BE1032251B1 - An acid-soluble protein isolate and its production process - Google Patents

Abstract

A protein isolate isolated from a grain material, preferably brewers' grains, having high water solubility at low pH, processes for producing the protein isolate, and foods and beverages comprising the protein isolate.

Description

1 BE2023/6033

An acid-soluble protein isolate and its production process

FIELD OF INVENTION

The present invention is directed to a protein isolate isolated from grain material having a high protein content and high water solubility, particularly at an acidic pH, i.e., a pH below 7. Furthermore, the protein isolate can remain soluble for extended periods of time at an acidic pH. Accordingly, the protein isolate of the present invention can be described as acid-soluble and/or acid-stable. The present invention is also directed to a process for producing the protein isolate and food or beverage products, particularly carbonated beverages, comprising the protein isolate.

BACKGROUND OF THE INVENTION

The use of protein isolates and supplements is well known in the art. For example, many people use protein isolates to make beverages or other foods as part of a training program to provide additional protein for muscle growth. People may use protein supplements when their daily diet is insufficient to meet the body's daily protein needs. Furthermore, individuals with specific diets that prohibit the consumption of traditional meat-based protein sources can supplement their diet with protein isolates to meet their daily needs.

Traditionally, protein isolates and supplements are generally based on whey, pea, soy, or casein. Whey and casein proteins are generally obtained as a byproduct of dairy production, with whey isolated from cheese production and casein from milk. Soy proteins are isolated from soybeans, and pea proteins from pea plants. While protein powders and supplements based on whey, pea, soy, and casein are used to successfully provide beneficial amounts of protein, the latter are not always suitable for people with food intolerances or allergies, such as lactose intolerance. While plant-based protein isolates have fewer immunogenic effects, the taste of these products is generally perceived as less pleasant and they are also less soluble than, for example, their whey counterparts. As such, the food and beverage industry, where high solubility at acidic pH levels is generally a requirement, is particularly susceptible to these issues.

2 BE2023/6033 is largely inaccessible to vegetable proteins. More recently, attempts have been made to improve the solubility of soy protein isolates for food and beverage applications (see, for example, WO 2005/044013 A2).

Brewers' spent grain (BSG) is the most common byproduct generated during the beer brewing process. This material comprises malt and grain husks, which are obtained as a solid fraction after filtration or lautering of the mash, where the insoluble BSG is separated from the soluble wort. BSG is rich in nutrients, particularly protein and fiber. The solubility of BSG proteins is generally low in the pH range relevant to food and beverage applications (pH 2-9). The solubility of BSG proteins is particularly low in the pH range of acidic carbonated beverages (pH 3-5). This is because the more soluble proteins dissolve in the wort and continue throughout the brewing process. Furthermore, BSG has a higher polyphenol content than other protein sources, and it is known that interactions between polyphenols and proteins, particularly the proline-rich hordeins found in BSG, can reduce its solubility.

Protein isolates were produced using BSG as described in

WO 2021/028509 A1. US 3,846,397 A and WO 2020/247363 A1 also describe the production of protein isolates from BSG by hydrolysis. Improving the solubility of BSG-based protein isolates is typically achieved by increasing the proportion of low molecular weight protein fragments, for example, by hydrolyzing the BSG for longer periods under more severe conditions and/or by removing the higher molecular weight protein fragments by filtration (see, for example, US 2012/0302731 A1). However, it is known that lower molecular weight protein fragments impart a bitter taste, which may be undesirable for food and beverage applications. Therefore, there remains a need in the art for protein isolates derived from grain material with a high protein content, high solubility at low pH, and a desirable organoleptic profile.

SUMMARY OF THE INVENTION

The present invention is directed to a protein isolate isolated from a grain material, in particular brewer's grain, having a high protein content and a high solubility in water, in particular at an acidic pH, i.e. a pH below 7. The protein isolate further has such taste, aroma and visual characteristics that it

3 BE2023/6033 can be easily incorporated into foods and beverages without significantly and/or negatively affecting the organoleptic properties of the food or beverage. The present invention is further directed to a process for producing the protein isolate and foods and beverages, particularly carbonated beverages, comprising the protein isolate.

From a first aspect, the present invention is directed to a protein isolate isolated from a grain material, preferably brewers’ grains, wherein the protein isolate: has a protein content of at least 60% dry matter by weight as determined by the Kjeldahl method using a conversion factor of 6.25; and has a solubility of at least 90%, preferably at least 95%, in water at a pH less than 7 and a protein concentration of 2% dry matter by weight as determined by the Kjeldahl method using a conversion factor of 6.25.

From a second aspect, the present invention is directed to a process for producing a protein isolate from a grain material, preferably brewers’ grains, the process comprising: a) subjecting an aqueous slurry of the grain material to enzymatic protein hydrolysis to produce a liquid protein stream comprising protein-containing material; b) removing solids from the liquid protein stream; c) subjecting the liquid protein stream to a first filtration process comprising one or more filtration steps; d) incubating the liquid protein stream at a pH of 2 to 5, preferably at a temperature of 5 to 70°C; e) removing solids from the liquid protein stream; f) optionally subjecting the liquid protein stream to a second filtration process comprising one or more filtration steps; and g) processing the liquid protein stream to produce the protein isolate.

From a third aspect, the present invention is directed to a protein isolate obtained or obtainable by the process described above.

4 BE2023/6033

From a fourth aspect, the present invention is directed to a food or beverage comprising the protein isolate described above.

Further advantageous features of the present invention are set forth in the description and the dependent claims.

DESCRIPTION OF THE DRAWINGS

Figure 1a shows the water solubility at pH 2 to 8 of sample A at 2.5% protein concentration, sample B at 2% protein concentration, and sample C at 2% protein concentration.

Figure 1b shows the water solubility of sample C at pH between 2 and 8 at protein concentrations of 2, 5, 10, 15, and 20% by weight.

Figure 1c shows the water solubility of the products from Examples 2, 4 and 5 at pH between 2 and 8 at a protein concentration of 2% by weight.

Figure 2a shows the molecular weight distribution of the products from Examples 2-4 and 14 and Sample B.

Figure 2b shows the molecular weight profile of the products from Examples 2-5 compared with a commercial pea protein isolate, a commercial rice protein isolate, and a commercial wheat protein isolate. The commercial isolates are marketed with high water solubility at acidic pH.

Figure 3a shows the clarity score (L* score) of sample A at 4 °C at pH 3 and 3.5 over time.

Figure 3b shows the turbidity score of sample A at 4 °C at pH 3 and 3.5 over time.

Figure 3c shows the clarity score (L* score) of sample A at 20°C at pH 2.5, 3, 3.5, and 4.2 over time. Figure 3d shows the turbidity score of sample A at 20°C at pH 2.5, 3, and 3.5 over time.

Figure 3e shows the clarity score (L* score) of sample A at 75°C at pH 3 and 3.5 over time. Figure 3f shows the turbidity score of sample A at 75°C at pH 3 and 3.5 over time. 5 DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that a highly water-soluble protein isolate with a high protein content can be isolated from grain material, particularly BSG. This isolate exhibits particularly improved water solubility at low pHs, such as below pH 7, while BSG proteins typically exhibit low water solubility at these pHs. This improvement in solubility is achieved by incubating proteinaceous material, isolated from BSG by hydrolysis, at a pH of 2 to 5, preferably at a temperature of 5 to 70°C. While not wishing to be bound by theory, the inventors believe that a portion of the proteinaceous material in this pH range may lack a net electrical charge, thereby reducing its solubility in aqueous media. The incubation conditions developed by the present inventors maximize the solubility of the remaining proteinaceous material when the material that precipitates under the incubation conditions is removed. The process of removing this material and further treating the proteinaceous material may further improve the solubility of the protein isolate and in particular the stability of the protein isolate in an acidic aqueous solution over extended periods of time, e.g., weeks, months or years.

The process of the present invention can improve solubility at low pH without the need for harsh or prolonged hydrolysis conditions common in the production of prior art protein isolates. Harsh or prolonged conditions can result in a higher proportion of lower molecular weight protein fragments in the product, which can contribute to a bitter taste in the protein isolate, which may be undesirable for food and beverage applications.

In view of the above, the protein isolate of the present invention is eminently suitable for food and beverage applications, particularly for the fortification of sour carbonated beverages.

6 BE2023/6033

In view of the foregoing, the present invention is directed in a first aspect to a protein isolate isolated from a grain material, preferably brewers’ grains, wherein the protein isolate: has a protein content of at least 60% dry matter by weight as determined by the Kjeldahl method using a conversion factor of 6.25; and has a solubility of at least 90%, preferably at least 95%, in water at a pH less than 7 and a protein concentration of 2% dry matter by weight as determined by the Kjeldahl method using a conversion factor of 6.25.

The grain material of the present invention can be brewer's spent grain, barley, barley malt, rice, corn, and combinations thereof. Preferably, the grain material is brewer's spent grain.

Brewer's spent grain (BSG) is a byproduct of the brewing industry after the mashing step. At this point in the brewing process, the soluble fraction (called wort) enters the further brewing steps, while the insoluble fraction is removed. This insoluble fraction is brewer's spent grain. The brewer's spent grain used in the process of the present invention is preferably obtained after brewing with grains, comprising barley and, optionally, one or more other grains or other starchy materials, for example, rice, oats, wheat, maize, sorghum, cassava, and/or millet, particularly rice, maize, sorghum, and/or cassava, more particularly rice and/or maize. Most preferably, the spent grain is obtained from brewing with barley or a mixture of barley and rice or maize, preferably rice.

In accordance with the above, the brewer's grains may comprise 100% barley grains.

Optionally, the spent grains may comprise 20% to 100% barley spent grains by weight, preferably 45% to 70% by weight, for example, 45%, 50%, 55%, 60%, 65%, or 70% barley spent grains by weight. If the spent grains are a mixture of barley spent grains and rice spent grains or corn spent grains, the rice spent grains or corn spent grains may be present in an amount of 0% to 80% by weight, preferably 30% to 55%, for example, 30%, 35%, 40%, 45%, 50%, or 55% by weight.

7 BE2023/6033

Protein content

As used herein, “protein content/concentration/amount” refers to the protein content as measured by the Dumas method (conversion factor 6.25), in particular AOAC 990.03 or AOAC 992.15. Other methods known in the art, such as the Kjeldahl method (conversion factor 6.25), may also be used to achieve essentially the same result. The protein content of the protein isolate may be at least 60% dry matter by weight, preferably 65 to 95% dry matter by weight, for example 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% dry matter by weight.

Solubility in water

The protein isolate is highly soluble in water, especially at low pH. The protein isolate may have a water solubility as shown in Figure 1b. At lower protein concentrations, the water solubility is very high. Thus, at a pH of 2 to 6 and a protein concentration of 2% dry matter by weight, the water solubility, as determined by the Kjeldahl method (conversion factor of 6.25), may be at least %, preferably at least 95%, more preferably at least 98%, for example 95, 96, 97, 98, 99 or 100%.

At higher protein concentrations, water solubility is very high. For example, at a protein concentration of up to 15% by weight, the solubility of the protein isolate in water can be at least 65% (e.g., 65, 70, 75, 80, 85, 90, or 95%) at a pH of 2 to 6. At a pH of about 3 to about 5, water solubility at a protein concentration of 15% can be at least 70%, and at a pH of 4 to 5, water solubility can be at least 90%.

At a protein concentration of up to 10% dry matter by weight, its solubility in water may be at least 80% (e.g., 80, 85, 90, or 95%) at a pH of 2 to 6. At a pH of about 3 to about 6, the water solubility at a protein concentration of up to 10% dry matter by weight may be at least 90%, preferably 90% to 95%.

Even at a protein concentration of 20% by weight, water solubility is very high. For example, at a pH of 2 to 6, water solubility can be at least 50%.

8 BE2023/6033, for example, can be 50, 55, 60, 65, 70, 75, 80, or 85%. At this concentration, the water solubility can be at least 75% at a pH of 4 to 8 and approximately 60% at pH 3. At a pH between 4 and 6, the water solubility can be at least 75%, for example, 75, 80, or 85%.

The water solubility of the protein isolate may be at least 5% higher (particularly at a pH of 2 to 6 and a temperature of 20°C and a protein concentration of 2% dry matter by weight as determined by the Kjeldahl method with a conversion factor of 6.25) than a protein isolate produced without the step of incubating the liquid protein stream at a pH of 2 to 5 and preferably at a temperature of 5 to 70°C (referred to herein as an acid solubilisation or stabilisation process). For example, the protein isolate may have a solubility in water at a pH of 2 to 5 which is at least 10% higher than that of a protein isolate produced without an acid solubilisation process, preferably 10 to 15% or 10 to 20%, for example 10, 11, 12, 13, 14, 15, 18, 17, 18, 19 or 20%, at a pH of 2 to 6 and a temperature of 20°C and a protein concentration of 2% dry matter by weight as determined by the Kjeldahl method (conversion factor of 6.25).

The high solubility of protein isolate in water at a wide range of protein concentrations makes it a versatile product for enriching foods and beverages. It is particularly well-suited for inclusion in beverages, especially sour carbonated drinks.

Soluble protein isolates commonly precipitate during storage. However, the high water solubility of the protein isolate of the present invention is retained even after storage in aqueous solution for extended periods. This is particularly advantageous for its use in beverages, which may be stored at refrigerated temperatures (e.g., 4°C) for days, weeks, or months prior to consumption. The protein isolate may be water-soluble at a concentration of 2.5% protein by weight and will remain water-soluble after storage in water at 4°C for at least one week, preferably one to four weeks, for example, one, two, three, or four weeks, more preferably at least one month, more preferably at least six months, for example, one, two, three, four, five, six, seven, eight, nine, 10, 11, or 12 months, more preferably at least one year. During these periods of time, there may be no or substantially no precipitation or settling of the protein isolate.

9 BE2023/6033 protein isolate in solution. After storage under the above conditions for these periods, the water solubility can be at least 95%.

Turbidity analysis

The protein isolate may have a turbidity value of less than 65% as measured by the ASTM D1003 method, preferably less than 40%, more preferably 5 to 35%, at a pH of 3.5 and a protein concentration of 2.5% as measured by the Kjeldahl method using a conversion factor of 6.25.

For example, the turbidity value may be 5, 10, 15, 20, 25, 30, or 35% as measured by the ASTM D1003 method at a pH of 3.5 and a protein concentration of 2.5% as measured by the Kjeldahl method using a conversion factor of 6.25.

Colour

The color of protein isolates that can be used to enrich or supplement foods and beverages is important because the protein isolate should preferably have minimal or no effect on the appearance of the food or beverage. The color of a material can be described using the CIELAB color space (or

L*a*“b* color space), defined by the International Commission for

Illumination Science (CIE). This expresses color in three values: L* for perceptual brightness and a* and b* for the four unique colors of human vision: red, green, blue, and yellow. An L* value of 0 produces black, and an L* value of 100 indicates diffuse white. For the present invention, the L* rating is the best indicator of the product's color and its suitability for mixing with other ingredients in foods and beverages, and is therefore used as such herein.

The BSG starting material can be dissolved at a dry matter content of 10%

L* scores of 10 to 50, as measured by the CIELAB method. Optionally, the BSG in solution at 10% dry matter can have an L* score of 30 to 50, for example, 30, 35, 40, 45, or 50. Consequently, the brewer's grain starting material in solution typically has a dark red/brown color. However, the protein isolate of the present invention can have an L* score of at least 50, resulting in a yellow/light brown solution. Consequently, the protein isolate is very suitable for mixing with foods and beverages without significantly altering its appearance and/or

10 BE2023/6033. The L* score may preferably be 75 to 100, more preferably 85 to 98, for example, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98, as measured by the CIELAB method. At these L* score values, the protein isolate in solution has a light off-white/yellow color and is thus readily miscible with other ingredients to produce food and beverage products without affecting their appearance. It may be preferable to achieve this higher L* score by incorporating the optional bleaching process into the process of the present invention.

To obtain L* score values.

The protein isolate may have an L* score of at least 70, preferably 85 to 98, as measured by the CIELAB method at pH 3.5 and a protein concentration of 2.5%, as measured by the Kjeldahl method using a conversion factor of 6.25. For example, the L* score may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98, as measured by the CIELAB method at pH 3.5 and a protein concentration of 2.5%, as measured by the Kjeldahl method using a conversion factor of 6.25.

Molecular weight

The molecular weight profile of the protein isolate may be as described in Figure 2a or 2b. The protein isolate may have an average molecular weight, preferably a mode average or number average molecular weight, in the range of 5,000 to 30,000 Da, preferably 5,000 to 10,000 Da.

Advantageously, the molecular weight profile of the protein isolate provides it with a desirable flavor profile. In this regard, the protein isolate may have a relatively high proportion of protein fragments with a molecular weight of at least 1,000 Da. The percentage of protein in the protein isolate with a molecular weight of 1,000 Da is at least 75% by weight of the protein isolate, preferably 75 to 98% by weight, for example, 75, 80, 85, 90, 95, or 98% by weight, as determined by the Kjeldahl method using a conversion factor of 6.25.

The protein isolate has a particularly high percentage of protein in the molecular weight range of 3,000 to 30,000 Da. This percentage can be at least 45% based on the weight of the protein isolate, preferably 50 to 75% based on the weight of the protein isolate. The percentage of the protein in the protein isolate with a molecular weight in the

11 BE2023/6033 range of 3,000 to 10,000 Da may be at least 30%, preferably 35 to 50%, by weight of the protein isolate. The percentage of protein with a molecular weight in the range of 3,000 to 10,000 Da may be at least 2% by weight higher, preferably 3 to 8% by weight higher, than a protein isolate produced without an acid solubilization step, e.g., sample B.

The protein isolate also has a high percentage of protein with a molecular weight in the range of at least 30,000 Da. This percentage can be at least 3%, preferably 5 to 10%. This percentage can be at least 5% lower, preferably 7.5 to 12.5% lower, than for a protein isolate produced without an acid solubilization step, e.g., sample B.

The protein isolate contains a low proportion of low molecular weight protein fragments, e.g., 1000 Da or less. For example, the percentage of protein in the protein isolate with a molecular weight of 500 to 1000 Da may be less than 15% by weight, preferably 2 to 12% by weight, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% by weight. The percentage of protein in the protein isolate with a molecular weight of 0 to 500 Da is less than 10% by weight, preferably 1 to 5% by weight, e.g., 1, 2, 3, 4, or 5% by weight. The low proportion of protein fragments with a molecular weight of 1000 Da or less, particularly 500 Da or less, has a beneficial effect on the flavour profile of the protein isolate and can reduce the bitterness of bitter flavours compared to protein isolates with a higher proportion of protein fragments in these molecular weight ranges.

Process

The advantageous properties of the protein isolate of the present invention may arise from the process by which it is made. Accordingly, in another aspect, the present invention is directed to a process for producing a protein isolate from a grain material, the process comprising: a) subjecting an aqueous slurry of the grain material to enzymatic protein hydrolysis to produce a liquid protein stream comprising proteinaceous material; b) removing solids from the liquid protein stream;

12 BE2023/6033 c) subjecting the liquid protein stream to a first filtration process comprising one or more filtration steps; d) incubating the liquid protein stream at a pH of 2 to 5, preferably at a temperature of 5 to 70 °C; e) removing solids from the liquid protein stream; f) optionally subjecting the liquid protein stream to a second filtration process comprising one or more filtration steps; and g) processing the liquid protein stream to produce the protein isolate.

Enzymatic protein hydrolysis

The aqueous slurry is formed by mixing the grain material and water. The ratio of water to grain material (dry weight) in the aqueous slurry is preferably 8:1 to 12:1, preferably 10:1 to 11:1. The aqueous slurry is preferably formed in a double-walled mixing vessel, preferably with heating means.

The aqueous slurry is subjected to enzymatic protein hydrolysis to produce a liquid protein stream. If desired, the grain material can be subjected to particle size reduction before and/or during this step. Any suitable size reduction technique can be used, such as milling.

Prior to enzymatic protein hydrolysis, the aqueous slurry is preferably subjected to enzymatic starch hydrolysis. The enzymatic starch hydrolysis preferably involves treatment with a glucoamylase enzyme. Suitable glucoamylase enzymes include those used in the brewing industry and can be obtained, for example, from EDC (Enzyme Development Corporation,

New York) or Novozymes.

Enzymatic starch hydrolysis is preferably carried out at the natural pH of the aqueous slurry. The pH can be, for example, about 4.5 to about 6.5 (e.g., 4.5, 5, 5.5, 6, or 6.5, or any value in between).

The enzymatic starch hydrolysis is preferably carried out at a temperature of about 50°C to about 65°C (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 65°C, or any intermediate temperature).

13 BE2023/6033

The enzymatic starch hydrolysis is preferably carried out for a period of at least about 15 minutes, preferably at least about 20 minutes, and up to about 60 minutes, preferably about 45 minutes. The enzymatic starch hydrolysis can be carried out, for example, for a period of 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or any intermediate period.

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., to glucose and/or to other water-soluble saccharides, including disaccharides and other short-chain oligosaccharides).

The enzymatic protein hydrolysis preferably involves treatment with a protease enzyme. The protease enzyme is preferably a food-grade protease enzyme, preferably a serine protease. Preferably, it is an alkaline protease, preferably an endopeptidase, preferably a serine endopeptidase.

Suitable protease enzymes can be obtained, for example, from Novozymes or

EDC (Enzyme Development Corporation, New York).

The enzymatic protein hydrolysis 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 in between), preferably at a pH of about 9. The target pH can be achieved by adding an alkali such as sodium and/or potassium hydroxide prior to treatment with the enzyme.

The enzymatic protein hydrolysis is preferably carried out at a temperature of about 50°C 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 in between), preferably about 55°C to about 68°C, preferably about 55°C to about 65°C.

The enzymatic protein hydrolysis is preferably carried out for a period of at least about 15 minutes, preferably at least about 20 minutes, and

14 BE2023/6033 to about 80 minutes, preferably about 60 minutes. The enzymatic protein hydrolysis can be carried out, for example, for a period of 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 minutes, or any intermediate period.

The 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 intermediate value) is reached, preferably between 2 and 8, more preferably 3 to 5. As used herein, the dH may be determined using the pH stat method, by adding alkali (e.g., NaOH) and applying the following formula:

BxN

A= MD C 2) My) * 100 wt% where B is the volume of alkali consumed (ml), Ns is the normality of the alkali, a is the average degree of dissociation of amino acids (typically 0.93 is used), hot is the total peptide bond content (or amino acid content) in 1 g of protein (meg/g; typically 9 meg/g is used) and Mp is the mass of protein present (g).

The enzymatic starch hydrolysis (if performed) and the enzymatic protein hydrolysis preferably take place in the double-walled mixing vessel in which the aqueous slurry is formed.

Following enzymatic protein hydrolysis, the enzyme(s) is/are preferably inactivated by increasing the temperature, for example to about 75 to about 90 °C (for example about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 °C, or any intermediate temperature), preferably to 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 for any intermediate time.

After enzymatic protein hydrolysis, solids are removed from the liquid protein stream. The removal of solids is preferably done by decantation, preferably using decantation centrifuges. Pressure can be applied to the solids.

15 BE2023/6033 are exercised to maximise the recovery of liquid protein flow, for example using a screw press.

The solids removed from the liquid protein stream are preferably washed with water and the resulting wash water is then combined with the liquid protein stream, again to maximize protein recovery.

First filtration process

The liquid protein stream is then subjected to microfiltration to obtain a protein-containing microfiltration permeate, which can be referred to as the liquid protein stream, and a microfiltration retentate. Microfiltration is preferably performed using a ceramic microfiltration membrane.

Microfiltration is preferably performed using a microfiltration membrane with 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 intermediate value), preferably 0.03 to 0.25 µm, preferably 0.05 to 0.2 µm, 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 intermediate value). Suitable microfiltration membranes are available from Pall.

Corporation. Microfiltration preferably includes a diafiltration step.

The microfiltration retentate can be subjected to enzymatic protein hydrolysis in a rehydrolysis step, and the liquid product of the rehydrolysis step can be combined with the liquid protein stream. Rehydrolysis of the microfiltration retentate can advantageously improve protein recovery.

The microfiltration permeate is subjected to nanofiltration at an applied pressure of 1.0 bar (100 kPa) to 8.0 bar (800 kPa) to obtain a nanofiltration permeate and a protein-containing nanofiltration retentate, which can be referred to as the liquid protein stream. The applied pressure is a common term in the filtration field and refers to the pressure at which the feed is applied to the filtration membrane. The pressure is typically controlled by a feed pump and monitored by pressure sensors to ensure a constant target feed pressure is maintained.

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Nanofiltration is typically performed at an applied pressure of at least about 10 bar (1,000 kPa) and up to about 40 bar (4,000 kPa). The present inventors have found that by performing nanofiltration at a lower applied pressure of 1 bar (100 kPa) to 10 bar (800 kPa), a protein isolate with a more favorable flavor and solubility profile can be produced.

The nanofiltration can be carried out at an applied pressure of 1.3 bar (130 kPa) to 3.3 bar (330 kPa), preferably 1.4 bar (140 kPa) to 3.2 bar (320 kPa), preferably 1.5 bar (150 kPa) to 3 bar (300 kPa). For example, the nanofiltration can be carried out at an applied pressure of 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 intermediate value.

Nanofiltration is preferably performed using a nanofiltration membrane with a molecular weight cutoff (MWCO) of 500 to 2,000 Da, preferably 800 to 2,000 Da, and preferably 800 to 1,200 Da. Nanofiltration can be performed, for example, using a nanofiltration membrane with a molecular weight cutoff (MWCO) 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 intermediate value. Suitable microfiltration membranes are available from MICRODYN-NADIR.

The nanofiltration retentate preferably has a total solids content of 15 to 25% by weight, preferably 18 to 22% by weight, and a protein content (% dry matter by weight) of at least 80%, preferably at least 85%, as determined by AOAC 990.03 or AOAC 992.15.

Optional bleaching process

Optionally, the process of the present invention may include a step of bleaching the liquid protein stream with a bleaching agent. Bleaching the liquid protein stream can increase the clarity of the protein isolate, potentially reducing the impact of the protein isolate on the visual properties of a food or beverage in which it is incorporated.

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The bleaching process can take place between steps (c) and (d), i.e. bleaching can take place between the initial filtration process (step c)) and the step of incubating the liquid protein stream (step d)).

The bleaching process may comprise the steps of: bleaching the liquid protein stream with a bleaching agent; and subjecting the liquid protein stream to nanofiltration to obtain a nanofiltration permeate and a nanofiltration retentate, the nanofiltration retentate comprising the liquid protein stream.

The bleaching step may comprise treating the liquid protein stream with a bleaching agent at a temperature of at least 50°C and a pH of at least 5. The temperature during the bleaching step may be from 50 to 100°C, preferably from 70 to 98°C, more preferably from 80 to 95°C, for example, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. High temperatures (i.e., 80°C and above) are preferred to shorten the bleaching time. The pH during the bleaching step may be important because a low pH can lead to precipitation of the proteinaceous material. The pH during the bleaching step may be from 5 to 10, preferably from 6 to 9.5, more preferably from 7 to 8.

In preferred embodiments, the bleaching agent is one or more selected from the group consisting of sodium metabisulfite, potassium metabisulfite, benzoyl hydrogen peroxide, urea hydrogen peroxide, and hydrogen peroxide, preferably wherein the bleaching agent is hydrogen peroxide. The hydrogen peroxide may be provided in a concentration of 20 to 45% (w/w) in aqueous solution, for example, 35% (w/w) in aqueous solution.

The amount of bleaching agent used in the bleaching step may be at least 0.25 moles of bleaching agent per kilogram of protein to be bleached (mol/kg protein) as determined by

AOAC 990.03 or AOAC 992.15. Preferably the amount of bleaching agent is from 0.5 to 100 mol/kg protein, more preferably from 1 to 75 mol/kg, more preferably from 2 to 50 mol/kg protein, more preferably from 2.5 to 5 mol/kg protein, for example 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,

18 BE2023/6033 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 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 intermediate value.

The amount of bleaching agent used in the bleaching step may also be described in grams of bleaching agent per kilogram of protein to be bleached (g/kg protein), as determined by AOAC 990.03 or AOAC 992.15. Accordingly, the amount of bleaching agent may be at least 10 g/kg protein, preferably from 17.5 to 1750 g/kg protein, preferably from 35 to 875 g/kg, preferably from 52.5 to 210 g/kg protein, more preferably from 85 to 175 g/kg protein, more preferably from 110 to 150 g/kg protein, for example 110, 115, 120, 125, 130, 135, 140, 145 or 150 g/kg protein, or any value in between.

As mentioned above, the bleaching agent may be added as a dilute solution, which may be a 20 to 45% w/w solution in water, preferably 30 to 40% w/w, more preferably 35% w/w. As a dilute solution, the bleaching agent may be added in an amount of at least 10 grams of bleach per kilogram of protein (g/kg protein) as determined by AOAC 990.03 or AOAC 992.15, preferably 17.5 to 1750 g/kg protein, preferably 50 to 750 g/kg protein, more preferably 250 g/kg to 500 g/kg protein, 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.

Incubating the liquid protein stream

The step of incubating the liquid protein stream is particularly important for imparting the beneficial characteristics of the protein isolate. This step aims to precipitate the protein fragments that have the greatest negative impact on water solubility at pH below 7, so that they can be removed by subsequent filtration steps. In other words, this step can be an acid solubilization or stabilization step. However, this objective must be balanced against the need to maintain a high protein content and a desirable flavor profile.

Protein solubility depends on several factors, such as temperature, pH, concentration, and the nature of the protein fragments. The present inventors have discovered a complex interrelationship between these factors, particularly pH and temperature.

19 BE2023/6033

In view of the foregoing, the incubation step may be carried out at a pH of 2 to 5, preferably 3 to 4, for example, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4. In one embodiment, the pH may be about 3.5. At pHs within these ranges, the acid-labile proteinaceous material may have no net electrical charge. The inventors have found that incubation at these pHs has a beneficial effect on the water solubility of the isolate.

The incubation step is preferably carried out at 5 to 70°C. It may be carried out at a temperature of 10 to 30°C, more preferably 15 to 25°C, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25°C. The present inventors tested a number of incubation temperatures before arriving at these preferred ranges. At lower temperatures, significant protein precipitation can occur, which can reduce the protein content of the isolate, while at higher temperatures, certain proteins become more soluble while others coagulate and precipitate, affecting the isolate's protein profile.

Incubation within the preferred pH and temperature ranges of the present process provides a solution with color and turbidity values that are stable over long periods of time (see Figures 3a-3f), indicating that they are beneficial in obtaining an isolate that has high solubility over extended periods of time.

Accordingly, in preferred embodiments, the incubating step may be carried out at a pH of 3 to 4 and a temperature of 15 to 25°C.

The incubating step may be carried out for at least 10 minutes, preferably from 30 minutes to 5 hours, more preferably from 45 to 90 minutes, for example 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 minutes. The concentration of the proteinaceous material as determined by the Kjeldahl method using a conversion factor of 6.25 during the incubating step may be up to 30% solids by weight, preferably from 1 to 20% solids by weight, more preferably from 5 to 15% solids by weight, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% solids by weight.

In accordance with the foregoing, in preferred embodiments, the step of incubating may be carried out at a pH of 3 to 4 and a temperature of 15 to 25°C for

20 BE2023/6033 45 to 90 minutes. In this embodiment, the protein concentration can be 5 to 15% solids by weight, as determined by the Kjeldahl method using a conversion factor of 6.25.

After the liquid protein incubation step, the solids are removed from the liquid protein stream. Solids can be removed by decantation or centrifugation.

After incubating the liquid protein stream, the liquid protein stream may be pasteurized, for example, by treating it at 65 to 95°C for 30 to 180 seconds, preferably at 77°C for 90 seconds.

Optional second filtration process

After the solids have been removed, the liquid protein stream may be subjected to a filtration process comprising one or more filtration steps, which may be referred to as the second filtration process. Therefore, in some embodiments, the second filtration process is not performed, and in some embodiments, the second filtration process is performed.

The second filtration process may include microfiltration. In a first variation, the second filtration process may include subjecting the liquid protein stream to microfiltration, which is performed using a ceramic membrane at a temperature of 4 to 70°C. Preferably, the temperature may be between 5 and 25°C, for example, 5, 10, 15, 20, or 25°C. Suitable ceramic membranes are marketed by, among others, TAMI Industries and Pall® Corporation.

The pore size of the ceramic membrane can be up to 2 µm, more preferably 0.1 to 1.5 µm, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 µm. In specific embodiments, the pore size can be 0.14, 0.2, 0.45, 0.8, or 1.2 µm. Microfiltration can be performed at a pressure of 0.5 to 2 bar, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 bar. In certain embodiments, the pressure can be 1 bar.

In a preferred embodiment of the first variation, the second filtration process comprises subjecting the liquid protein stream to microfiltration, which is performed

21 BE2023/6033 using a ceramic membrane at a temperature of 5 to 25°C, with the ceramic membrane having a pore size of 0.1 to 0.3 µm. This can be combined with an incubation step, which is carried out at a pH of 3 to 4 and a temperature of 15 to 25°C.

In accordance with the preferred embodiment of the first variation, the present invention is directed to a process for producing a protein isolate from a grain material, the process comprising: a) subjecting an aqueous slurry of the grain material to enzymatic protein hydrolysis to produce a liquid protein stream comprising proteinaceous material; b) removing solids from the liquid protein stream; c) subjecting the liquid protein stream to a first filtration process comprising one or more filtration steps; d) incubating the liquid protein stream at a pH of 3 to 4 at a temperature of 15 to 25°C; e) removing solids from the liquid protein stream; f) subjecting the liquid protein stream to a second filtration process comprising subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 5 to 25 °C, the ceramic membrane having a pore size of from 0.1 to 0.3 µm; and g) processing the liquid protein stream to produce the protein isolate.

In a second variation, the second filtration process involves subjecting the liquid protein stream to filtration at less than 4°C. The filtration can be performed using cellulose filter sheets, such as those marketed by Pall.

Corporation. The filtration temperature should be low, preferably -2 to 4 °C, more preferably -0.5 to 1.5 °C, e.g., -0.5, 0.4, -0.3, -0.2, 0.1, 0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 °C.

The filtration can be carried out with one or more stabilizing agents. These can be selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrrolidone, silica gel, colloidal silica, and tannic acid.

22 BE2023/6033 diatomaceous earth and tannic acid are used. These stabilizers can be added to the solution to be filtered before filtration. Alternatively, they can be impregnated into the filter, for example, Pall Seitz® filter sheets.

Diatomaceous earth (also known as kieselguhr or celite) is typically composed of silica, alumina, and iron oxide as minor components. When diatomaceous earth is mixed with the liquid protein stream, it is mixed to a concentration of 1 g/L to 10 g/L, more preferably 3 to 9 g/L, for example, 3, 4, 5, 6, 7, 8, or 9 g/L.

Tannic acid may be included in addition to diatomaceous earth or on its own, preferably at a concentration of 1 to 20 g/hl, more preferably 10 to 15 g/hl, for example 10, 11, 12, 13, 14 or 15 g/hl.

In a preferred embodiment of the second variation, the second filtration process comprises subjecting the liquid protein stream to filtration, preferably using a cellulose filter at -2 to 2°C with diatomaceous earth as a stabilizing agent. This can be combined with an incubation step, which is carried out at a pH of 3 to 4 and a temperature of 15 to 25°C.

In the preferred embodiment of the second variation, the present invention is directed to a process for producing a protein isolate from a grain material, the process comprising: a) subjecting an aqueous slurry of the grain material to enzymatic protein hydrolysis to produce a liquid protein stream comprising proteinaceous material; b) removing solids from the liquid protein stream; c) subjecting the liquid protein stream to a first filtration process comprising one or more filtration steps; d) incubating the liquid protein stream at a pH of 3 to 4 at a temperature of 15 to 25°C; e) removing solids from the liquid protein stream; f) subjecting the liquid protein stream to a second filtration process comprising subjecting the liquid protein stream to filtration, preferably using a cellulose filter at -2 to 2°C with diatomaceous earth as a stabilizing agent; and

23 BE2023/6033 g) processing the liquid protein stream to produce the protein isolate.

The second filtration process may (further) include a step of filtration with a stabilizing agent such as activated carbon. In other words, the second filtration process may include a step of filtration with activated carbon. In other embodiments, the stabilizing agent may be one or more selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrrolidone, silica gel, colloidal silica, and tannic acid.

Additionally, a filtration step may be performed with one or more stabilizers selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrrolidone, silica gel, colloidal silica, and tannic acid, as a supplement to or as part of either the first or second variation of the second filtration process described above. The stabilizer is preferably activated carbon. The activated carbon may be added to the liquid protein stream prior to filtration or impregnated into the filter. Suitable impregnated filters include the Pall Seitz® AKS4

Activated Carbon Sheets.

Taken together, the incubation and second filtration steps can be described as an acid solubilization/stabilization process or a process to increase the solubility of the protein isolate in acidic pH water.

Processing to produce the protein isolate

Processing the liquid protein stream to produce the protein isolate may include a step of nanofiltration of the liquid protein stream with diafiltration to provide a protein-containing nanofiltration retentate. Processing the liquid protein stream to produce the protein isolate preferably involves evaporation to increase the total solids content to a total solids content of 10 to 55% (e.g., to 10, 15, 20, 25, 30, 35, 40, 45, or 50%, or any intermediate value), preferably 25 to 55%, and then spray-drying to produce the protein isolate, which may be in the form of a powder.

The protein isolate produced by the process preferably has a total solids content of at least 90% by weight, preferably at least 93% by weight (for example at least 90, 91, 92, 93 or 94%, or any

24 BE2023/6033 intermediate value), and a protein content (% dry matter by weight) of at least 60%, preferably 65 to 95%, more preferably 70 to 80% (for example at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 or any intermediate value), as determined by AOAC 990.03 or AOAC 992.15.

In another embodiment, the present invention is directed to a protein isolate obtained or obtainable by the process described above. The protein isolate produced by this process is the protein isolate discussed above, and thus the features and embodiments described with respect to that isolate apply equally to the protein isolate obtained or obtainable by the process described above.

Food and beverage products

The present invention is also directed to food or beverage products comprising the protein isolate defined above. The beneficial properties of the protein isolate of the present invention mean that it is ideally suited for inclusion in foods and beverages to increase their protein content and that it does not significantly and/or negatively affect the taste, appearance, or physical or chemical properties of the product.

The protein isolate is particularly suitable for inclusion in beverages due to its high water solubility. Due to its improved solubility at low pH, the protein isolate of the present invention is suitable for inclusion in acidic beverages with a pH of 2 to 6, preferably 3 to 5.

Another advantage of the beneficial properties of protein isolate is that it can be incorporated into food or beverage products in large quantities, such as up to 50% dry matter based on the weight of the food or beverage. Preferably, the food or beverage may comprise the protein isolate in an amount of 0.1% to 30%, more preferably 0.5% to 20%, even more preferably 1% to 10%, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% dry matter based on the weight of the food or beverage.

In preferred embodiments, the beverage, preferably a carbonated beverage, comprises the protein isolate in an amount of 0.5 to 20% dry matter on a per serving basis

25 BE2023/6033 the weight of the beverage. The beverage may be a "soft drink," e.g., an energy drink, a hydrating sports drink, a flavored water drink, a ready-to-drink (RTD) beverage such as RTD coffee, cola, or a fruit-flavored carbonated drink, or a ready-to-mix (RTM) beverage. The beverage may be an alcoholic beverage, which can be a fermented alcoholic beverage, such as beer or cider, or a distilled alcoholic beverage. The beverage may also be a non-alcoholic or low-alcohol beverage, such as a non-alcoholic or low-alcohol fermented beverage. The alcohol content of these beverages may be less than 1% alcohol by volume (ABV) or less than 0.5% ABV, e.g., 0% ABV. The carbonated beverage may be supplied in a bottle, can, keg, or any other suitable container.

The food or beverage may further comprise additives commonly used in food or beverage products, such as one or more of the following components: sweeteners, such as stevia, monk fruit extract, sucralose and/or acesulfame K; sugar; juices or juice concentrates, taurine, caffeine, an acid such as phosphoric acid, citric acid, tartaric acid, malic acid, folic acid or fumaric acid; colourings; flavourings, such as raspberry, strawberry, berries, banana, lemon-lime, lychee, peach, cherry, mango, blackcurrant, orange, passion fruit, guava and/or guarana; fruit or vegetable juice, vitamins and minerals.

The present invention will now be further described with reference to the following non-limiting examples.

EXAMPLES

Sample A is a protein isolate prepared from BSG according to the general method described in WO 2021/028509 A1. Sample B is a protein isolate prepared according to the general method described in WO 2021/028509 A1 with the additional step of bleaching the permeate from the microfiltration step using hydrogen peroxide (35% w/w) at 4 mol/kg protein at 90°C for 100 minutes, followed by nanofiltration and spray drying of the nanofiltration retentate. Sample C is prepared according to Example 3 below.

26 BE2023/6033

Optimization of acid solubilization conditions

The stability of the isolates with lower solubility at acidic pH was investigated at various temperatures and pHs to determine the appropriate incubation conditions. Sample A was combined with water to a concentration of 3.5% by weight and incubated under stirring under the conditions shown in Table 1.

Table 1

The samples were taken at 0 min, 30 min, 3 h, 6 h, 8 h, 14 h and 24 h and centrifuged (15 min at 4000 rpm). The supernatant was analyzed for brightness (L*) using the CIELAB method (CR-5

Konica Minolta) and turbidity (EBC method). The results are shown in Figures 3a-3f.

Incubation at 75°C resulted in a darker supernatant and higher turbidity than at 4°C or 20°C. This may be due to increased solubility of unstable proteins at the higher temperature, which then precipitated after the sample returned to ambient temperature. Incubation at a lower temperature resulted in higher turbidity than at 20°C. It is thought that the lower temperatures caused unstable proteins to precipitate. At 20°C, turbidity was low and stable, and each pH value at this temperature gave similar turbidity and clarity results.

This indicates that these conditions are favorable to obtain an isolate with high solubility at low pH.

General procedure for producing acid-soluble protein powder from BSG

Protein isolates were prepared according to the following general method using brewer's grains comprising barley grains and either corn grains or rice grains.

27 BE2023/6033

The grain feed was collected in a double-walled mixing vessel with water to achieve a water-to-dry weight ratio of 10.5:1. To hydrolyze the starch, the resulting slurry was heated to 55°C and treated with a glucoamylase enzyme (Novozymes® glucoamylase) for 45 minutes. The pH was then raised to 9 with alkali and held at that value for 45 minutes.

The mixture was then treated with a food-grade protease enzyme (Novozymes® alkaline protease) at 60°C for 20 to 60 minutes to hydrolyze the protein component. The enzymes were then inactivated by heating the mixture to 80°C and holding it at this temperature for up to 25 minutes.

The solids were separated from the liquid protein stream by decanter centrifuges. The liquid protein stream was fed into a microfiltration system (0.1 µm membranes; 70 to 80 °C; suitable membranes are available from

Pall Corporation).

The permeate from the microfiltration was processed into a nanofiltration system (MWCO of approximately 1,000 Da; suitable membranes available from MICRODYN-NADIR).

Optionally, the resulting retentate was bleached using hydrogen peroxide (35% w/w) at 4 mol/kg protein at 90°C for 100 min. The bleached retentate was further processed in a nanofiltration system (MWCO of approximately 1,000 Da; suitable membranes are available from MICRODYN-NADIR).

The retentate obtained from the nanofiltration system was adjusted to pH 3.5 and incubated for 1 hour at 15-20°C with a protein content of 10% by weight. Loose solids were removed from the resulting suspension using a disk-stack centrifuge or decanter (suitable decanters are available from GEA, among others). Optionally, the filtrate from the removal of loose solids was pasteurized for 90 seconds at 77°C.

28 BE2023/6033

Second filtration process

The present inventors have discovered that the long-term solubility of protein isolate at acidic pH can be improved by performing a secondary/polishing filtration at low temperatures after the incubation process.

In Examples 1-6, the filtrate from the removal of loose solids was subjected to a second filtration according to the conditions in Table 3 below. Step a) of the second filtration was performed using cellulose filter sheets (Pall Seitz® D-400 filter sheets). Step b), if performed, was performed using filter sheets impregnated with activated carbon (Pall Seitz®

AKS4 Activated Carbon Sheets).

In Examples 7-17, the filtrate from the removal of the loose solids was subjected to a second filtration according to the conditions in Tables 4 and 5 below. Step a) of the second filtration was performed using a ceramic microfilter at a pressure of 1 bar. Step b), if performed, was performed using filter sheets impregnated with activated carbon (Pall Seitz®

AKS4 Activated Carbon Sheets). In Examples 13 through 17, the filtrate from the removal of loose solids was pasteurized at 77°C for 90 seconds.

The filtrate obtained from the secondary filtration was spray dried to a powdery product.

Stability assessment

The stability of the products in Table 3 was assessed by mixing the powdered product with deionized water at a concentration of 2.5% protein concentration by weight at pH 3.5 and 4 and visually inspecting the mixture after the indicated time.

The stability of the products in Tables 4 and 5 was assessed by mixing the powdered product with deionized water at a concentration of 2.5% protein concentration by weight at pH 3.5 and 4 and visually inspecting the mixture.

29 BE2023/6033 after 3-4 weeks of storage. The symbols used to describe product stability are listed in Table 2 below.

Table 2 eee eee

Table 3

Retentate Type Bleached Bleached Bleached Bleached Bleached Bleached

Acid Sulphuric acid Phosphoric acid (min. Sulphuric acid:phosphoric acid | Sulphuric acid:phosphoric acid | Sulphuric acid:phosphoric acid | Sulphuric acid sulphuric acid) 1:1 (v/v) 1:1 (v/v) 1:1 (v/v)

Solid Removal | Centrifugation Disc Stack - Disc Stack - Disc Stack - Decanter Centrifugation substances centrifugation centrifugation centrifugation

Polishing - First Cellulose filter + | Cellulose filter + | Cellulose filter + | Cellulose filter + | Cellulose filter + filtration filtration Diatomite Diatomite (8.5 g/l) | Diatomite (7 g/l) Diatomite (7 g/l) Diatomite (7 g/l) Diatomite (3 g/l) + tannic acid (3 g/l) + tannic acid (12 g/hl) (12 g/hl)

Temp. -1 0,3 0,3 0,3 4 4 &

PC

Second | No Cellulose filter No No No No filtration impregnated with activated carbon

Stability assessment

After filtration Clear Clear Clear Clear Clear Clear solution solution solution solution solution solution

Long term - No settling | No settling No settling No settling Settling Settling stability (1 week) (3 weeks) (3 weeks) (3 weeks) (1 week) (1 week) w mM

N

©

N œ a © co oo

Table 4

Sample No. | Pore size | Temperature | Average flux | Protein yield | Stability assessment flow rate (lhr/m?) ([/hr)

R

Table 5

Example No. Pore size | Temperature | Average flux | Protein yield | Flow rate stability (lhr/m?) (lhr)

N

©

N œ a © co oo

32 BE2023/6033

Composition analysis

A sample prepared according to the general procedure above, including the bleaching process, (Example 3, Sample C) and a commercial pea protein product adapted for low pH applications were analyzed, the results of which can be found in Table 6 below.

Table 6 [°° [vewseewe [EvrememE

Protein (Kjeldahl method, conversion factor | 75.4 78.7 pq

Cattle |8 8

Gekes |E

Solubility analysis

The solubility of samples A and C was measured as a function of pH and protein concentration according to the following procedure. A suspension of the sample with the desired protein concentration was prepared at 20°C and the pH was adjusted to the desired value using 1 M HCl or 1 M NaOH. The samples were centrifuged for 10 minutes at 4000 rpm (Eurolabs centrifuge). A control sample with the desired protein concentrations without pH adjustment (Native) was prepared. The protein content of the Native sample and the supernatants of the test samples was measured using the Kjeldahl method, conversion factor 6.25. The solubility was calculated according to the equation below:

Solubility (%) = Sample protein content x 100

Protein content Native

33 BE2023/6033

The results are shown in Figures 1a and 1b. Sample C exhibits high solubility (295%) across the entire pH range at 2 and 5% protein concentrations. Solubility remains high (90% or higher) at 10% protein concentration by weight at pH 3-8. Lower solubility is observed at 15% and 20% protein concentrations by weight.

Turbidity analysis and CIELAB (or L*a*b*) color measurement

The measurements were performed using a Konica Minolta benchtop spectrophotometer CM-5. Liquid samples were prepared with a protein concentration by weight and a pH as listed in the table below, as determined by the

Kjeldahl method (conversion factor 6.25), transferred to a 10 ml cuvette (plastic cell – CM A131, 50 x 38, optical path length 10 mm – front thickness 0.1 mm/side thickness 0.23 mm) and measured in transmission mode. The protein concentration described here is 5% by weight unless otherwise stated. Solid samples were measured in reflection mode.

Turbidity analysis was performed according to the ASTM D1003 method at the protein concentration by weight and pH as listed in Tables 7 and 8 below.

Table 7

Table 8

34 BE2023/6033

Molecular weight characterization

Molecular weight analysis was performed according to the following procedure using a Thermofisher Vanquish high-performance liquid chromatography (HPLC) apparatus. A sample (200 µl) was injected into two columns in series (Superdex® 75 GL (molecular weight range 70,000–3,000 Da) and Superdex® 30 GL (molecular weight range 7,000–100 Da)) using a mobile phase of dipotassium hydrogen phosphate (125 mM) and potassium dihydrogen phosphate (125 mM) at pH 6.8. The flow rate was 0.50 ml/min, and the procedure was run at room temperature for 90 to 160 min. Ultraviolet detection was performed at 218 nm.

Claims (27)

35 BE2023/6033 CONCLUSIONS 1. A protein isolate isolated from a grain material, preferably brewer's grains, wherein the protein isolate: has a protein content of at least 60% dry matter by weight as determined by the Kjeldahl method using a conversion factor of 6.25; and has a solubility of at least 95% in water at a pH of 2 to 6 and a protein concentration of 2% dry matter by weight as determined by the Kjeldahl method using a conversion factor of 6.25. The protein isolate of claim 1, wherein the protein isolate has a turbidity value, as measured by the ASTM D1003 method, of less than 65%, preferably less than 40%, more preferably 5% to 35%, at a pH of 3.5 and a protein concentration of 2.5%, as measured by the Kjeldahl method using a conversion factor of 6.25. Protein isolate according to claim 1 or 2, wherein the protein isolate has an L* score of at least 60, preferably 75 to 100, more preferably 85 to 98, as measured by the CIELAB method. A protein isolate according to any preceding claim, wherein the protein content is 65 to 95% dry matter by weight. A protein isolate according to any preceding claim, wherein the solubility of the protein isolate in water is at least 70% at a pH of 2 to 6 and a protein concentration of up to 15% by weight, as determined by the Kjeldahl method using a conversion factor of 6.25. A protein isolate according to any preceding claim, wherein the solubility in water is at least 80%, preferably 85%, at a pH of 2 to 6 and a protein concentration of up to 10% dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25. 7. Protein isolate according to any one of the preceding claims, wherein the water solubility of the protein isolate is at least 5% higher, preferably 10 to 20% 36 BE2023/6033 higher, may be than a protein isolate that is produced without a process to increase the solubility of the protein isolate in water at a pH lower than 7. A protein isolate according to any preceding claim, wherein the protein isolate has an L* score of at least 70, preferably 85 to 98, as measured by the CIELAB method at a pH of 3.5 and a protein concentration of 2.5%, as measured by the Kjeldahl method using a conversion factor of 6.25. 9. A protein isolate according to any preceding claim, wherein the percentage of protein in the protein isolate having a molecular weight of 3,000 to 30,000 Da is at least 45%, preferably 50 to 75%, based on the weight of the protein isolate. 10. A protein isolate according to any preceding claim, wherein the percentage of protein in the protein isolate having a molecular weight of 500 to 1000 Da is less than 15% by weight of the protein isolate, preferably 2 to 12% by weight. A protein isolate according to any preceding claim, wherein the percentage of protein in the protein isolate having a molecular weight of 0 to 500 Da is less than 10% by weight of the protein isolate, preferably 1 to 5% by weight. A protein isolate according to any preceding claim, wherein the protein isolate remains soluble after storage in water at a pH of 3.5 and a temperature of 4°C for at least 1 week, preferably at least one month, more preferably at least 6 months, even more preferably at least 1 year, at a concentration of 2.5% protein by weight. 13. A process for producing a protein isolate from a grain material, preferably brewers' grains, the process comprising: a) subjecting an aqueous slurry of the grain material to enzymatic protein hydrolysis to produce a liquid protein stream comprising proteinaceous material; b) removing solids from the liquid protein stream; c) subjecting the liquid protein stream to a first filtration process comprising one or more filtration steps; 37 BE2023/6033 d) incubating the liquid protein stream at a pH of 2 to 5, preferably at a temperature of 5 to 70 °C; e) removing solids from the liquid protein stream; f) subjecting the liquid protein stream to a second filtration process comprising one or more filtration steps, the second filtration process comprising: subjecting the liquid protein stream to microfiltration performed using a ceramic membrane at a temperature of 5 to 25°C, the ceramic membrane having a pore size of 0.1 to 0.3 um; and subjecting the liquid protein stream from the microfiltration to activated carbon filtration; and g) processing the liquid protein stream to produce the protein isolate. 14. The process of claim 13, wherein incubating the liquid protein stream is carried out at a pH of 2 to 5, preferably 3 to 4, more preferably about 3.5. The process of claim 13 or 14, wherein incubating the liquid protein stream is carried out at a temperature of 10 to 30°C, more preferably 15 to 25°C. A process according to any one of claims 13 to 15, wherein the concentration of the proteinaceous material during the incubating step is up to 30% solids by weight, preferably 1 to 20% solids by weight, more preferably 5 to 15% solids by weight. Process according to any one of claims 13 to 16, wherein the step of incubating is carried out at a pH of 3 to 4 and a temperature of 15 to 25°C for 45 to 90 minutes. The process of any one of claims 13 to 17, wherein removing solids from the liquid protein stream comprises centrifugation or decantation. 38 BE2023/6033 Process according to claim 13, wherein the microfiltration is carried out at a pressure of 0.5 to 2 bar, more preferably about 1 bar. The process of any one of claims 13 to 19, wherein: the step of incubating is carried out at a pH of 3 to 4 and a temperature of 15 to 25°C. A process according to any preceding claim, wherein processing the liquid protein stream to produce the protein isolate comprises evaporating to increase the total solids content to a total solids content of from 10 to 55%, preferably from 25 to 55%, and then spray drying to produce the protein isolate. Process according to any one of the preceding claims, wherein the protein isolate has a total solids content of at least 90% by weight, preferably at least 93% by weight, and a protein content (% dry matter by weight) of at least 60%, preferably from 65 to 95%, more preferably from 70 to 80%, as determined by AOAC 990.03 or AOAC 992.15. 23. Protein isolate obtained or obtainable by the process of any one of claims 13 to 22. 24. A food or beverage comprising the protein isolate according to any one of claims 1 to 12 or according to claim 23. A food or beverage according to claim 24 comprising the protein isolate in an amount of up to 50% dry matter based on the weight of the food or beverage product, preferably 0.1% to 30%, more preferably 0.5 to 20% dry matter based on the weight of the food or beverage product. A food or beverage according to claim 24 or 25, wherein the food or beverage is a drink, preferably a carbonated drink, comprising the protein isolate in an amount of 0.5 to 20% dry matter based on the weight of the drink. 39 BE2023/6033 27. A beverage according to claim 26, wherein the beverage has a pH of 2 to 6, preferably 3 to 5.