treated with different enzymes together with α-amylase at 2° C. for 2 hours and followed by slow (non-steam) heat inactivation of the enzymes.

[0245] [Table 23]

[0246] Fibrous slurries were prepared as disclosed above. Viscosity was measured with a viscometer as described above. Sensory attributes were evaluated as described above. Samples were heated to 77°C in a water bath for 7 minutes and then heated to boiling in a microwave oven for less than 2 minutes as described above for enzyme inactivation. Sample concentrations were based on initial raw material weight. Sieving was rated using a 5-point scale: (1) very easy to sieve, (3) neither difficult nor easy to sieve, and (5) very difficult to impossible to sieve.

[0247] Example 20 Example 20 shows the viscosity and other properties of secondary oat milk slurries treated with different enzymes for 2 hours at 2°C in the absence of α-amylase and following slow (non-steam) heat inactivation of the enzymes.

[0248] [Table 24]

[0249] Fibrous slurries were prepared as disclosed above. Viscosity was measured with a viscometer as described above. Samples were heated to 80°C for 0.5 minutes by direct steam injection into the slurry using a Nuova Simonelli Appia II V GR1, followed by further heating to boiling in a microwave oven for less than 1 minute as described above. Measurements were based on the initial material weight. Sieving was rated using a 5-point scale as described above: (1) very easy to sieve, (3) neither difficult nor easy to sieve, and (5) very difficult to impossible to sieve.

[0250] Example 21 Example 21 shows the effect of rapid enzyme inactivation (steam treatment) on the viscosity and other properties of secondary oat milk obtained from a fibrous slurry treated with NPL and trypsin for 2 hours at 2°C in the absence of α-amylase.

[0251] [Table 25]

[0252] Fibrous slurries were prepared as disclosed above. Viscosity was measured with a viscometer as described above. Samples were heated to 80°C for 0.5 minutes by direct steam injection into the slurry using a Nuova Simonelli Appia II V GR1, followed by further heating to boiling in a microwave oven for less than 1 minute as described above. Measurements were based on the initial material weight. Sieving was rated using a 5-point scale as described above: (1) very easy to sieve, (3) neither difficult nor easy to sieve, and (5) very difficult to impossible to sieve.

[0253] Example 22 Example 22 shows the starting viscosity, pH and solids content of untreated dilute fibrous slurries of different materials for texture analysis at 2°C. Steam injection gave a somewhat superior product compared to slower heat enzyme inactivation. Table 25 shows that in the absence of α-amylase, the combination of NEUTB and steam inactivation (or rapid inactivation) gave a product with superior organoleptic properties and easier sieving. NEUTB also gave a lower viscosity, but not as much as the trypsin protease; otherwise, all were effective in the present disclosure. Microbial trypsin was shown to be effective, but not as effective as the metalloproteases in terms of viscosity reduction and yield according to the present disclosure; in some respects, NEUTB and Neutrase® were more effective, as can be seen from Table 25.

[0254] [Table 26]

[0255] The fibrous slurries were prepared as disclosed above. Measurements are expressed as mean ± standard deviation.

[0256] The data in Table 26 shows the starting concentrations of the untreated fibrous slurry and can be used as a general reference for other data provided in this disclosure.

[0257] Example 23 Example 23 shows the relative viscosity change of a 10% chickpea protein isolate slurry treated with different enzymes at 2°C or 50°C.

[0258] [Table 27]

[0259] Protein slurries were prepared as described above. Temperature is the incubation temperature during texture analysis. Initial viscosity before enzyme addition and texture analysis.

[0260] The results show that the neutral proteases and trypsin of the present disclosure, which are effective in reducing the viscosity of oats, barley and other plant materials, are completely ineffective in reducing the viscosity of a 10% chickpea protein isolate slurry over a 20 minute reaction time at 2° C. At 50° C., NEUTB reduced the viscosity of the chickpea material by approximately 25%.

[0261] Example 24 Example 24 discloses the relative viscosity change of a 21% pea protein isolate slurry treated with different enzymes at 2°C or 50°C.

[0262] [Table 28]

[0263] Protein slurries were prepared as described above. Temperature is the incubation temperature during texture analysis. Initial viscosity before enzyme addition and texture analysis.

[0264] The results show that the neutral proteases and trypsin of the present disclosure, which are effective in reducing the viscosity of oats, barley and other plant materials, are completely ineffective in reducing the viscosity of the pea protein isolate slurry at 2° C. At 50° C., NEUTB reduced the viscosity of the chickpea material by approximately 8% after 20 minutes of incubation.

[0265] Other embodiments While the present invention has been described as a detailed description of the invention, it is to be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined in the appended claims. Other aspects, advantages, and modifications are intended to be encompassed by the appended claims.

Claims

1. A method of producing a feedstock slurry from a ground plant material; separating the feed slurry to produce a primary liquor and a fibrous slurry; treating the fibrous slurry with a protease that is an endoprotease; treating the fibrous slurry with the protease under suboptimal protease activity conditions to produce a treated fibrous slurry; and separating the treated fibrous slurry to produce a secondary liquor and clean fibers; The method includes:

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

3. 2. The method of claim 1, wherein the suboptimal protease activity conditions are a suboptimal protease activity temperature of less than 10°C and an incubation time for treatment with the protease of less than 30 minutes.

4. 2. The method of claim 1, wherein the suboptimal protease activity condition is a suboptimal protease activity pH.

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

6. 2. The method of 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. 10. The method of claim 1, further comprising combining the secondary liquid with the primary liquid to produce a combined liquid.

8. 10. The method of claim 1, wherein the β-glucan concentration in the secondary liquor is at least twice the β-glucan concentration of the primary liquor on a dry solids basis.

9. 10. The method of claim 1, wherein the combination of the first liquor and the second liquor comprises at least half of the total β-glucan contained in the plant material on a dry solids basis.

10. 2. The method of claim 1, wherein the protease is Bacillolysin.

11. 10. The method of claim 1, wherein the fibrous slurry is rapidly heated to limit protein hydrolysis during heat inactivation.

12. 10. The method of claim 1, wherein the method further comprises treating the secondary liquid at a high temperature sufficient for shelf-life extension or aseptic packaging without the addition of additional enzymes for liquefaction.

13. 10. The method of claim 1, wherein the fibrous slurry is rapidly heated by direct or indirect steam treatment to thermally inactivate the metal endoprotease and to limit proteolysis during heat inactivation, thereby producing a secondary liquor having at least one superior organoleptic property, which secondary liquor has improved sievability compared to secondary liquor produced using slower heat inactivation methods.

14. The method of claim 1, comprising: grinding untreated cereal grain containing β-glucan and producing a feedstock slurry from the ground untreated cereal grain containing β-glucan; sieving the feed slurry to produce a primary liquor and a fibrous slurry; treating the fibrous slurry with a protease that is a metalloendoprotease; treating the fibrous slurry with the metalloendoprotease at a temperature of less than 5° C. for an incubation time of less than 30 minutes to produce a treated fibrous slurry; and separating the treated fibrous slurry to produce a secondary liquor and clean fibers; The method includes:

15. 20. The method of claim 18, further comprising treating the secondary liquid at a high enough temperature for shelf-life extension or aseptic packaging without the addition of additional enzymes for liquefaction.

16. The method of claim 1, comprising grinding a β-glucan-containing unprocessed feedstock and producing a feedstock slurry from the ground β-glucan-containing unprocessed feedstock; sieving the feed slurry to produce a primary liquor and a fibrous slurry; treating the fibrous slurry with a metal endoprotease by incubating for less than 60 minutes to produce a treated fibrous slurry; 1. A method comprising: wherein the viscosity of the treated fibrous slurry is at least 30% lower than the viscosity of the fibrous slurry; method.

17. The method of claim 1, comprising: grinding a plant material and producing a feedstock slurry from the ground plant material; separating the feed slurry to produce a primary liquor and a fibrous slurry; treating the fibrous slurry with the enzyme under suboptimal enzyme conditions to produce a treated fibrous slurry; and separating the treated fibrous slurry to produce a secondary liquor and clean fibers; The method includes:

18. A method for producing a feedstock slurry from the ground β-glucan-containing untreated cereal grain; sieving the feed slurry to produce a primary liquor and a fibrous slurry; treating the fibrous slurry with the enzyme at a temperature of less than 10° C. for an incubation time of less than 30 minutes to produce a treated fibrous slurry; and separating the treated fibrous slurry to produce a secondary liquor and clean fibers; The method includes:

19. A composition comprising a secondary liquid comprising a liquid portion separated from a solid portion of a fibrous slurry, said secondary liquid being obtained from an enzymatic extraction of bran of a cereal grain containing β-glucan, and said secondary liquid comprising a modifying enzyme.

20. The composition of claim 19, wherein the modified enzyme is bacillolysin.

21. The method of claim 1, wherein the milling step is carried out by aqueous wet milling.

22. The method of claim 1, wherein the suboptimal protease activity condition is a suboptimal protease activity temperature.

23. The method of claim 1, wherein the endoprotease is a metalloendoprotease.

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