Method for producing protein isolate from cereal material and protein isolate produced from cereal material

Abstract

The present invention provides a method for producing a protein isolate from a grain material, which comprises treating a liquid protein stream with a bleaching agent to reduce the color of the protein isolate. The present invention also provides the protein isolate and food and beverage products containing the protein isolate.

Description

[Technical Field]

[0001] The present invention relates to a method for isolating a protein isolate from grain material, particularly brewer's spent grain, by enzymatic hydrolysis, which method comprises treating a liquid protein stream with a bleaching agent. The present invention also relates to the protein isolate isolated from the grain material and to food and beverage products comprising the protein isolate. [Background technology]

[0002] The use of protein isolates and supplements is widely known in the art. For example, many people utilize protein isolates to create beverages or other foods as part of their training regimen to provide additional protein for muscle growth. In addition, people may utilize protein supplements when their daily diet is insufficient to meet the daily protein needs of the human body. Furthermore, individuals with certain dietary restrictions that prevent them from consuming traditional meat-based protein sources may supplement with protein isolates to meet their daily needs.

[0003] Traditionally, protein isolates and supplements are generally whey-, soy-, or casein-based products. Whey and casein proteins are typically recovered as by-products of dairy production, with whey isolated from cheese production and casein isolated from milk. Soy protein is isolated from soybeans. Whey-, soy-, and casein-based protein powders and supplements can provide beneficial amounts of protein, but the latter are not always suitable for people with food intolerances or allergies, such as lactose intolerance. While some plant-based protein isolates are less immunogenic, these products are generally perceived as less palatable and have lower solubility than comparable whey products, for example. This may discourage consumers from choosing these alternatives.

[0004] Brewer's grains sold as slag (BSG) is the most abundant by-product produced in the beer brewing process. This material contains malt and grain husks and is obtained as the solid fraction after mash filtration or lautering processes. To date, this brewery by-product has been used primarily for low-value applications, particularly as livestock feed.

[0005] BSG is highly nutritious, particularly rich in protein and fiber. Protein isolates using BSG have been produced, as disclosed in International Publication No. 2021 / 0258509A1, U.S. Patent No. 2018 / 0199593A1, and U.S. Patent No. 2018 / 0199594A1. Protein isolates isolated from BSG have been found to be dark in color and sometimes have a bitter taste, which may reduce their usefulness in preparing protein-fortified foods and beverages. Attempts have been made to bleach protein isolates obtained from other protein sources, such as whey (Jervis et al., Effect of bleaching whey on sensory and functional properties, J. Dairy Sci., 2012, 95(6), 2848-2862). However, the present inventors have found that the conditions used in the prior art do not sufficiently reduce the brightness and color of protein isolates isolated from BSG. Therefore, there remains a need for a method to isolate protein isolates from BSG that are light in color and have a low flavor and aroma. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to a method for isolating a protein isolate from grain material, particularly BSG, which involves enzymatic hydrolysis and treating the liquid protein stream with a bleaching agent. The present invention also relates to protein isolates that are light in color and have a low overall flavor and aroma, and which can be easily blended with other ingredients to produce protein-enriched edibles. The present invention further relates to food and beverage products comprising the protein isolate of the present invention. [Means for solving the problem]

[0007] According to a first aspect, the present invention provides a method for isolating a protein isolate from a cereal material, comprising the steps of: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c) subjecting the liquid protein stream to one or more filtration steps; and d) treating a liquid protein stream to produce a protein isolate, The method may further comprise, during or after step (c), treating the liquid protein stream with a bleaching agent at a temperature of 50° C. or greater and a pH of 5 or greater.

[0008] According to a second aspect, the present invention relates to a protein isolate obtainable by the method of the invention.

[0009] According to a third aspect, the present invention relates to a protein isolate isolated from cereal material, preferably BSG, said protein isolate having a L 1 value measured by the CIELAB method in a 5% by weight protein solution in water. * The score is 60 or more, preferably 70 to 100, and more preferably 75 to 98.

[0010] According to a fourth aspect, the present invention relates to a food or beverage comprising a protein isolate obtainable by the method of the present invention.

[0011] According to a fifth aspect, the present invention relates to a food or beverage comprising the protein isolate of the present invention.

[0012] Further advantageous features of the invention are set out in the present specification and in the respective dependent claims. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a flow chart that schematically illustrates a particular embodiment of the present invention in which bleaching is performed between the microfiltration and nanofiltration steps. [Figure 2] 1 is a flow chart illustrating a schematic diagram of a particular embodiment of the present invention in which bleaching is performed between two nanofiltration steps. [Figure 3] 1 shows the brightness (L* score) during the bleaching process described in Example 1. [Figure 4] 1 shows the brightness (L* score) during the bleaching process described in Example 2. [Figure 5] Figures 5A and 5B show brightness (L* score) during the bleaching process described in Example 3. Figure 5A shows brightness during the bleaching process at a 10X dosage (175 g / kg protein) and the indicated temperature, and Figure 5B shows brightness during the bleaching process at a 5X dosage (87.5 g / kg protein) and the indicated temperature. [Figure 6] The results of the sensory profile test described in Table 15 of Example 7 are shown. [Figure 7] 1 shows the solubility of a protein isolate of the present invention, a commercial soy protein product, and a commercial pea protein product at 2% protein concentration. [Figure 8] FIG. 8A shows the viscosity of a protein isolate of the present invention at a 2% protein concentration, and FIG. 8B shows the viscosity of a protein isolate of the present invention at a 2% protein concentration compared to a commercial soy protein product, a commercial pea protein product, and a commercial whey protein product. [Figure 9] 1 shows the molecular weight distribution of the protein isolate of the present invention. [Figure 10] Table 16 in Example 7 shows the results of a sensory profile test of a protein isolate of the present invention compared to a typical commercial pea protein product. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Detailed Description of the Invention] The present inventors have discovered that conditions used in the prior art to bleach protein isolates do not significantly reduce the brightness and color of brewer's grains (BSG)-derived protein isolates, nor do they produce BSG-derived protein isolates with flavors ideal for use in food and beverage applications. The inventors hypothesize that this is due to the unique chemical composition of BSG and the conditions used to extract proteins from BSG, which may include enzymatic hydrolysis under caustic conditions. As a result, considerable innovation and ingenuity were required to devise a process that reduces the color, flavor, and aroma of BSG-derived protein isolates without adversely affecting the taste profile, protein content, and amino acid profile.

[0015] In light of the above, the present invention provides a method for isolating a protein isolate from a grain material, comprising the steps of: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c) subjecting the liquid protein stream to one or more filtration steps; and d) treating a liquid protein stream to produce a protein isolate, The method may further comprise, during or after step (c), treating the liquid protein stream with a bleaching agent at a temperature of 50° C. or greater and a pH of 5 or greater.

[0016] The grain material in the present invention may be brewer's grains, barley, malted barley, rice, corn, and combinations thereof. Preferably, the grain material is brewer's grains.

[0017] "Brewers grains" (BSG) are a by-product of the brewing process that occurs after the mashing step. At this stage, the soluble portion (known as the "wort") is sent to a later brewing step, and the insoluble portion is removed. This insoluble portion is the brewers grains. The brewers grains used in the process of the present invention are preferably obtained after brewing with cereals that include barley, and may optionally further comprise one or more other cereals or other starchy ingredients, such as rice, oats, wheat, maize, sorghum, cassava, and / or millet, particularly rice, maize, sorghum, and / or cassava, more particularly rice and / or maize. Most preferred are brewers grains obtained after brewing with barley or brewers grains obtained after brewing with a mixture of barley and rice or maize, preferably a mixture of barley and rice.

[0018] According to the above, the brewer's grains may comprise 100% spent barley. Optionally, the brewer's grains may comprise 20-100% spent barley by weight, preferably 45-70% by weight, for example 45%, 50%, 55%, 60%, 65%, or 70% by weight, based on the weight of the brewer's grains. When the brewer's grains is a mixture of spent barley and spent rice or spent corn, the spent rice or spent corn can be present in an amount of 0-80% by weight, preferably 30-55% by weight, for example 30%, 35%, 40%, 45%, 50%, or 55% by weight, based on the weight of the brewer's grains.

[0019] The color of the material is measured in the CIELAB color space (also known as L * a * b * In this color system, colors are expressed by three values: L * denotes the perceived lightness, and a and b represent the four unique colors in human vision: red, green, blue, and yellow.* A score of 0 indicates black, and a score of 100 indicates diffuse white. * The score indicates a position between red and green, with negative values ​​indicating green and positive values ​​indicating red, and b * The score indicates a position between yellow and blue, with negative values ​​indicating blue and positive values ​​indicating yellow. In the present invention, the best indicator of product color and its compatibility with other ingredients in foods and beverages is L. * Since it is a score, this value is the main reference.

[0020] The L of the starting material, brewer's grains, measured by the CIELAB method in a 10% dry matter solution * The score can be between 10 and 60. In some cases, the L of the brewer's grains * The score may be 30 to 50 in a 10% dry matter solution, for example, 30, 35, 40, 45 or 50. The a of the brewer's grains of the present invention measured by the CIELAB method in a 10% dry matter solution * The score can be between 5 and 50. * The score may be 20 to 40 in a 10% dry matter solution, for example, 20, 25, 30, 35, or 40. The b of the brewer's grains of the present invention measured by the CIELAB method in a 10% dry matter solution * The score can be between 40 and 90. * The score may be between 50 and 80 in a 10% dry matter solution, for example 50, 55, 60, 65, 70, 75 or 80. Distiller's grains therefore typically appear dark red / brown in solution.

[0021] L of the starting material, brewer's grains, measured by the CIELAB method in solid state and in reflectance mode. * The score can be 30 to 70, preferably 40 to 60, for example 40, 45, 50, 55 or 60. The a of the brewer's grains of the present invention measured by the CIELAB method in the solid state in reflectance mode *The score can be between -10 and 20, preferably between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The b of the brewer's grains of the present invention measured by the CIELAB method in the solid state and in the reflectance mode * The score can be 0 to 40, preferably 10 to 30, for example 10, 15, 20, 25 or 30.

[0022] The aqueous slurry is formed by mixing the grain material with water. The ratio of water to grain material (dry weight) is preferably 8:1 to 12:1, more preferably 10:1 to 11:1. The aqueous slurry is preferably formed in a jacketed mixing tank, which is preferably equipped with heating means.

[0023] The aqueous slurry is subjected to enzymatic proteolysis to produce a liquid protein stream. Optionally, particle size reduction of the cereal material may be carried out prior to and / or during this step. Particle size reduction may be achieved by any suitable technique, such as milling.

[0024] Prior to enzymatic protein hydrolysis, the aqueous slurry is preferably subjected to enzymatic starch hydrolysis. The enzymatic starch hydrolysis preferably comprises treatment with a glucoamylase enzyme. Suitable glucoamylase enzymes include those used in the brewing industry and are available, for example, from EDC (Enzyme Development Corporation, New York) or Novozymes.

[0025] The enzymatic starch hydrolysis is preferably carried out at the natural pH of the aqueous slurry, which can be, for example, from about 4.5 to about 6.5 (e.g., 4.5, 5.5, 6, or 6.5, or any value within the range).

[0026] The enzymatic starch hydrolysis is preferably carried out at a temperature of about 50 to about 65°C (eg, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 54°C, or any temperature within that range).

[0027] The enzymatic starch hydrolysis is preferably carried out for at least about 15 minutes, preferably from 20 to 60 minutes, and preferably for about 45 minutes. For example, the enzymatic starch hydrolysis can be carried out for 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or any time within that range.

[0028] The enzymatic starch hydrolysis is preferably carried out until at least about 90% by weight, preferably at least about 95% by weight, of the initial starch content has been hydrolyzed to sugars (i.e., water-soluble sugars, including glucose and / or disaccharides and other short-chain oligosaccharides).

[0029] Preferably, enzymatic protein hydrolysis involves treatment with a protease enzyme. The protease enzyme is preferably a food-grade protease enzyme, more preferably a serine protease. The protease enzyme is preferably an alkaline protease, more preferably an endopeptidase, more preferably a serine endopeptidase. Suitable protease enzymes are available, for example, from Novozymes or EDC (Enzyme Development Corporation, New York).

[0030] Enzymatic proteolysis is preferably carried out at a pH of about 7 to about 10 (e.g., 7, 7.5, 8, 8.5, 9, 9.5, or any value within the range), more preferably at a pH of about 9. The desired pH can be achieved by adding an alkali such as sodium hydroxide and / or potassium hydroxide prior to enzymatic treatment.

[0031] The enzymatic protein hydrolysis is preferably carried out at a temperature of about 50 to about 75°C (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C, or any temperature within that range), preferably at about 55 to about 68°C, and more preferably at about 55 to about 65°C.

[0032] The enzymatic proteolysis is preferably carried out for at least about 15 minutes, preferably from at least about 20 minutes to at most about 20 minutes, and preferably for about 60 minutes. For example, the enzymatic proteolysis can be carried out for 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 minutes, or any time within that range.

[0033] Enzymatic protein hydrolysis is preferably carried out until a degree of hydrolysis (dH) of between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any value within that range) is reached, and preferably until a dH of between 4 and 8 is reached. As used herein, dH can be calculated using the pH-stat method by adding an alkali (e.g., NaOH) and using the following formula:

number

[0034] The enzymatic starch hydrolysis (if performed) and the enzymatic protein hydrolysis are preferably carried out in a jacketed mixing tank in which an aqueous slurry is formed.

[0035] After enzymatic proteolysis, the enzymes are preferably inactivated by raising the temperature, for example at about 75 to about 90°C (e.g., about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90°C, or any temperature within that range), preferably at about 80°C for up to about 35 minutes, for example up to about 25 minutes, for example up to about 10, 15, 20 or 25 minutes, or a time within that range.

[0036] After enzymatic protein hydrolysis, solids are removed from the liquid protein stream. Solids removal is preferably accomplished by decantation, preferably using a decantation centrifuge. To maximize recovery of the liquid protein stream, pressure may be applied to the solids, for example, using a screw press.

[0037] The solids removed from the liquid protein stream are preferably washed with water, and the resulting wash water is mixed with the liquid protein stream, similarly maximizing protein recovery.

[0038] The solids removed from the liquid protein stream may be further processed to provide a fibrous product.

[0039] The liquid protein stream is then subjected to one or more filtration steps while undergoing a bleach treatment step at a temperature of 50° C. or greater and a pH of 5 or greater.

[0040] <Bleaching conditions> Bleaching is a process used to remove color from products or whiten products. In the present invention, the bleaching agent can be an oxidizing agent or a reducing agent. More specifically, the bleaching agent can be one or more selected from peroxide bleaches, chlorine bleaches, nitrogen bleaches, and sulfur bleaches. Along with removing color, the L of the product is also reduced. * The score increases. * Decrease in score and / or b * In some cases, an increase in scores may be observed.

[0041] If the bleaching agent is a peroxide bleaching agent, the peroxide bleaching agent is Hydrogen peroxide peroxide salts, preferably calcium peroxide; organic peroxides, preferably benzoyl peroxide; peroxide adducts, preferably sodium percarbonate or urea hydrogen peroxide; perborate, preferably sodium perborate; persulfates, preferably sodium persulfate; peracetic acid; and Permanganate, preferably potassium permanganate It can be one or more selected from the group consisting of: Preferably, the peroxide bleaching agent is one or more selected from the group consisting of hydrogen peroxide, benzoyl peroxide, or urea hydrogen peroxide.

[0042] When the bleaching agent is a chlorine bleaching agent, it may be one or more selected from the group consisting of chlorine, chlorine dioxide, and chlorite, and is preferably sodium hypochlorite.

[0043] When the bleaching agent is a nitrogen-based bleaching agent, it can be azodicarbonamide or nitrogen dioxide.

[0044] When the bleaching agent is a sulfur bleach, it can be one or more selected from the group consisting of sulfur dioxide, dithionite salts, preferably sodium dithionite, and sulfite salts, preferably ammonium bisulfite, magnesium bisulfite, potassium bisulfite, sodium bisulfite, potassium metabisulfite, and sodium metabisulfite. Preferably, the sulfur bleach is potassium metabisulfite or sodium metabisulfite.

[0045] In a preferred embodiment, the bleaching agent is one or more selected from the group consisting of sodium metabisulfite, potassium metabisulfite, benzoyl peroxide, urea hydrogen peroxide, and hydrogen peroxide, preferably hydrogen peroxide. The hydrogen peroxide may be supplied in the form of a 20 to 45 wt % aqueous solution, for example, a 35 wt % aqueous solution.

[0046] The amount of bleaching agent used in the bleaching step can be 0.25 moles per kg of protein to be bleached (mol / kg protein) or more, as measured by AOAC 990.03 or AOAC 992.15. The amount of bleaching agent is preferably 0.5 to 50 mol / kg protein, more preferably 1 to 25 mol / kg protein, more preferably 1.5 to 6 mol / kg protein, and even more preferably 2.5 to 5 mol / kg protein, for example, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol / kg protein, or any value within that range. In certain embodiments, the amount of bleaching agent is between 2.5 and 50 mol / kg protein, for example between 2.5 and 25 mol / kg protein.

[0047] The amount of bleaching agent used in the bleaching step can also be described as grams of bleaching agent per kilogram of protein to be bleached (g / kg protein), as measured by AOAC 990.03 or AOAC 992.15. Thus, the amount of bleaching agent can be 10 g or more per kilogram of protein, preferably 17.5 to 1750 g / kg protein, more preferably 35 to 875 g / kg, more preferably 52.5 to 210 g / kg protein, more preferably 85 to 175 g / kg protein, more preferably 110 to 150 g / kg protein, e.g., 110, 115, 120, 125, 130, 135, 140, 145, or 150 g / kg protein, or any value within that range.

[0048] As mentioned above, the bleaching agent may be added as a dilute solution, in which case the weight ratio in water can be 20-45% by weight, preferably 30-40% by weight, more preferably 35% by weight. As a dilute solution, bleach can be added in an amount of 10 g or more per kilogram of protein (g / kg protein) as measured by AOAC 990.03 or AOAC 992.15, preferably 17.5 to 1750 g / kg protein, more preferably 50 to 750 g / kg protein, even more preferably 250 to 500 g / kg protein, and even more preferably 325 to 425 g / kg protein, for example, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, or 425 g / kg protein.

[0049] The temperature in the bleaching step can be 50 to 100° C., preferably 70 to 98° C., more preferably 80 to 95° C., for example, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C. To shorten the bleaching time, a high temperature (i.e., 80° C. or higher) is preferred.

[0050] The pH during the bleaching step of the liquid protein stream can be important for the present invention, since a low pH can cause precipitation of proteinaceous materials. The pH during bleaching can be between 5 and 10, preferably between 6 and 9.5, and more preferably between 7 and 8. The pH can be adjusted by adding an acid or alkali. Since the pH decreases as bleaching progresses, potentially causing protein precipitation, it is preferable to maintain a constant pH during bleaching. Therefore, the pH during the bleaching step can be maintained at a value of 5 or higher, preferably between 5 and 10, more preferably between 6 and 9.5, and even more preferably between 7 and 7.5. The pH can be maintained by adding an alkaline agent, such as sodium hydroxide or potassium hydroxide. The alkaline agent can be added during bleaching as a 2 molar aqueous solution to maintain a constant pH.

[0051] The present inventors aimed to provide an efficient bleaching method that can be easily adapted to existing methods, and as a result, the duration of the step of treating a liquid protein stream with a bleaching agent is up to 5 hours, preferably 5 minutes to 5 hours, more preferably 10 minutes to 3 hours, more preferably 15 minutes to 1 hour, and more preferably 20 to 45 minutes.

[0052] In the step of treating a liquid protein stream with a bleaching agent, the protein concentration, measured according to AOAC 990.03 or AOAC 992.15, can be 0.1 to 40 wt %, preferably 0.5 to 15 wt %, more preferably 1 to 20 wt %, and even more preferably 2 to 15 wt %, based on the weight of the liquid protein stream. The inventors have found that the above ranges are preferable because if the protein concentration during the bleaching step is too high, excessive foaming occurs, which may interfere with subsequent processing of the liquid protein stream.

[0053] To achieve the desired protein concentration, the liquid protein stream may need to be concentrated or diluted. In other words, a step of concentration or dilution to obtain a concentrated or diluted liquid protein stream may be performed before the step of treating with a bleaching agent. Concentration can be achieved by any method known to those skilled in the art, for example, one or more methods selected from the group consisting of filtration (preferably nanofiltration), centrifugation, dehydration, and evaporation. Dilution can be achieved by adding water.

[0054] The inventors have found that treating a liquid protein stream with bleach under the following conditions results in significant reduction in color and flavor / aroma in a practical timeframe and constitutes a particularly preferred embodiment. bleaching agent in an amount of 2.5 to 50 moles / kg protein, for example, bleaching agent in an amount of 2.5 to 5 moles / kg protein, or bleaching agent in an amount of 85 to 175 g / kg protein; The pH is 7 to 8, and can be preferably maintained within this range by adding alkali; Temperatures between 80 and 95 ° C; Duration: 20-40 minutes; Liquid protein flow ratio of 1 to 20 wt% protein concentration.

[0055] <Bleaching method> A further important feature of the present invention is that a step of treating the liquid protein stream with a bleaching agent is carried out during or after step (c).

[0056] The step of treating the liquid protein stream with a bleaching agent can be carried out during step (c), i.e., during the step of subjecting the liquid protein stream to one or more filtration steps. Preferably, step (c) comprises at least one nanofiltration step. More preferably, the filtration steps comprise a microfiltration step and one or more nanofiltration steps, and the step of treating the liquid protein stream with a bleaching agent can be carried out prior to the one or more nanofiltration steps.

[0057] The microfiltration step yields a protein-containing microfiltration permeate and a microfiltration retentate. Microfiltration is preferably carried out using ceramic microfiltration membranes. Surprisingly, ceramic microfiltration membranes have been found to be more effective than polymeric membranes in the process of the present invention.

[0058] Microfiltration can be carried out using a microfiltration membrane preferably having a pore size of 0.03 to 0.5 μm (e.g., 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 μm, or any value within that range), preferably 0.03 to 0.25 μm, more preferably 0.05 to 0.2 μm, and even more preferably 0.07 to 0.13 μm (e.g., 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, or 0.13 μm, or any value within that range). Suitable microfiltration membranes are available from Pall Corporation. Microfiltration preferably includes a diafiltration step.

[0059] The microfiltration permeate may be subjected to nanofiltration at a relatively low operating pressure of 1.0 bar (100 kPa) to 8.0 bar (800 kPa) to obtain a nanofiltration permeate and a protein-containing nanofiltration permeate. Operating pressure is a well-known concept in the filtration field and refers to the pressure at which a feed solution is fed to a filtration membrane. The inventors have found that performing nanofiltration at a relatively low operating pressure of 1.0 bar (100 kPa) to 8.0 bar (800 kPa) can produce protein powders with better taste and solubility profiles.

[0060] Nanofiltration can be carried out at operating pressures starting from 1.0 bar (100 kPa), preferably 1.3 bar (130 kPa), up to 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 bar, or any value within that range. Nanofiltration is preferably carried out from 1.3 bar (130 kPa) to 4.0 bar (400 kPa), for example, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, It may be carried out at 3.9 or 4.0 bar (130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 kPa), or any value within that range.

[0061] More preferably, nanofiltration is carried out at 1.3 bar (130 kPa) to 3.3 bar (330 kPa), even more preferably at 1.4 bar (140 kPa) to 3.2 bar (320 kPa), and even more preferably at 1.5 bar (150 kPa) to 3 bar (300 kPa). For example, nanofiltration can be carried out at 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2 or 3.3 bar (130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320 or 330 kPa), or any value within that range.

[0062] Nanofiltration is preferably carried out using nanofiltration membranes with a molecular weight cutoff (MWCO) of 500 to 2000 Da, more preferably 800 to 2000 Da, and even more preferably 800 to 1200 Da. For example, nanofiltration can be carried out using nanofiltration membranes with a molecular weight cutoff of 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 Da, or any value within these ranges. Suitable microfiltration membranes are available from MICRODYN-NADIR.

[0063] The step of treating the liquid protein stream with a bleaching agent is preferably carried out by branching the liquid protein stream into a jacketed tank equipped with a heating means and adjusting the bleaching conditions to the above-mentioned conditions. The step of treating with a bleaching agent may be carried out in a batch mode. The step of treating with a bleaching agent may be carried out on the microfiltration permeate between one or more nanofiltration steps. In this embodiment, it is preferred to carry out a single nanofiltration step. Alternatively, the step of treating with a bleaching agent on the nanofiltration retentate may be carried out between two nanofiltration steps, i.e., the filtration process includes a microfiltration step and two or more nanofiltration steps, and the bleaching step is carried out on the nanofiltration retentate between these two nanofiltration steps. Specific forms of these two embodiments are shown schematically in Figures 1 and 2, respectively.

[0064] When step (c) incorporates treating the liquid protein stream with a bleaching agent, the step of subjecting the liquid protein stream to one or more filtration steps comprises: c1) microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2) treating the microfiltration permeate with a bleaching agent to provide a bleached microfiltration permeate; and c3) nanofiltering the bleached microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; Step d) is d) processing the nanofiltration retentate to produce a protein isolate.

[0065] As a result, the method for isolating a protein isolate from a grain material comprises: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c1) microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2) treating the microfiltration permeate with said bleaching agent to provide a bleached microfiltration permeate; and c3) nanofiltering the bleached microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; Step (d) is d) processing the nanofiltration retentate to produce a protein isolate; Furthermore, the step of treating with a bleaching agent includes a step of treating at a temperature of 50° C. or higher and at a pH of 5 or higher.

[0066] The inventors have found that bleaching the microfiltration permeate is particularly advantageous because subsequent filtration steps, such as the nanofiltration step described above, can remove by-products of the bleaching step (and optionally by-products of additional steps performed to inactivate the bleaching agent), such as salts and short peptide chains. This can result in a purer protein isolate and potentially eliminate flavor anomalies (especially bitterness) in the protein isolate. The bleaching step can be performed under any of the conditions described above, but is preferably performed under the bleaching conditions of the preferred embodiments.

[0067] The microfiltration permeate before bleaching was measured by the CIELAB method in a 1.28 wt% protein aqueous solution. * The score may be less than 70, preferably 20 to 70, and more preferably 45 to 65. The a of the microfiltration permeate measured by the CIELAB method in a 1.28 wt % protein aqueous solution * The score can be 20 to 50, preferably 30 to 40. The b of the microfiltration permeate measured by the CIELAB method in a 1.28 wt % protein aqueous solution * The score can be 40 to 80, and is preferably 55 to 75.

[0068] The bleached microfiltration permeate, i.e., the microfiltration permeate after the bleaching step, had a L measured by the CIELAB method in a 1.28 wt % protein aqueous solution. * The score can be 60 or more, preferably 70 to 100, and more preferably 75 to 98. The a of the bleached microfiltration permeate measured by the CIELAB method in a 1.28 wt % protein aqueous solution. * The score can be from -10 to +10, preferably from -5 to 0. The b of the microfiltration permeate measured by the CIELAB method in a 1.28 wt % protein aqueous solution * The score can be 20 to 60, and is preferably 30 to 50.

[0069] In another embodiment, the step of treating the liquid protein stream with a bleaching agent may be carried out between the two nanofiltration steps. In this embodiment, the method comprises: c1') microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing the protein; c3') treating the nanofiltration concentrate with a bleaching agent to provide a bleached nanofiltration concentrate; and c4') nanofiltering the bleached nanofiltration retentate again to obtain a second nanofiltration permeate and a second nanofiltration retentate; Step (d) is d) processing the second nanofiltration retentate to produce a protein isolate.

[0070] As a result, the method for isolating protein isolates from grain materials includes: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c1') microfiltering the liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; c3') treating the nanofiltration concentrate with a bleaching agent to provide a bleached nanofiltration concentrate; c4') nanofiltration of the bleached nanofiltration retentate again to obtain a second nanofiltration permeate and a second nanofiltration retentate, Step (d) is d) processing the second nanofiltration retentate to produce a protein isolate; Furthermore, the step of treating with a bleaching agent includes a step of treating at a temperature of 50° C. or higher and at a pH of 5 or higher.

[0071] The inventors have found that this process is particularly advantageous because the subsequent nanofiltration step removes by-products of the bleaching step (and optionally by-products of additional steps performed to inactivate the bleaching agent), such as salts and short peptide chains. This can result in a purer protein isolate and potentially eliminates flavor anomalies (especially bitterness) in the protein isolate. The bleaching step can be performed under any of the conditions described above, but is preferably performed under the bleaching conditions of the preferred embodiment.

[0072] Preferably, before subjecting the bleached nanofiltration concentrate to nanofiltration, the bleached nanofiltration concentrate is diluted to a protein concentration of 0.1 to 10% by weight, preferably 1 to 5%, more preferably 2 to 4%, and even more preferably 3% based on the weight of the bleached nanofiltration concentrate. The inventors have found that this improves the effectiveness of removing off-notes from the protein isolate, improving its flavor and aroma. Preferably, the step of subjecting the bleached nanofiltration concentrate to nanofiltration includes diafiltration.

[0073] Additionally, treating the liquid protein stream with a bleaching agent may occur after step (c), i.e., subjecting the liquid protein stream to one or more nanofiltration steps. That is, the bleaching step may occur after filtration is complete. These filtration steps may include a microfiltration step and one or more nanofiltration steps.

[0074] In this embodiment, the method further comprises: c1') microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing the protein; and d1') treating the nanofiltration concentrate with a bleaching agent to obtain a bleached nanofiltration concentrate; Step d) is d) processing the nanofiltration retentate to produce a protein isolate.

[0075] As a result, the method for isolating protein isolates from grain materials includes: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c1') microfiltering the liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; and d1') treating the nanofiltration concentrate with a bleaching agent to obtain a bleached nanofiltration concentrate; Step d) is d) processing the nanofiltration retentate to produce a protein isolate; Furthermore, the step of treating with a bleaching agent includes a step of treating at a temperature of 50° C. or higher and at a pH of 5 or higher.

[0076] In accordance with the above, a bleaching step may be carried out on the nanofiltration concentrate. The inventors have found that carrying out bleaching at this stage of the process is particularly advantageous because the nanofiltration concentrate is concentrated and highly pure. This advantage also applies to the embodiment in which bleaching is carried out on the nanofiltration concentrate between the two nanofiltration steps. The bleaching step can be carried out under any of the conditions described above, but is preferably carried out under the bleaching conditions of the particularly preferred embodiment.

[0077] The nanofiltration concentrate before the bleaching process had a L measured by the CIELAB method in a 5 wt% protein aqueous solution. * The score may be less than 50, preferably 5 to 40, more preferably 10 to 30. The a of the nanofiltration concentrate before the bleaching step, measured by the CIELAB method in a 5 wt % protein aqueous solution, * The score can be 20 to 50, preferably 30 to 40. The b of the nanofiltration concentrate before the bleaching step measured by the CIELAB method in a 5 wt % protein aqueous solution * The score can be 20 to 60, and is preferably 30 to 40.

[0078] The bleached nanofiltration concentrate had a L of 5 wt. % protein in water measured by the CIELAB method. * The score can be 60 or more, preferably 70 to 100, and more preferably 75 to 98. The a of the bleached nanofiltration concentrate measured by the CIELAB method in a 5 wt % protein aqueous solution * The score can be −10 to +10, and preferably −5 to 0. The b of the microfiltration permeate measured by the CIELAB method in a 5 wt % protein aqueous solution * The score can be between 20 and 60, preferably between 30 and 50. The above properties of the nanofiltration concentrate and the bleached nanofiltration concentrate also apply to the embodiment in which bleaching is performed on the nanofiltration concentrate between the two nanofiltration steps.

[0079] Optionally, the nanofiltration concentrate may be subjected to an activated carbon filtration step. Activated carbon may be added to the liquid protein stream prior to filtration, or may be impregnated into the filter used in this additional filtration step. An example of a suitable impregnated filter is Pall Seitz® AKS4 activated carbon sheet. Incorporating this step into the process can further reduce the overall flavor of the protein isolate. Accordingly, any of the embodiments described herein above or below may include an activated carbon filtration step prior to, and preferably immediately prior to, step (d) (i.e., treating the liquid protein stream to produce the protein isolate).

[0080] The nanofiltration concentrate is treated to produce a protein isolate. When an activated carbon filtration step is performed, the nanofiltration concentrate is filtered through activated carbon, and the permeate from the activated carbon filtration is treated to produce a protein isolate. The process of treating the nanofiltration concentrate (or the activated carbon filtration permeate) to produce a protein isolate preferably includes an evaporation step to increase the total solids concentration to 20 to 55 wt % (e.g., 20, 25, 30, 35, 40, 45, 50 wt %, or any value within this range), followed by a spray-drying step. The total solids concentration is preferably 25 to 55 wt %, more preferably 35 to 55 wt %, more preferably 45 to 55 wt % (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 wt %, or any value within this range), and particularly preferably 48 to 52 wt %.

[0081] <Bleach inactivation> The method may further include a step of inactivating the bleaching agent (hereinafter, sometimes simply referred to as "inactivation"). Inactivation of the bleaching agent can be achieved by heating or boiling, ascorbic acid treatment, or enzyme treatment, and is preferably achieved by enzyme treatment. This inactivation method may be carried out in the same jacketed vessel as the bleaching agent treatment step. Once the bleaching agent concentration in the liquid protein stream is less than 1 ppm, preferably less than 0.5 ppm, and more preferably substantially free of bleaching agent, the liquid protein stream may be removed from the vessel and proceed to the next step.

[0082] When inactivation is achieved by enzyme treatment, the enzyme treatment may include treatment with an enzyme composition containing catalase. The enzyme treatment can be carried out under conditions of pH 4 to 12, temperature 40 to 90°C, and dosage of the enzyme composition 0.005 to 1 ml / L, preferably pH 8, temperature 30 to 65°C, and dosage of the enzyme composition 0.02 to 0.3 ml / L, more preferably 0.04 to 0.25 ml / L.

[0083] When inactivation is achieved by ascorbic acid treatment, the treatment temperature can be 45°C or higher, preferably 65 to 95°C, the dosage of ascorbic acid can be 0.5 g / L or higher, preferably 0.6 to 1 g / L, and the treatment time can be up to 40 minutes, preferably 5 to 15 minutes.

[0084] When inactivation is achieved by heating or boiling, the treatment can be performed at 100°C for 5 minutes or more, preferably 10 to 60 minutes, and more preferably 20 to 50 minutes.

[0085] In embodiments in which the bleaching process is performed on a microfiltration permeate, the step of inactivating the bleaching agent may be performed between the bleaching process and one or more nanofiltration processes, i.e., between treating the microfiltration permeate with a bleaching agent to obtain a bleached microfiltration permeate and subjecting the bleached microfiltration permeate to nanofiltration to obtain a protein-containing nanofiltration permeate and nanofiltration retentate. In other words, when the bleaching process is performed on a microfiltration permeate, the inactivation may be performed on the bleached microfiltration permeate or prior to the subsequent nanofiltration process. The inventors have found that performing the inactivation process at this time is advantageous because it allows for the removal of by-products of the inactivation process and unreacted bleaching agent by filtration, thereby eliminating off-flavor components in the protein isolate. Additionally, this avoids degradation of the filtration membrane, which may be caused by residual bleaching activity.

[0086] Based on the above, the method comprises: c1) microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2) treating the microfiltration permeate with a bleaching agent to provide a bleached microfiltration permeate; c2a) inactivating the bleaching agent; and c3) nanofiltering the bleached microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; Step d) is d) processing the nanofiltration retentate to produce a protein isolate.

[0087] As a result, the method for isolating a protein isolate from a grain material comprises: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c1) microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2) treating the microfiltration permeate with said bleaching agent to provide a bleached microfiltration permeate; c2a) deactivating the bleaching agent, and c3) nanofiltering the bleached microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; Step (d) is d) processing the nanofiltration retentate to produce a protein isolate; Furthermore, the step of treating with a bleaching agent includes a step of treating at a temperature of 50° C. or higher and at a pH of 5 or higher.

[0088] In embodiments where the bleaching process is performed on a nanofiltration retentate, the step of deactivating the bleaching agent may be performed between steps (c3') and (c4'), i.e., between the steps of bleaching the nanofiltration retentate to obtain a bleached nanofiltration retentate and subjecting the bleached nanofiltration retentate to nanofiltration to obtain a second nanofiltration retentate containing protein and a second nanofiltration retentate.

[0089] Based on the above, in this embodiment, the method comprises: c1') microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing the protein; c3') treating the nanofiltration concentrate with a bleaching agent to provide a bleached nanofiltration concentrate; c3a) inactivating the bleaching agent; and c4') nanofiltering the bleached nanofiltration retentate again to obtain a second nanofiltration permeate and a second nanofiltration retentate; Step d) is d) processing the second nanofiltration retentate to produce a protein isolate.

[0090] As a result, the method for isolating protein isolates from grain materials is a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c1') microfiltering the liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; c3') treating the nanofiltration concentrate with a bleaching agent to provide a bleached nanofiltration concentrate; c3a) deactivating the bleaching agent; and c4') nanofiltration of the bleached nanofiltration retentate again to obtain a second nanofiltration permeate and a second nanofiltration retentate, Step (d) is d) processing the second nanofiltration retentate to produce a protein isolate; Furthermore, the step of treating with a bleaching agent includes a step of treating at a temperature of 50° C. or higher and at a pH of 5 or higher.

[0091] In embodiments, when the nanofiltration retentate is subjected to a bleaching treatment, the step of inactivating the bleaching agent can be carried out between shifts (d1') and (d), i.e., between the steps of bleaching the nanofiltration retentate to provide a bleached nanofiltration retentate and treating the bleached nanofiltration retentate to produce the protein isolate.

[0092] In accordance with the above, the method may comprise the steps of: c1') microfiltering a liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing the protein; d1') treating the nanofiltration concentrate with a bleaching agent to obtain a bleached nanofiltration concentrate; and d2') inactivating the bleaching agent; Step d) is d) processing the nanofiltration retentate to produce a protein isolate.

[0093] As a result, the method for isolating a protein isolate from a grain material comprises: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from the liquid protein stream; c1') microfiltering the liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; d1') treating the nanofiltration concentrate with a bleaching agent to obtain a bleached nanofiltration concentrate; and d2') deactivating the bleaching agent; Step d) is d) processing the nanofiltration retentate to produce a protein isolate; Furthermore, the step of treating with a bleaching agent includes a step of treating at a temperature of 50° C. or higher and at a pH of 5 or higher.

[0094] Between the bleach treatment step and the bleach inactivation step, there may be a temperature change step, preferably carried out by a heat exchanger, to raise or lower the temperature of the liquid protein stream to the temperature required for the inactivation method, preferably below 60°C, more preferably between 40 and 55°C, particularly when the inactivation is carried out by enzymatic treatment.

[0095] <Protein Isolate> The protein isolate of the present invention has a high protein content, which is muted in color and milder in flavor than a protein isolate produced from a grain starting material and by the same method but without the step of treating the liquid protein stream with bleach. This makes the protein isolate more suitable for blending with other ingredients to provide protein-enriched foods, with minimal impact on the taste and appearance of the food. The protein isolate also has a high polyphenol content, but the polyphenol content is reduced compared to a protein isolate produced by the same method but without the step of treating the liquid protein stream with bleach.

[0096] The protein isolate of the present invention is obtained or obtainable by the methods described above.

[0097] The protein isolate has a protein content (by weight of dry matter) of 80% or more, preferably 85% or more (e.g., 80, 81, 82, 83, 84, or 85% or more, or any value within a range thereof) as measured by AOAC 990.03 or AOAC 992.15. The present process devised by the inventors does not affect the protein content of the protein isolate compared to an isolate produced by the same process without a bleaching step. Importantly, the present process does not significantly affect the amino acid profile of the protein isolate compared to an isolate produced by the same process without a bleaching step, as shown in Table 1 below.

[0098] [Table 1]

[0099] The L of this protein isolate in a 5 wt% protein aqueous solution measured by the CIELAB method *The score is usually 60 or higher, preferably 70 to 100, more preferably 75 to 98, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98. The a of the present protein isolate in a 5 wt % protein aqueous solution measured by the CIELAB method * The score is usually 5 to 15, preferably 0 to 10. The b of the present protein isolate in a 5 wt % protein aqueous solution measured by the CIELAB method is * The score is typically between 50 and 80, preferably between 60 and 75. The protein isolate of the present invention therefore has a pale off-white / yellow color, which allows it to be easily mixed with other ingredients to produce food and beverage products without affecting their appearance.

[0100] The L * The score is at least 1.25 times higher than that of a protein isolate isolated by a method not including the bleaching step described above, preferably 1.25 to 3.5 times higher, and more preferably 1.5 to 3 times higher.

[0101] Protein isolates obtained from BSG by enzymatic hydrolysis, without bleaching, have polyphenol contents approximately 10-100 times higher than other protein isolates of plant and animal origin (see Table 2).

[0102] [Table 2] * Measured by the Folin-Ciocalteu method (or gallic acid equivalent).

[0103] Bleaching can result in a protein isolate having a reduced polyphenol content compared to an unbleached protein isolate isolated from BSG. Thus, the bleaching step can reduce the polyphenol content by up to 90%, preferably 10-60%, and more preferably 20-40%, as measured by the Folin-Ciocalteu method, compared to the polyphenol content of a protein isolate produced by a process that does not include treating the liquid protein stream with a bleaching agent.

[0104] The total polyphenol content of the present protein isolate, as measured by the Folin-Ciocalteu method, is 10,000 to 50,000 mg / kg, preferably 15,000 to 35,000 mg / kg, and more preferably 20,000 to 30,000 mg / kg per protein isolate.

[0105] The total polyphenol content of the protein isolate, as measured by the Folin-Ciocalteu method, can be 40-80%, preferably 55-75%, of the total polyphenol content of a protein isolate obtained from cereal material, preferably distiller's grains, and produced by the aforementioned method, which does not include treating the liquid protein stream with bleach.

[0106] The protein isolate has a water solubility of 70% or more, preferably 75% or more, in an aqueous solution at a pH of 8 or less, at a protein concentration of 2% by weight on a dry matter basis (measured by the Kjeldahl method using a conversion factor of 6.25). At a protein concentration of 2% by weight on a dry matter basis, the water solubility can be 90% or more, preferably 95%, at a pH of 5 or more. At a protein concentration of 2% by weight on a dry matter basis, the water solubility can be 70% or more, preferably 75-90%, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90%, at a pH of 2-5. This solubility means that the protein isolate is suitable for incorporation into foods and beverages, particularly beverages. Its solubility at low pH makes it suitable for incorporation into acidic carbonated beverages.

[0107] The viscosity of the protein isolate in an aqueous solution having a protein concentration of 5% by weight on a dry matter basis can be less than 5 centipoise (or mPa·s), preferably 0.5 to 4 mPa·s, and more preferably 2 to 3.5 mPa·s (measured by the Kjeldahl method using a conversion factor of 6.25). At a protein concentration of 10% by weight on a dry matter basis, the viscosity of the protein isolate is 4 to 10 mPa·s, more preferably 4.5 to 6.5 mPa·s. At a protein concentration of 20% by weight on a dry matter basis, the viscosity of the protein isolate is 10 to 25 mPa·s, more preferably 12 to 18 mPa·s. Therefore, the protein isolate does not impart body or viscosity to liquid products containing the protein isolate.

[0108] The present protein isolate may have a molecular weight distribution as shown in Figure 9. The majority of the proteinaceous substance may have a molecular weight of 3,000 to 30,000 Da, preferably 5,000 to 30,000 Da. For example, the proportion of proteins in the present protein isolate having a molecular weight of 3,000 to 30,000 Da, preferably 5,000 to 30,000 Da, may be 51% or more, preferably 51 to 70%, more preferably 55 to 65%. The present protein isolate may have an average molecular weight of 5,000 to 10,000 Da, preferably 5,500 to 8,000 Da, more preferably 6,500 to 7,500 Da, for example, 6,500, 6,750, 7,000, 7,250, or 7,500 Da.

[0109] The molecular weight profile of the protein isolate is advantageous in providing a desired flavor profile. In this regard, the protein isolate may have a relatively high proportion of protein fragments having a molecular weight of 1,000 Da or greater. The proportion of proteins in the protein isolate having a molecular weight of 1,000 Da or greater, as measured by the Kjeldahl method with a conversion factor of 6.25, of the total protein is 85% by weight or greater, preferably 90-98% by weight, e.g., 90, 92, 94, 96, or 98% by weight. Correspondingly, the protein isolate contains a low proportion of low molecular weight protein fragments, i.e., those having a molecular weight of 1,000 Da or less. For example, the proportion of proteins having a molecular weight of 1,000 Da or less of the total protein in the protein isolate may be less than 15% by weight, preferably 5-12% by weight, e.g., 5, 6, 7, 8, 9, 10, 11, or 12% by weight. The proportion of proteins with molecular weights of 0 to 500 Da of the total protein in the protein isolate is less than 10% by weight, preferably 1 to 7% by weight, for example, 1, 2, 3, 4, 5, 6, or 7% by weight. A low proportion of protein fragments with molecular weights of 1,000 Da or less, particularly 500 Da or less, has an advantageous effect on the flavor profile of the protein isolate, and may contribute to a reduction in bitterness compared to other protein isolates that have a higher proportion of protein fragments in these molecular weight ranges.

[0110] The in vitro digestibility of the present protein isolate can be 80% or more, preferably 80 to 100%, and most preferably 100%.

[0111] The protein isolate is substantially free of bleaching agents. When the bleaching agent is hydrogen peroxide, the protein isolate is substantially free of hydrogen peroxide. The hydrogen peroxide concentration of the protein isolate can be less than 1 ppm, preferably less than 0.5 ppm, and more preferably 0 ppm, as measured by Quantofix® Peroxide 100 test strips.

[0112] The total solids content of the protein isolate produced by the present method is 90% by weight or greater, preferably 93% by weight or greater (eg, 90, 91, 92, 93, or 94% by weight or greater, or any value within the range).

[0113] The protein isolate can be used in or incorporated into food or beverage preparations. The protein isolate can be incorporated in any amount, for example, up to 75% by weight of the food or beverage product without affecting its flavor, preferably 0.5-50% by weight, and more preferably 20-40% by weight. In some embodiments, the food or beverage product is a beverage or liquid food product, including energy drinks, shakes, smoothies, coffee and coffee-based beverages (lattes, mochas, etc.), teas, and plant-based milk substitutes. In some embodiments, the protein isolate can be provided as a ready-to-mix (RTM) powder for beverage preparation or as a coffee creamer powder. In other embodiments, the food or beverage product includes muscle-building supplements, including meal replacement bars and workout drinks. In some embodiments, the food or beverage product includes meat substitutes, including meat and meat binder substitutes and extruded meat substitutes. In some embodiments, the (protein-enriched) food or beverage product includes coatings and / or binding agents for granola, nutrition bars, and muesli. In other embodiments, the protein-enriched food or beverage product may include seasonings for preparing bases, gravies, soups, and sauces. In some embodiments, the (protein-enriched) food or beverage product may include baked goods such as brownies, cakes, cookies, breads, crackers, etc. In yet other embodiments, the (protein-enriched) food or beverage product may include breakfast products such as waffles, pancakes, quick breads, pastries, etc. In some embodiments, the protein-enriched food or beverage product may include dairy products such as yogurt, cheese bread, cheese-based products, etc. In some embodiments, the (protein-enriched) food or beverage product may include cocoa powder extender. In some embodiments, the protein-enriched food or beverage product may include chocolate, candy, or sweets.In some embodiments, examples of such (protein-enriched) foods include carbohydrate-based main dishes such as pasta (macaroni and cheese), rice, and grains. In some embodiments, examples of protein-enriched food or beverage products include dips, spreads, and toppings (hummus).

[0114] The food or beverage product is suitable for both human and animal use. In certain embodiments, the composition is suitable for use as a pet food or pet food formulation.

Example

[0115] The present invention is further illustrated based on the following examples. However, the present invention is not limited to the following examples or the illustrated embodiments.

[0116] <Folin-Ciocalteu (or gallic acid equivalent) method> The polyphenol content was measured by the Folin-Ciocalteu method described in ISO 14502:2005.

[0117] <CIELAB (or L * a * b * ) color measurement> Measurements were performed using a Konica Minolta benchtop spectrophotometer CM-5. Liquid samples were taken from the reaction mixture, transferred to a 10 mL cuvette (plastic cell CM A131, 50×38, optical path length 10 mm - front pressure 0.1 mm / side thickness 0.23 mm), and measured in transmission mode. Solid samples were measured in reflection mode.

[0118] <Protein content> The protein content was measured using a LECO FP928 protein analyzer based on AOAC 990.03 or AOAC 992.15 with a sample (0.1 g of solids or 1 mL of solution).

[0119] <Amino acid content> Amino acid content was measured according to ISO 13903:2005.

[0120] <Solubility analysis> Solubility was measured as a function of pH and protein concentration using the following method. Sample suspensions were prepared at 20°C to the desired protein concentration, and the pH was adjusted to the desired value using hydrochloric acid (1 M) or sodium hydroxide (1 M). The samples were centrifuged at 4,000 rpm for 10 minutes. A control sample (native) was also prepared at the desired protein concentration without pH adjustment. The protein content of the supernatants of the native and test samples was measured by the Kjeldahl method using a conversion factor of 6.25. The solubility was calculated using the following formula:

number

[0121] <Viscosity> The viscosity was measured using an Anton Paar rheometer MCR102e with a double gap spindle at a shear rate of 1 sec -1 (@ rpm 1 / sec) at room temperature.

[0122] <Molecular weight characterization> Molecular weight analysis was performed using a ThermoFisher Vanquish high-performance liquid chromatography (HPLC) according to the following method. Samples (200 μL) were injected onto two columns: Superdex® 75GL (molecular weight range: 70,000-3,000 Da) and Superdex® 30GL (molecular weight range: 7,000-100 Da). The mobile phase used was 125 mM dipotassium hydrogen phosphate and 125 mM potassium dihydrogen phosphate at pH 6.8. The flow rate was 0.50 mL / min, and measurements were performed for 90-160 minutes at room temperature. UV detection was performed at 218 nm.

[0123] <Example of bleaching treatment> (Conventional method) Protein isolate was prepared according to the following conventional method: The brewer's grains used, which included spent barley and corn or rice, were added to a jacketed mixing tank with water to a water to dry weight ratio of 10.5:1. The resulting slurry was heated to 55°C and treated with glucoamylase enzyme (EDC Enzeco® Glucoamylase) for 45 minutes to hydrolyze the starch. The pH was then raised to 9 with alkali and maintained for 45 minutes.

[0124] The mixture was then treated with food-grade protease enzyme (EDC Enzeco® Alkaline Protease L-660) at 60°C for 20-60 minutes to hydrolyze the protein components, after which the mixture was heated to 80°C and held for up to 25 minutes to inactivate the enzyme.

[0125] The solids were isolated from the liquid protein stream using a decanter centrifuge. The liquid protein stream was subjected to a microfiltration system (0.1 μm membrane cutoff, 70-80°C, suitable membranes available from Pall Corporation). The microfiltration permeate was processed in a nanofiltration system (MWCO 1000 Da, operating pressure: 1.5-3 bar, suitable membranes available from MICRODYN-NADIR). The resulting nanofiltration concentrate was subjected to vacuum evaporation to remove water prior to spray drying.

[0126] <Bleaching with hydrogen peroxide> Example 1 Bleaching of Nanofiltration Concentrate The following examples were prepared from brewer's grains containing spent barley and spent rice by conventional methods. The spray-dried product was dissolved in water to a protein concentration of 5% by weight and treated with hydrogen peroxide (Solvay Interox® ST-35, 35% by weight) at 66°C for 300 minutes. The hydrogen peroxide dosages listed in Tables 1 and 2 are defined as multiples of X, where X = hydrogen peroxide (35% by weight) per kg of protein, i.e., 17.5 g of pure hydrogen peroxide per kg of protein. This dosage was chosen based on the method used to bleach whey proteins by Jervis et al. in "Effect of bleaching whey on sensory and functional properties of 80% whey protein concentrate," J. Dairy Sci., 2012, 95(6), 2848-2862, in which 5 g of hydrogen peroxide (35% by weight) was administered per kg of whey.

[0127] The color of the solution changes during the bleaching process. * a * b * The lightness (L) at the start and end of treatment is shown in Table 3 below. * a * b * The data from the measurements are shown in Table 4 and are illustrated in FIG.

[0128] [Table 3]

[0129] [Table 4]

[0130] These results indicate that concentrations of hydrogen peroxide suitable for bleaching whey proteins are insufficient to significantly reduce / brighten the color of this BSG-derived protein isolate. Doses of 5X or greater are preferred to provide significant bleaching within a practical timeframe.

[0131] Example 2: Bleaching of Microfiltration Permeate The following examples were prepared from brewer's grains containing spent barley and spent corn by conventional methods, except that after microfiltration, the microfiltration permeate was concentrated to a protein concentration of 1.28 wt % and treated with hydrogen peroxide (Solvay Interox® ST-35 35 wt %) under the conditions shown in Table 5. Hydrogen peroxide dosages are expressed as multiples of X, where X is defined as 50 g hydrogen peroxide (35 wt %) / kg protein, or 17.5 g hydrogen peroxide / kg protein. After bleach treatment, the bleached microfiltration permeate was nanofiltered, concentrated, and spray-dried by conventional methods.

[0132] During the bleaching process, the color of the solution changes to L * a * b * The lightness (L * ) are shown in Table 5 below, and all measured L * a * b * The data from the measurements are shown in Table 6 and are illustrated in Figure 4. The pH was monitored during the bleaching process and maintained at the desired pH by the addition of 2M aqueous potassium hydroxide solution.

[0133] [Table 5]

[0134] [Table 6]

[0135] Treatment of the microfiltration permeate with hydrogen peroxide significantly improved the brightness of the aqueous solution compared to unbleached barley / corn flour protein isolate and unbleached barley / corn 5% protein aqueous solution. Experiments performed at 90°C proceeded at similar rates, while experiments performed at 80°C showed slower reaction rates and did not achieve high brightness values.

[0136] Example 3: Effect of temperature on bleaching treatment Protein isolates in the following examples were prepared by conventional methods from brewer's grains containing spent barley and spent rice. The spray-dried product was dissolved in an aqueous solution with a protein concentration of 5% by weight and bleached with hydrogen peroxide (Solvay Interox® ST-35, 35% by weight) under the conditions shown in Table 7. The hydrogen peroxide dosage is expressed as a multiple of X, where X is defined as 50 g of hydrogen peroxide (35% by weight) per kg of protein, or 17.5 g of hydrogen peroxide per kg of protein.

[0137] [Table 7]

[0138] [Table 8]

[0139] [Table 9]

[0140] [Table 10]

[0141] The results showed that the bleaching rate increased with increasing temperature, reaching equilibrium in 120 minutes at 90°C, 150 minutes at 80°C, and 270 minutes at 70°C. The high-temperature bleaching treatment did not significantly deactivate hydrogen peroxide.

[0142] Example 4: Bleaching with Potassium Metabisulfite The following examples were prepared by conventional methods from brewer's grains containing spent barley and spent rice. The spray-dried product was dissolved in a 5% protein aqueous solution and treated with potassium metabisulfite (KMS) (VINOFERM Campden) at 66°C for 180 minutes. The potassium metabisulfite dosage is expressed as a multiple of X, where X is defined as 100g KMS / kg protein.

[0143] The color of the solution during the bleaching process is * a * b * The L at the start and end of treatment was measured. * a * b * The scores are shown in Table 11 below.

[0144] [Table 11]

[0145] (Example 5) Bleaching with urea peroxide The following examples were prepared by conventional methods from brewer's grains containing spent barley and spent rice. The spray-dried product was dissolved in a 5% protein aqueous solution and treated with a 35% by weight solution of urea peroxide (UHP) (ThermoFisher Scientific, urea peroxide ≥ 97%) in water at 66°C for 120 minutes. The dosage of urea peroxide is expressed as a multiple of X, where X is defined as 3 g (35% by weight) UHP / kg protein.

[0146] The color of the solution during the bleaching process is * a * b * The L at the start and end of treatment was measured. * a * b * The scores are shown in Table 12 below.

[0147] [Table 12]

[0148] Example 6: Determining the effect of bleaching on amino acid and polyphenol content and in vitro digestibility of bleached products Protein isolates in the following examples were prepared conventionally from brewer's grains containing spent barley and spent rice. The spray-dried product was used as an unbleached sample. The spray-dried product was dissolved in an aqueous solution containing 5% protein by weight and treated with hydrogen peroxide (34% by weight) at an 8X dosage, where X is defined as 50 g hydrogen peroxide (34% by weight) per kg protein, for 180 minutes.

[0149] The amino acid content of each sample is shown in Table 13.

[0150] [Table 13]

[0151] The data in Table 13 above show that the bleaching treatment has minimal effect on the amino acid content of the product.

[0152] The polyphenol content of the product obtained by the bleaching process (bleached) and the product produced in the same steps without bleaching (unbleached) was measured by the Folin-Ciocalteu method (or gallic acid equivalent), and the results are shown in Table 14 below.

[0153] [Table 14]

[0154] According to the data presented in the table above, the bleaching process significantly reduces the polyphenol content, resulting in a reduction of approximately one-third.

[0155] The in vitro digestibility of the bleached product was determined by Medallion Labs to be 1.00 (100%) on a 150g sample using the method described in US Patent No. 14 / 599,050.

[0156] (Example 7 Sensory Evaluation Test) Sensory evaluation was performed by 10 trained descriptive panelists using the Spemtrum™ method for appearance, aroma, and mouthfeel. Each product sample was evaluated at a protein concentration of 5% by weight (protein purity of 85%) in water.

[0157] Bleached: A product prepared according to the process of the present invention. Unbleached: A product prepared by the conventional method described above without the bleaching step using bleaching agents.

[0158] [Table 15] *The strength of the attribute indicates no significant difference at the 95% confidence level.

[0159] As shown in Figure 6, the data in Table 15 demonstrate that the products of the present invention have significantly reduced color intensity, opacity, foaming, and overall flavor compared to products made using the same manufacturing method but without the bleaching process. Additionally, certain aroma and flavor attributes are significantly reduced. Therefore, the products of the present invention have less impact on flavor, aroma, and appearance when mixed with other ingredients to prepare edible products compared to unbleached products.

[0160] Similar sensory evaluation methods were used to compare the bleached product with a commercial pea protein product, and the results are shown in Table 16 and Figure 10.

[0161] [Table 16]

[0162] The results show that the bleached product has less overall flavor, grainy flavor, and bitterness compared to the pea protein product. Additionally, the green, pea, plant-based milk, clay-like, and chalky flavors found in the pea protein product are absent in the bleached product.

[0163] <Example of hydrogen peroxide inactivation> Example 9: Inactivation by Enzyme Treatment Protein isolates in the following examples were prepared conventionally from brewer's grains containing spent barley and spent rice by dissolving the spray-dried product in an aqueous solution with a protein concentration of 5% by weight on a dry matter basis and treating it with hydrogen peroxide (Solvay Interox® ST-35 (35% by weight)) at a pH of 7 for 30 minutes at 90°C, using a 7X dosage, where X = 50 g hydrogen peroxide (34% by weight) / kg protein.

[0164] The bleached solution was treated with catalase enzyme (Catazyme® 25L, manufactured by Novozymes®, 25,000 CIU / g) to inactivate the hydrogen peroxide under the conditions shown in Table 17. The inactivation time was determined as the time required for the hydrogen peroxide concentration to reach less than 0.5 ppm, as measured using Quantofix® Peroxide 100 test strips.

[0165] [Table 17] *Native indicates that no pH adjustment was performed, and the pH was 7.4 to 7.5. #No inactivation of hydrogen peroxide was observed under these conditions.

[0166] Example 10: Inactivation with Ascorbic Acid Protein isolates in the following examples were prepared conventionally from brewer's grains containing spent barley and spent rice by dissolving the spray-dried product in an aqueous solution with a protein concentration of 5% by weight on a dry matter basis and treating it with hydrogen peroxide (Solvay Interox® ST-35 (35% by weight)) at a pH of 7 for 30 minutes at 90°C, using a 7X dosage, where X = 50 g hydrogen peroxide (34% by weight) / kg protein.

[0167] The bleached solution was treated with ascorbic acid under the conditions in Table 18. The inactivation time was the time until the hydrogen peroxide concentration reached less than 0.5 ppm using Quantofix® Peroxide 100 test strips.

[0168] [Table 18]

[0169] Increasing the dosage of ascorbic acid has a greater effect on inactivation time than increasing the temperature, but increasing the temperature also indicates an acceleration of the inactivation process. Doses of ascorbic acid greater than 1 g / L may decrease the pH, potentially leading to protein precipitation.

[0170] Example 11 Inactivation by Boiling Protein isolates in the following examples were prepared conventionally from brewer's grains containing spent barley and spent rice. The spray-dried product was dissolved to an aqueous solution containing 5% protein by weight and bleached according to the conditions in Table 19. After the designated bleaching times listed in Table 19, with an intermediate rest period, hydrogen peroxide inactivation was performed by raising the temperature to 100°C and holding for the indicated time. The hydrogen peroxide (Solvay Interox® ST-35 (35% by weight)) dosage was defined as X = 50 g hydrogen peroxide (34% by weight) / kg protein. Inactivation was performed by continuing boiling until the hydrogen peroxide concentration reached less than 0.5 ppm using Quantofix® Peroxide 100 test strips.

[0171] [Table 19] As the hydrogen peroxide dosage increased, the boiling time required also increased.

[0172] Example 12: Foods using the protein isolate of the present invention [Coffee creamer] Ingredients: Inventive Protein Isolate, Medium Chain Triglyceride (MCT) Oil, Maltodextrin, Acacia Gum, Sunflower Lecithin, Natural Flavors. Add 20g per cup of coffee.

[0173] [Plant-based milk substitute] Ingredients: Water, Broad Bean Protein, Inventive Protein Isolate, Coconut Cream, Canola Oil, Sugar, Calcium, Natural Flavors, Gellan Gum, Locust Bean Gum, Potassium Sulfate, Sunflower Lecithin.

[0174] [Plant-based milk substitute] Ingredients: Water, Oat Powder, Pea Protein, Inventive Protein Isolate, Coconut Cream, Sugar, Soluble Corn Fiber, Calcium, Natural Flavors, Soy Protein Isolate, Gellan Gum, Locust Bean Gum, Dipotassium Phosphate, Sunflower Lecithin.

[0175] [Ready-to-mix vanilla beverage] Ingredients: Pea protein, inventive protein isolate, medium chain triglyceride (MCT) oil, maltodextrin, acacia gum, sugar, natural flavors, xanthan gum, stevia extract, sucralose. Blend 32g in 1 cup (8 oz) of water or plant-based milk alternative.

[0176] [Ready-to-drink strawberry beverage] Ingredients: Water, Pea Protein, Inventive Protein Isolate, Sugar, Sunflower Oil, Natural Flavors, Calcium Carbonate, Cellulose Gel,

[0177] [Ready-to-drink vanilla beverage] Ingredients: Water, Inventive Protein Isolate, Pea Protein, Sugar, Canola Oil, Natural Flavors, Calcium Carbonate, Cellulose Gel, Sunflower Lecithin, Sucralose®.

Claims

1. 1. A method for isolating a protein isolate from a grain material, comprising: a) subjecting an aqueous slurry of cereal material to enzymatic protein hydrolysis to produce a liquid protein stream; b) removing solids from said liquid protein stream; c) subjecting said liquid protein stream to one or more filtration steps; and d) processing said liquid protein stream to produce said protein isolate, The method further comprises, during or after step (c), treating the liquid protein stream with a bleaching agent at a temperature of 50° C. or greater and a pH of 5 or greater.

2. 10. The method of claim 1, wherein the grain material is selected from brewer's grains, barley, malted barley, rice, corn, and combinations thereof, and preferably the grain material is brewer's grains.

3. 3. The method of claim 1 or 2, wherein the brewer's grains comprise spent barley, and optionally spent rice or spent corn.

4. The method according to any one of claims 1 to 3, wherein the bleaching agent is an oxidizing agent or a reducing agent.

5. The method according to any one of claims 1 to 4, wherein the bleaching agent is one or more selected from the group consisting of potassium metabisulfite, benzoyl peroxide, urea hydrogen peroxide, and hydrogen peroxide.

6. The method of any one of claims 1 to 5, wherein the bleaching agent is hydrogen peroxide.

7. 7. The method according to any one of claims 1 to 6, wherein the amount of bleaching agent is at least 0.25 mol / kg protein, preferably 0.5 to 50 mol / kg protein, more preferably 1.5 to 6 mol / kg protein, and even more preferably 2.5 to 5 mol / kg protein.

8. 8. The method according to any one of claims 1 to 7, wherein the amount of bleaching agent is from 2.5 to 50 moles / kg protein.

9. 9. The method according to any one of claims 1 to 8, wherein the amount of bleaching agent is at least 17.5 g / kg protein, preferably 17.5 to 1750 g / kg protein, more preferably 52.5 to 210 g / kg protein, and even more preferably 85 to 175 g / kg protein.

10. 10. A method according to any one of the preceding claims, wherein the temperature during the step of treating the liquid protein stream with a bleaching agent is from 50 to 100°C, preferably from 70 to 98°C, more preferably from 80 to 95°C.

11. 11. A method according to any one of the preceding claims, wherein the pH during the step of treating the liquid protein stream with a bleaching agent is between 5 and 10, preferably between 6 and 9.5, more preferably between 7 and 8.

12. 12. A method according to any one of claims 1 to 11, wherein the pH during the step of treating the liquid protein stream with a bleaching agent is maintained at a value above 5, preferably between 5 and 10, more preferably between 6 and 9.5, and even more preferably between 7 and 7.

5.

13. 13. A method according to any one of claims 1 to 12, wherein the step of treating the liquid protein stream with a bleaching agent is carried out for a duration of 5 hours or less, preferably from 5 minutes to 5 hours, more preferably from 10 minutes to 3 hours, even more preferably from 15 minutes to 1 hour, and even more preferably from 20 to 45 minutes.

14. 14. The method according to any one of claims 1 to 13, wherein during the step of treating the liquid protein stream with a bleaching agent, the protein concentration in the liquid protein stream is 0.1 to 40 wt%, preferably 1 to 20 wt%, more preferably 5 to 15 wt%.

15. The step of treating the liquid protein stream with a bleaching agent comprises treating the liquid protein stream with a bleaching agent under the following conditions: the amount of bleaching agent is 2.5 to 50 moles / kg protein, for example 2.5 to 5 moles / kg protein, or 85 to 175 g / kg protein; the pH is between 7 and 8, and preferably the pH is maintained at a value between 7 and 8 during the step of treating the liquid protein stream with a bleaching agent; The temperature is 80 to 95°C, The processing time is 20 to 45 minutes, and 15. The method of any one of claims 1 to 14, wherein the protein concentration in the liquid protein stream is 1 to 20% by weight.

16. 16. The method of any one of claims 1 to 15, wherein step (c) of subjecting the liquid protein stream to one or more filtration steps comprises a microfiltration step and one or more nanofiltration steps.

17. 17. The method of any one of claims 1 to 16, wherein the step of treating the liquid protein stream with a bleaching agent is carried out during step (c).

18. The step (c) is c1) microfiltering said liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2) subjecting the microfiltration permeate to a step of treating with the bleaching agent to provide a bleached microfiltration permeate; and c3) nanofiltering the bleached microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; The step (d) 18. The method of any one of claims 1 to 17, comprising d) processing said nanofiltration retentate to produce said protein isolate.

19. 20. The method of claim 18, further comprising concentrating the microfiltration permeate to obtain the concentrated microfiltration permeate prior to the bleaching step, preferably wherein the concentrating the microfiltration permeate to obtain the concentrated microfiltration permeate is performed by one or more methods selected from the group consisting of filtration, centrifugation, dewatering, and evaporation.

20. The step (c) is c1') microfiltering said liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; c3') subjecting the nanofiltration concentrate to a step of treating with the bleaching agent to obtain a bleached nanofiltration concentrate; and c4') nanofiltering the bleached nanofiltration retentate to obtain a second nanofiltration permeate and a second nanofiltration retentate; The step (d) 18. The method of any one of claims 1 to 17, comprising d) processing the second nanofiltration retentate to produce a protein isolate.

21. 17. The method of any one of claims 1 to 16, wherein a step of treating the liquid protein stream with a bleaching agent occurs after step (c).

22. The step (c) is c1') microfiltering said liquid protein stream to obtain a protein-containing microfiltration permeate and a microfiltration retentate; c2') nanofiltration of the microfiltration permeate to obtain a nanofiltration permeate and a nanofiltration retentate containing proteins; and d1′) subjecting the nanofiltration concentrate to a step of treating with the bleaching agent to obtain the bleached nanofiltration concentrate; The step d) 22. The method of any one of claims 1 to 16 and claim 21, comprising d) treating the bleached nanofiltration retentate to produce a protein isolate.

23. The step d) increasing the total solids content of the liquid protein stream to 20-55% by evaporation; and 23. The method of any one of claims 1 to 22, comprising producing the protein isolate by spray drying.

24. 24. The method of any one of claims 1 to 23, further comprising a filtration step using activated carbon prior to, preferably immediately before, step (d).

25. 25. The method according to any one of claims 1 to 24, wherein the method further comprises the step of inactivating the bleaching agent by heating, ascorbic acid treatment or enzyme treatment, preferably by enzyme treatment.

26. 26. The method of claim 25, wherein the enzymatic treatment comprises treatment with an enzyme composition comprising catalase.

27. 27. A method according to claim 25 or 26, wherein the temperature of the liquid protein stream is adjusted by heat exchange, preferably to below 60°C, prior to the step of inactivating the bleaching agent.

28. 28. The method according to any one of claims 25 to 27, wherein the method is carried out according to claim 18, and wherein a step of deactivating the bleaching agent is carried out between step (c2) and step (c3).

29. 28. The method according to any one of claims 25 to 27, wherein the method is carried out according to claim 20, and wherein a step of deactivating the bleaching agent is carried out between step (c3') and step (c4').

30. 28. The method according to any one of claims 25 to 27, wherein the method is carried out according to claim 22, and wherein a step of deactivating the bleaching agent is carried out between step (d1') and step (d).

31. 31. A method according to any one of claims 25 to 30, wherein at the end of the step of deactivating the bleaching agent the concentration of the bleaching agent in the liquid protein stream is less than 1 ppm, preferably less than 0.5 ppm.

32. The protein isolate L ＊ The L score was measured by the CIELAB method in a 5 wt% protein aqueous solution. ＊ The method of any one of claims 1 to 31, wherein the score is 60 or higher, preferably 70 to 100, more preferably 75 to 98.

33. The protein isolate comprises: L in the 5 wt % aqueous protein solution ＊ The score is the L of the protein isolate isolated from said cereal material, preferably from said brewer's grain, by the method of claims 1 to 32, which does not include the step of treating the liquid protein stream with said bleaching agent. ＊ the score is 1.25 times or more, preferably 1.25 to 3.5 times or more, and more preferably 1.5 to 3 times or more, the total polyphenol content in the protein isolate, as measured by the Folin-Ciocalteu method, is 10,000 to 50,000 mg / kg, preferably 15,000 to 40,000 mg / kg, and more preferably 25,000 to 30,000 mg / kg; the total polyphenol content in the protein isolate, measured by the Folin-Ciocalteu method, is 40-80%, preferably 55-75%, of the total polyphenol content in the protein isolate isolated from the cereal material, preferably from the brewer's grain, by a method according to any one of claims 1 to 29, which does not include the step of treating a liquid protein stream with a bleaching agent; The average molecular weight is 3,000 to 30,000 Da, preferably 5,000 to 10,000 Da, more preferably 5,500 to 8,000 Da, and even more preferably 6,500 to 7,500 Da; and the bleach concentration is less than 1 ppm, preferably less than 0.5 ppm; 33. The method according to any one of claims 1 to 32, having one or more of the following features:

34. The protein isolate comprises: the solubility in water at a pH of 8 or less, measured by the Kjeldahl method using a conversion factor of 6.25 with a protein concentration of 2% on a dry matter basis, is 70% or more, preferably 75% or more; the viscosity in water when the protein concentration is 5% on a dry matter basis is less than 5 mPa s, preferably 0.5 to 4 mPa s, and more preferably 2 to 3.5 mPa s; the viscosity in water is 10 to 25 mPa s, more preferably 12 to 18 mPa s, when the protein concentration is 20% on a dry matter basis; the proportion of proteins in the protein isolate having a molecular weight of 1000 Da or less is less than 15%, preferably 5-12%, by weight of the proteins in the protein isolate; and / or an in vitro digestibility of 80% or more, preferably 80 to 100%, and most preferably 100%; 34. The method according to any one of claims 1 to 33, having one or more of the following features:

35. A protein isolate obtainable according to any one of claims 1 to 34.

36. A protein isolate isolated from cereal material, preferably from brewer's grain, having an L 100% or less of saturation, as measured by the CIELAB method, in a 5% by weight protein solution in water. ＊ A protein isolate having a score of 60 or greater, preferably 70-100, more preferably 75-98.

37. the protein isolate L in a 5 wt% aqueous protein solution ＊ 10. The method of claim 1, wherein the step of treating the liquid protein stream with a bleaching agent is not included. ＊ the score is 1.25 times or more, preferably 1.25 to 3.5 times or more, and more preferably 1.5 to 3 times or more, the total polyphenol content in the protein isolate, as measured by the Folin-Ciocalteu method, is 10,000 to 50,000 mg / kg, preferably 15,000 to 40,000 mg / kg, and more preferably 25,000 to 30,000 mg / kg; the total polyphenol content in said protein isolate, measured by the Folin-Ciocalteu method, is 40-80%, preferably 55-75%, of the total polyphenol content in a protein isolate isolated from cereal material, preferably from brewer's grain, by a method according to any one of claims 1 to 34, which does not include a step of treating the liquid protein stream with a bleaching agent; The average molecular weight is 3,000 to 30,000 Da, preferably 5,000 to 10,000 Da, more preferably 5,500 to 8,000 Da, and even more preferably 6,500 to 7,500 Da; and the concentration of said bleaching agent is less than 1 ppm, preferably less than 0.5 ppm; 37. The protein isolate of claim 36, having one or more of the following characteristics:

38. the protein isolate the solubility in water at a pH of 8 or less, measured by the Kjeldahl method using a conversion factor of 6.25 with a protein concentration of 2% on a dry matter basis, is 70% or more, preferably 75% or more; the viscosity in water when the protein concentration is 5% on a dry matter basis is less than 5 mPa s, preferably 0.5 to 4 mPa s, and more preferably 2 to 3.5 mPa s; the viscosity in water is 10 to 25 mPa s, more preferably 12 to 18 mPa s, when the protein concentration is 20% on a dry matter basis; the proportion of proteins in the protein isolate with a molecular weight of 1000 Da or less is less than 15%, preferably 5-12%, by weight of the proteins in the protein isolate; and / or an in vitro digestibility of 80% or more, preferably 80 to 100%, and most preferably 100%; 38. The method of claim 36 or 37, comprising one or more of the following features:

39. A food or beverage containing a protein isolate obtainable by the method according to any one of claims 1 to 34.

40. 39. A food or beverage comprising the protein isolate of any one of claims 36 to 38.

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