An acid soluble protein isolate and its process of production

Abstract

A protein isolate isolated from a grain material, preferably brewer's spent grain, having a high water solubility at low pH, processes for producing the protein isolate and food and beverages comprising the protein isolate.

Description

[0001] An acid soluble protein isolate and its process of production

[0002] FIELD OF THE INVENTION

[0003] The present invention is directed to a protein isolate isolated from a grain material having a high protein content and high solubility in water, particularly at acidic pH, i.e. a pH of below 7. Furthermore, the protein isolate may remain soluble at acidic pH over extended time periods. Accordingly the protein isolate of the present invention may 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, in particular carbonated beverages, comprising the protein isolate.

[0004] BACKGROUND TO THE INVENTION

[0005] The use of protein isolates and supplements is well known in the art. For example, many people utilise protein isolates to make beverages or other foodstuffs as part of a training regimen to provide additional protein for muscle growth. People may utilise protein supplements when their daily diet is insufficient to satisfy the human body's daily protein requirements. In addition, individuals with specific diets that do not allow for the consumption of traditional meat-based protein sources may supplement their diets with protein isolates to meet their daily requirements.

[0006] Traditionally, protein isolates and supplements have generally been whey-, pea- soy- or casein-based products. Whey and casein proteins are generally recovered as a byproduct from dairy production, with whey being isolated from cheese production and casein being isolated from milk. Soy proteins are isolated from soybeans and pea proteins from pea plants. While whey, pea, soy and casein-based protein powders and supplements are used to successfully provide beneficial amounts of protein, the latter are not always suitable for people having food intolerances or allergies such as lactose intolerance. Plant-based protein isolates exist that provide less immunogenic effects, but these products are typically perceived to have a less pleasant taste and are also less soluble than, for instance, their whey counterparts. As such, a large part of the food and beverage sector, which typically requires high solubility at acidic pHs, has not been accessible for plant-based 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). Brewer’s spent grain (BSG) is the most abundant by-product generated in the beerbrewing process. This material comprises malt and grain husks obtained as a solid fraction after the mash filtration or lautering step, which separates the insoluble BSG from the soluble wort. BSG is rich in nutrients, particularly protein and fibre. BSG proteins generally have low solubility in the pH range that is relevant to food and beverage applications (pH 2-9). In particular, BSG proteins have low solubility in the pH range of acidic carbonated beverages (pH 3-5). This is because the more soluble proteins dissolve in the wort and proceed through the brewing process. Furthermore, BSG has a higher polyphenol content than other protein sources and it is known that interactions between polyphenols and proteins, in particular the proline-rich hordeins found in BSG, can reduce solubility.

[0007] Protein isolates have been produced using BSG, such as disclosed in WO 2021 / 028509 A1. US 3,846,397 A and WO 2020 / 247363 A1 also disclose the production of protein isolates from BSG by hydrolysis. Improvements in the solubility of BSG-based protein isolates is usually achieved by increasing the proportion of low molecular weight protein fragments by, for example, hydrolysing the BSG for longer periods, under harsher conditions and / or by removing the higher molecular weight protein fragments by filtration (see, for example, US 2012 / 0302731 A1 ). However, lower molecular weight protein fragments are known to impart bitterness, which may not be desirable for food and beverage applications. As such there remains a need in the art for protein isolates derived from grain material having a high protein content, high solubility at low pH and a desirable organoleptic profile.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is directed to a protein isolate isolated from a grain material, in particular brewer’s spent grain, having a high protein content and high solubility in water, particularly at acidic pH, i.e. below pH 7. The protein isolate additionally has taste, flavour and visual characteristics that allow it to be readily incorporated into foods and beverages without significantly and / or negatively impacting the organoleptic properties of the food or beverage. The present invention is additionally directed to a process for producing the protein isolate and food and beverages, in particular carbonated beverages, comprising the protein isolate. Viewed from a first aspect, the present invention is directed to a protein isolate isolated from a grain material, preferably brewer’s spent grain, wherein the protein isolate has: a protein content of at least 60% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25; and, a solubility of at least 90%, preferably at least 95%, in water at a pH of below 7 and a protein concentration of 2% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25.

[0010] Viewed from a second aspect, the present invention is directed to a process for producing a protein isolate from a grain material, preferably brewer’s spent grain, 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 from 2 to 5, preferably at a temperature of from 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.

[0011] Viewed from a third aspect, the present invention is directed to a protein isolate obtained or obtainable by the process described hereinabove.

[0012] Viewed from a fourth aspect, the present invention is directed to a food or beverage comprising the protein isolate described hereinabove.

[0013] Further beneficial features of the present invention are set out in the description and the dependent claims. DESCRIPTION OF THE DRAWINGS

[0014] Figure 1a shows the water solubility between pH 2 and 8 of Sample A at 2.5% protein concentration, Sample B at 2% protein concentration, and Sample C at 2% protein concentration.

[0015] Figure 1 b shows the water solubility of Sample C between pH 2 and 8 at protein concentrations of 2, 5, 10, 15, and 20% by weight.

[0016] Figure 1 c shows the water solubility of the products of Examples 2, 4 and 5 between pH 2 and 8 at a protein concentration of 2% by weight.

[0017] Figure 1 d shows the water solubility of the product of Example 13 between pH 2 and 6 at 2%, 5%, and 10% protein concentration as measured using the Kjeldahl method, with a conversion factor of 6.25.

[0018] Figure 2a shows the molecular weight distribution of the products of Examples 2-4 and 14 and Sample B.

[0019] Figure 2b shows the molecular weight profile of the products of Examples 2-5 compared to a commercial pea protein isolate, a commercial rice protein isolate, and a commercial wheat protein isolate. The commercial isolates are marketed as having high solubility in water at acidic pH.

[0020] Figure 3a shows the lightness (L*) score of Sample A at 4°C at pH 3 and 3.5 over time. Figure 3b shows the haze score of Sample A at 4°C at pH 3 and 3.5 over time.

[0021] Figure 3c shows the lightness ( / _*) score of Sample A at 20°C at pH 2.5, 3, 3.5 and 4.2 over time. Figure 3d shows the haze score of Sample A at 20°C at pH 2.5, 3, and 3.5 over time.

[0022] Figure 3e shows the lightness ( / _*) score of Sample A at 75°C at pH 3 and 3.5 over time. Figure 3f shows the haze score of Sample A at 75°C at pH 3 and 3.5 over time. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present inventors have discovered that a highly water-soluble protein isolate having a high protein content can be isolated from grain material, in particular BSG. This isolate has particularly improved solubility in water at low pHs, such as below pH 7, where BSG proteins usually exhibit low water solubility. This improvement in solubility is achieved by incubating proteinaceous material, that has been isolated from BSG by hydrolysis, at a pH of from 2 to 5 and, preferably, at a temperature of from 5 to 70°C. Without wishing to be bound by theory, the inventors understand that a portion of the proteinaceous material may have no net electrical charge in this pH range, which reduces its solubility in aqueous media. The incubation conditions developed by the present inventors maximise the solubility improvement seen in the remaining proteinaceous material when the material that precipitates under the incubation conditions is removed. The method of its removal and the subsequent treatment of the proteinaceous material can further improve the solubility of the protein isolate and, in particular, the stability of the protein isolate in acidic aqueous solution over extended time periods, e.g. weeks, months or years.

[0024] The process of the present invention can improve solubility at low pH without the need for using harsh or extended hydrolysis conditions, which are commonly used in the production of prior art protein isolates. Harsh or extended conditions can result in a higher proportion of lower molecular weight protein fragments in the product, which may contribute to a bitter taste in the protein isolate which may not be desirable for food and beverage applications.

[0025] In view of the above, the protein isolate of the present invention is ideally suited to food and beverage applications, in particular the fortification of acidic carbonated beverages.

[0026] With the above in mind, in a first aspect, the present invention is directed to a protein isolate isolated from a grain material, preferably brewer’s spent grain, wherein the protein isolate has: a protein content of at least 60% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25; and, a solubility of at least 90%, preferably at least 95%, in water at a pH of below 7 and a protein concentration of 2% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25. The grain material of the present invention may be brewer’s spent grain, barley, barley malt, rice, corn and combinations thereof. Preferably, the grain material is brewer’s spent grain.

[0027] Brewer’s spent grain

[0028] “Brewer’s spent grain” (BSG) is a by-product of the brewing industry following the mashing step. At this point of the brewing process, the soluble fraction (known as ‘wort’) is taken forward for 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, corn, sorghum, cassava and / or millet, particularly rice, corn, sorghum and / or cassava, more particularly rice and / or corn. It is most preferred that the brewer’s spent grain is obtained after brewing with barley or a mixture of barley and rice or corn, preferably rice.

[0029] According to the above, the brewer’s spent grain may comprise 100% spent barley. Optionally, the brewer’s spent grain may comprise from 20% to 100% spent barley by weight of the brewer’s spent grain, preferably from 45% to 70% by weight, for example 45%, 50%, 55%, 60%, 65%, or 70% spent barley by weight of the brewer’s spent grain. Where the brewer’s spent grain is a blend of spent barley and spent rice or spent corn, the spent rice or the spent corn may be present in an amount of from 0% to 80% by weight of the brewer’s spent grain, preferably from 30% to 55%, for example 30%, 35%, 40%, 45%, 50% or 55% by weight of the brewer’s spent grain.

[0030] Protein content

[0031] “Protein content / concentration / amount” as used herein refers to the protein content as measured according to the Dumas method (conversion factor 6.25), in particular according to 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 obtain essentially the same result. The protein content of the protein isolate may be at least 60% of dry matter by weight, preferably from 65 to 95% of 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% of dry matter by weight. Solubility in water

[0032] The protein isolate is highly soluble in water, especially at low pH. The protein isolate may have a water solubility as shown in Figures 1 a to 1d. In the following, where a solubility in water is defined as being, for example, at least 95% at a pH of from 2 to 6, the solubility is at least 95% at all pHs from 2 to 6.

[0033] At lower protein concentrations, the water solubility is very high. For example, at a pH of from 2 to 6 and a protein concentration of 2% of dry matter by weight, as determined by the Kjeldahl method (conversion factor 6.25), the solubility in water may be at least 95%, preferably at least 98%, for example 95, 96, 97, 98, 99, or 100%. In particular, at a pH of from 2 to 5 and a protein concentration of 2% or 2.5% of dry matter by weight, as determined by the Kjeldahl method (conversion factor 6.25), the solubility in water may be at least 95%, preferably at least 97%, more preferably at least 98%. Additionally, at a pH of from 2 to 4 and a protein concentration of 2% or 2.5% of dry matter by weight, as determined by the Kjeldahl method (conversion factor 6.25), the solubility in water may be at least 95%, preferably at least 97%.

[0034] At higher protein concentrations, the water solubility is very high. For example, at a protein concentration of or up to 15% by weight, the solubility of the protein isolate in water may be at least 65% (for example 65, 70, 75, 80, 85, 90, or 95%) at a pH of from 2 to 6. At a pH of about 3 to about 5, the water solubility may be at least 70% at 15% protein concentration, and at pH 4 to 5 the water solubility may be at least 90%.

[0035] At a protein concentration of or up to 10% of dry matter by weight, its solubility in water may be at least 80% (for example 80, 85, 90, or 95%) at a pH of from 2 to 6. From a pH of about 3 to about 6, the water solubility at a protein concentration of up to 10% of dry matter by weight may be at least 90%, preferably from 90% to 95%.

[0036] Even at 20% protein concentration by weight, the water solubility is very high. For example, from pH 2 to 6, the water solubility may be at least 50%, for example, 50, 55, 60, 65, 70, 75, 80, or 85%. At this concentration, the water solubility may be at least 75% from pH 4 to 8 and about 60% at pH 3. Between a pH of from 4 to 6, the water solubility may be at least 75%, for example 75, 80, or 85%. The solubility in water of the protein isolate may be at least 5% higher (especially at a pH of from 2 to 6 and a temperature of 20°C and a protein concentration of 2% of dry matter by weight, as determined by the Kjeldahl method conversion factor 6.25) than a protein isolate produced without the step of incubating the liquid protein stream at a pH of from 2 to 5 and preferably at a temperature of from 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 from 2 to 5 may be at least 10% higher than a protein isolate produced without an acid solubilisation process, preferably from 10% to 15% or 10 to 20%, for example 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%, at a pH of from 2 to 6 and a temperature of 20°C and a protein concentration of 2% of dry matter by weight, as determined by the Kjeldahl method (conversion factor 6.25).

[0037] The high solubility of the protein isolate in water at a variety of protein concentrations means the protein isolate is a versatile product for fortifying foods and beverages. In particular the protein isolate is very well suited to incorporation into beverages, particularly acidic carbonated beverages.

[0038] It is common for soluble protein isolates to precipitate during storage. However, the protein isolate of the present invention maintains its high solubility in water 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 before being consumed. The protein isolate may be and remain soluble in water following storage in water at 4°C for at least 1 week, preferably from 1 to 4 weeks, for example 1 , 2, 3, or 4 weeks, more preferably at least one month, more preferably at least 6 months, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months, more preferably at least a year, at a concentration of 2.5% protein by weight. There may be no or substantially no precipitation or sedimentation of the protein isolate in solution over these time periods. The water solubility may be at least 95% after storage under the above conditions for these time periods.

[0039] Haze analysis

[0040] The protein isolate may have a haze value of less than 65 Formazin Nephelometric Units (FNU), as measured by the ISO 7027 turbidity method, preferably less than 40 FNU, more preferably from 5 FNU to 35 FNU 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 haze value may be 5, 10, 15, 20, 25, 30, or 35 FNU, as measured by the ISO 7027 turbidity 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. In a preferred embodiment, the protein isolate has a haze value of from 2 FNU to 20 FNU, preferably from 4 FNU to 17 FNU, more preferably from 5 FNU to 15 FNU, for example, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 FNU as measured by the ISO 7027 turbidity 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.

[0041] Colour

[0042] The colour of protein isolates that can be used to fortify / supplement foods and beverages is important because it is preferable for the protein isolate to have minimal or no impact on the visual appearance of the food or beverage. The colour of a material can be described by the Cl ELAB colour space (or L*a*b* colour space), defined by the International Commission on Illumination (CIE). It expresses colour as three values: L* for perceptual lightness and a* and b* for the four unique colours of human vision: red, green, blue and yellow. An L* value 0 yields black and an L* value of 100 indicates diffuse white. For the present invention, the L* score provides the best indicator of the colour of the product and its suitability for mixing with other ingredients in foods and beverages and so this value will be referred to herein.

[0043] The BSG starting material may have an L* score of from 10 to 50 in solution at 10% dry matter, as measured by the CIELAB method. Optionally, the BSG may have an L* score of from 30 to 50, for example 30, 35, 40, 45, or 50 in solution at 10% dry matter. Accordingly, the brewer’s spent grain starting material is typically a dark red / brown colour in solution. However, the protein isolate of the present invention may have an L* score of at least 50, which produces a yellow / light brown solution. Accordingly, the protein isolate is well-suited to blending with foods and beverages without significantly and / or negatively impact their appearance. The L* score may preferably be from 75 to 100, more preferably from 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 is a light off-white / yellow colour in solution, which allows it to be readily blended with other ingredients to produce food and beverage products without affecting their appearance. It may be preferable to achieve these higher L* score values by incorporating the optional bleaching process into the process of the present invention. The protein isolate may have an L* score of at least 70, preferably from 85 to 98, as measured by the Cl ELAB 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. 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 Cl ELAB 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.

[0044] Molecular weight

[0045] The molecular weight profile of the protein isolate may be that described in Figure 2a or 2b. The protein isolate may have an average molecular weight, preferably a modal average or a number average molecular weight, in the range of from 5,000 to 30,000 Da, preferably from 5,000 to 10,000 Da.

[0046] The molecular weight profile of the protein isolate is advantageous in providing the isolate with a desirable taste profile. In this regard, the protein isolate may have a relatively high proportion of protein fragments having a molecular weight of at least 1 ,000 Da. The percentage of protein in the protein isolate having a molecular weight of at least 1 ,000 Da is at least 75% by weight of the protein isolate, preferably from 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.

[0047] The protein isolate has a particularly high percentage of protein in the molecular weight range of from 3,000 to 30,000 Da range. This percentage may be at least 45% by weight of the protein isolate, preferably from 50 to 75% by weight of the protein isolate. The percentage of the protein in the protein isolate having a molecular weight in the range of from 3,000 to 10,000 Da may be at least 30%, preferably from 35 to 50% by weight of the protein isolate. The percentage of protein having a molecular weight in the range of from 3,000 to 10,000 Da may be at least 2% by weight higher, preferably from 3 to 8% by weight than a protein isolate produced without an acid solubilisation step, e.g. Sample B.

[0048] The protein isolate also has a high percentage of protein having a molecular weight in the range of at least 30,000 Da. This percentage may be at least 3%, preferably from 5 to 15%, more preferably from 5 to 10%. This percentage may be at least 5% lower, preferably from 7.5 to 12.5% lower, than for a protein isolate produced without an acid solubilisation step, e.g. Sample B. The amount of high molecular weight protein in the isolate of the present invention is high compared to protein isolates of the prior art. It is common to improve solubility in protein isolates by simply digesting the protein into smaller, more soluble fragments. However, this can have a negative impact upon the flavour profile. The process of the present invention provides a protein isolate having high solubility at low pH while maintaining a relatively high proportion of high molecular weight protein.

[0049] 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 having a molecular weight of 500 to 1000 Da may be less than 15% by weight of the protein isolate, preferably from 2 to 12% by weight, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12% by weight. 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 from 1 to 5% by weight, for example 1 , 2, 3, 4, 5% by weight. The low proportion of protein fragments having a molecular weight of 1000 Da or less, in particular 500 Da or less, has an advantageous impact on the taste profile of the protein isolate and may reduce bitterness of bitter flavours compared to protein isolates having a higher proportion of protein fragments in these molecular weight ranges.

[0050] Process

[0051] The advantageous properties of the protein isolate of the present invention may arise from the process by which it is made. Accordingly, in a further 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; 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 from 2 to 5, preferably at a temperature of from 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.

[0052] Enzymatic Protein hydrolysis

[0053] The aqueous slurry is formed by mixing the grain material and water. The ratio of water to grain material (dry matter weight) in the aqueous slurry is preferably from 8:1 to 12:1 , preferably from 10:1 to 11 :1. The aqueous slurry is preferably formed in a jacketed, mixed tank, preferably with heating means.

[0054] The aqueous slurry is subjected to enzymatic protein hydrolysis to produce a liquid protein stream. If desired, the grain material may be subjected to particle size reduction before and / or during this step. Any suitable size reduction technique may be used, for example milling.

[0055] 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 may be obtained from EDC (Enzyme Development Corporation, New York) or Novozymes, for example.

[0056] The enzymatic starch hydrolysis is preferably carried out at the natural pH of the aqueous slurry. The pH may be, for example, from about 4.5 to about 6.5 (for example 4.5, 5, 5.5, 6 or 6.5, or any intermediate value).

[0057] The enzymatic starch hydrolysis is preferably carried out at a temperature of from about 50 °C to about 65 °C (for example 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63 or 65 °C, or any intermediate temperature).

[0058] 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. For example, the enzymatic starch hydrolysis may be carried out 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 hydrolysed to sugars (i.e. to glucose and / or to other water-soluble saccharides, including di-saccharides and other short-chain oligosaccharides).

[0059] The enzymatic protein hydrolysis preferably comprises treatment with a protease enzyme. The protease enzyme is preferably a food grade protease enzyme, preferably a serine protease. It is preferably an alkaline protease, preferably an endopeptidase, preferably a serine endopeptidase. Suitable protease enzymes may be obtained from Novozymes or EDC (Enzyme Development Corporation, New York), for example.

[0060] The enzymatic protein hydrolysis is preferably carried out at a pH of from about 7 to about 10 (for example 7, 7.5, 8, 8.5, 9, 9.5, or any intermediate value), preferably at a pH of about 9. The target pH can be achieved by the addition of an alkali such as sodium and / or potassium hydroxide prior to the treatment with the enzyme.

[0061] The enzymatic protein hydrolysis is preferably carried out at a temperature of from about 50 °C to about 75 °C (for example 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 intermediate temperature), preferably about 55 °C to about 68 °C, preferably about 55 °C to about 65 °C.

[0062] The enzymatic protein hydrolysis is preferably carried out for a period of at least about 15 minutes, preferably at least about 20 minutes, and up to about 80 minutes, preferably about 60 minutes. For example, the enzymatic protein hydrolysis may be carried out for a period of 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 minutes, or any intermediate period.

[0063] The enzymatic protein hydrolysis is preferably carried out until a degree of hydrolysis (dH) of between 1 and 10 (for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, or any intermediate value) has been reached, preferably until a dH of between 2 and 8, more preferably from 3 to 5, has been reached. As used herein, dH may be determined using the pH-stat method, by adding alkali (e.g. NaOH) and applying the following formula:

[0064] (B x NB~) dH = — — - — x 100wt%

[0065] (a x htotx Mp) where B is the volume of alkali (mL) consumed, NB is the normality of the alkali, or is the average degree of dissociation of amino acids (0.93 is typically used herein), htot is the total peptide bond content (or amino acid content) in 1 g of protein (meq / g; 9 meq / g is typically used herein) and MP is the mass of the protein present (g).

[0066] The enzymatic starch hydrolysis (if carried out) and the enzymatic protein hydrolysis preferably take place in the jacketed, mixed tank in which the aqueous slurry is formed.

[0067] Subsequent to enzymatic protein hydrolysis, the enzyme(s) is / are preferably deactivated 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 period of time.

[0068] Subsequent to enzymatic protein hydrolysis, solids are removed from the liquid protein stream. The removal of solids preferably takes place by decantation, preferably using decantation centrifuges. Pressure may be applied to the solids in order to maximise the recovery of liquid protein stream, for example using a screw press.

[0069] 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 maximise recovery of proteins.

[0070] First filtration process

[0071] The liquid protein stream is then be subjected to microfiltration to obtain a microfiltration permeate comprising protein, which may be referred to as the liquid protein stream, and a microfiltration retentate. The microfiltration is preferably carried out using a ceramic microfiltration membrane.

[0072] The microfiltration is preferably carried out using a microfiltration membrane having a pore size of from 0.03 to 0.5 pm (for example 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 pm, or any intermediate value), preferably from 0.03 to 0.25 pm, preferably from 0.05 to 0.2 pm, preferably from 0.07 to 0.13 pm (for example 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12 or 0.13 pm, or any intermediate value). Suitable microfiltration membranes may be obtained from Pall Corporation. The microfiltration preferably comprises a diafiltration step.

[0073] The microfiltration retentate may 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 may advantageously improve recovery of proteins.

[0074] The microfiltration permeate is subjected to nanofiltration at an applied pressure of from 1.0 bar (100 kPa) to 8.0 bar (800 kPa)) to obtain a nanofiltration permeate and a nanofiltration retentate comprising protein, which may be referred to as the liquid protein stream. Applied pressure is a well-known concept in the field of filtration and relates to the pressure at which the feed is fed to the filtration membrane. It is typically controlled by a feed pump and regulated by pressure sensors to ensure that a constant target feed pressure is maintained.

[0075] Nanofiltration is typically carried out 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 carrying out nanofiltration at a lower applied pressure of from 1 bar (100 kPa) to 10 bar (800 kPa), a protein isolate having a more favorable taste and solubility profile can be produced.

[0076] The nanofiltration may be carried out at an applied pressure of from 1.3 bar (130 kPa) to 3.3 bar (330 kPa), preferably from 1 .4 bar (140 kPa) to 3.2 bar (320 kPa), preferably from 1 .5 bar (150 kPa) to 3 bar (300 kPa). For example, nanofiltration may 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.

[0077] The nanofiltration is preferably carried out using a nanofiltration membrane having a molecular weight cut-off (MWCO) of from 500 to 2,000 Da, preferably from 800 to 2,000 Da, preferably from 800 to 1 ,200 Da. For example, nanofiltration may be carried out using a nanofiltration membrane having a molecular weight cut-off (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 may be obtained from MICRODYN-NADIR.

[0078] The nanofiltration retentate preferably has a total solids content of from 15 to 25% by weight, preferably from 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.

[0079] Optional bleaching process

[0080] Optionally, the process of the present invention may comprise a step of bleaching the liquid protein stream with a bleaching agent. Bleaching the liquid protein stream may increase the lightness of the protein isolate, which may reduce the impact the protein isolate has on the visual properties of a food or beverage into which it is incorporated.

[0081] The bleaching process may occur between steps (c) and (d), i.e. the bleaching may take place between the first filtration process (step (c)) and the step of incubating the liquid protein stream (step (d)).

[0082] The bleaching process may comprise 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, wherein the nanofiltration retentate comprises the liquid protein stream.

[0083] The step of bleaching 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 step of bleaching 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 reduce the timeframe over which bleaching takes place. The pH during the step of treating the liquid protein stream with a bleaching agent can be important because a low pH may cause precipitation of the proteinaceous material. The pH during the bleaching may be from 5 to 10, preferably 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 at a concentration of from 20 to 45% (w / w) in aqueous solution, for example 35% (w / w) in aqueous solution.

[0084] The amount of bleaching agent used in the bleaching step may be at least 0.25 moles of bleaching agent per kilogram of protein (mol / kg protein) to be bleached 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 1 to 75 mol / kg, more preferably from 2 to 50 mol / kg of protein, more preferably 2.5 to 5 mol / kg of protein, for example 2.5, 2.6, 2.7,

[0085] 2.8, 2.9, 3, 3.1 , 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,

[0086] 4.9, or 5 mol / kg of protein, or any intermediate value.

[0087] 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 of protein, preferably from 17.5 to 1750 g / kg of protein, preferably from 35 to 875 g / kg, preferably from 52.5 g / kg protein to 210 g / kg protein, more preferably from 85 to 175 g / kg of protein, more preferably to 110 to 150 g / kg protein for example 110, 115, 120, 125, 130, 135, 140, 145, or 150 g / kg, of protein, or any intermediate value.

[0088] As mentioned above, the bleaching agent may be added as a dilute solution, which may be a solution in water at from 20 to 45% w / w, 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 bleaching agent per kilogram of protein (g / kg protein) as determined by AOAC 990.03 or AOAC 992.15, preferably from 17.5 to 1750 g / kg of protein, preferably from 50 to 750 g / kg of protein, more preferably from 250 g / kg to 500 g / kg of protein, more preferably 325 to 425 g / kg of 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 or protein.

[0089] Incubating the liquid protein stream The step of incubating the liquid protein stream is particularly important for providing the advantageous features of the protein isolate. The aim of this step is to cause the protein fragments that have the greatest negative impact on water solubility below pH 7 to precipitate, so that they can be removed by subsequent filtration steps. In other words, this step may be an acid solubilisation or stabilisation step. However, this aim must be balanced with the need to maintain a high protein content and to maintain a desirable flavour profile.

[0090] Protein solubility is dependent on a number of factors, such as temperature, pH, concentration and the nature of the protein fragments. The present inventors have observed that there is a complex interrelationship between these factors, in particular pH and temperature.

[0091] With the above in mind, the step of incubating may be carried out at a pH of from 2 to 5, preferably from 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 unstable proteinaceous material may have no net electrical charge. The inventors have observed that incubation at these pHs has a beneficial impact on the water solubility of the isolate.

[0092] The step of incubating is preferably carried out at from 5 to 70°C. It may be carried out at a temperature of from 10 to 30°C, more preferably from 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 may occur which may reduce the protein content of the isolate, while at higher temperatures, certain proteins may become more soluble while others coagulate and precipitate, which affects the profile of the isolate.

[0093] Incubation within the pH and the preferable temperature ranges of the present process provides a solution having colour and haze values that are stable for long time periods (see Figures 3a-3f), indicating that they are favourable for obtaining an isolate having high solubility for extended time periods. Accordingly, in preferred embodiments, the step of incubating may be carried out at a pH of from 3 to 4 and a temperature of from 15 to 25°C. The step of incubating 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 step of incubating 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.

[0094] According to the above, in preferred embodiments, the step of incubating may be carried out at a pH from 3 to 4, a temperature from 15 to 25°C for from 45 to 90 minutes. In this embodiment, the protein concentration may be from 5 to 15% solids by weight, as determined by the Kjeldahl method using a conversion factor of 6.25. No enzymatic treatment is performed during the incubation step.

[0095] Subsequent to the step of incubating the liquid protein, solids are removed from the liquid protein stream. The removal of solids may take place by decantation or centrifugation.

[0096] The liquid protein stream may be pasteurised after incubating the liquid protein stream, for example by treating it at from 65 to 95°C for from 30 to 180 seconds, preferably at 77°C for 90 seconds.

[0097] Optional second filtration process

[0098] Following removal of the solids from the liquid protein stream, it may be subjected to a filtration process comprising one or more filtration steps, which may be referred to as the second filtration process. Accordingly, in certain embodiments, the second filtration process is not performed and, in certain embodiments, the second filtration process is performed. Preferably, the second filtration process is performed.

[0099] The second filtration process may comprise microfiltration. In a first variation of the second filtration process, it may comprise subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 4 to 70°C. Preferably the temperature may be from 5 to 25°C, preferably from 4 to 20°C for example, 4, 5, 10, 15, 20, or 25°C. Suitable ceramic membranes are those marketed by TAMI Industries or Pall® Corporation. The pore size of the ceramic membrane may be up to 2 pm, preferably from 0.1 to 1.5 pm, for example 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, or 1 .5 pm. In specific embodiments, the pore size may be 0.14, 0.2, 0.45, 0.8, or 1.2 pm. In a preferred embodiment, the pore size of the ceramic microfiltration membrane is from 0.1 pm to 0.2 pm, for example 0.1 , 0.14, or 0.2 pm. The microfiltration may be carried out at a pressure of from 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 may be 1 bar.

[0100] In a preferred embodiment of the first variation, the second filtration process comprises subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 5 to 25°C, or from 4 to 20°C, wherein the ceramic membrane has a pore size of from 0.1 to 0.3 pm, optionally from 0.1 pm to 0.2 pm. This may be combined with a step of incubating carried out at a pH of from 3 to 4, a temperature of from 15 to 25°C.

[0101] According to 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 from 3 to 4 at a temperature of from 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, or from 4 to 20°C, wherein the ceramic membrane has a pore size of from 0.1 to 0.3 pm, optionally from 0.1 pm to 0.2 pm; and, g) processing the liquid protein stream to produce the protein isolate. In a second variation of the second filtration process, the second filtration process comprises subjecting the liquid protein stream to filtration at below 4°C. The filtration may be carried out using cellulose filter sheets, such as those marketed by Pall Corporation. The temperature of the filtration should be low, preferably from -2 to 4°C, more preferably from -0.5 to 1.5°C, for example, -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.

[0102] The filtration may be carried out with one or more stabilisation agents. These may be selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrolidone, silica gel, colloidal silica, and tannic acid. For example, diatomaceous earth and tannic acid may be used. These stabilisation agents may be added to the solution to be filtered prior to filtration. Alternatively they may be impregnated into the filter, for example Pall Seitz® filter sheets.

[0103] Diatomaceous earth (which is also known as Kieselguhr or celite) is typically composed of silica, with minor components of alumina and iron oxide. If the diatomaceous earth is mixed with the liquid protein stream, it is mixed to a concentration of from 1 g / l to 10 g / l, more preferably from 3 to 9 g / L, for example 3, 4, 5, 6, 7, 8, or 9 g / L. Tannic acid may be included either in addition to diatomaceous earth or alone, preferably at a concentration of from 1 to 20 g / hL, more preferably from 10 to 15 g / hL, for example 10, 11 , 12, 13, 14, or 15 g / hL.

[0104] 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 from -2 to 2°C with diatomaceous earth as a stabilisation agent. This may be combined with a step of incubating carried out at a pH of from 3 to 4, a temperature of from 15 to 25°C.

[0105] According to 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 from 3 to 4 at a temperature of from 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 from -2 to 2°C with diatomaceous earth as a stabilisation agent; and, g) processing the liquid protein stream to produce the protein isolate.

[0106] The second filtration process may comprise or further comprise a step of filtration with a stabilisation agent such as activated carbon. In other words, the second filtration process may comprise a step of filtration with activated carbon. In other embodiments, the stabilisation agent may be one or more selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrolidone, silica gel, colloidal silica, and tannic acid.

[0107] Additionally, a step of filtration with one or more stabilisation agents selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrolidone, silica gel, colloidal silica, and tannic acid may be performed in addition to or as part of either the first or second variation of the second filtration process described above. Preferably the stabilisation agent is activated carbon. In other words, the first or second variation of the second filtration process may comprise a step of filtration with activated carbon. The activated carbon may be added to the liquid protein stream prior to filtration, or it may be impregnated in the filter. Suitable impregnated filters include the Pall Seitz® AKS4 Activated Carbon Sheets.

[0108] According to the above, in a preferred embodiment of the first variation, the second filtration process comprises: subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 5 to 25°C, or from 4 to 20°C, wherein the ceramic membrane has a pore size of from 0.1 to 0.3 pm, for example from 0.1 pm to 0.2 pm; and, subjecting the liquid protein stream from the microfiltration to filtration with activated carbon. This preferred embodiment may be combined with a step of incubating carried out at a pH of from 3 to 4, a temperature of from 15 to 25°C.

[0109] Together, the steps of incubating and the second filtration process may be described as an acid solubilisation / stabilisation process or a process for increasing the solubility of the protein isolate in water at acidic pH.

[0110] Processing to produce the protein isolate

[0111] Processing the liquid protein stream to produce the protein isolate may comprise a step of nanofiltration of the liquid protein stream with diafiltration to provide a nanofiltration retentate comprising protein. Processing the liquid protein stream to produce the protein isolate preferably comprises evaporation to increase the total solids content to a total solids content of from 10 to 55% (for example to 10, 15, 20, 25, 30, 35, 40, 45 or 50%, or any intermediate value), preferably from 25 to 55%, and then spray drying to produce the protein isolate, which may be in the form of a powder.

[0112] 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 intermediate value), and a protein content (% dry matter by weight) of at least 60%, preferably from 65% to 95%, more preferably from 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.

[0113] In a further embodiment, the present invention is directed to a protein isolate obtained or obtainable by the process described hereinabove. The protein isolate produced by this process is the protein isolate discussed hereinabove and so the features and embodiments disclosed in respect of that isolate apply equally to the protein isolate obtained or obtainable by the above process.

[0114] Food and beverage products

[0115] The present invention is additionally directed to food or beverage products comprising the protein isolate defined hereinabove. The beneficial properties of the protein isolate of the present invention mean that it is ideally suited for incorporation into foods and beverage products to increase their protein content and it may not significantly and / or negatively impacting the taste, appearance, physical or chemical properties of the product.

[0116] The protein isolate is particularly suited to incorporation into beverages because of its high water solubility. Due to the improved solubility at low pH, the protein isolate of the present invention is suitable for incorporation into acidic beverages having a pH of from 2 to 6, preferably from 3 to 5.

[0117] A further benefit of the beneficial properties of the protein isolate is that it can be incorporated into food or beverage products in high amounts, such as up to 50% of dry matter by weight of the food or beverage product. Preferably the food or beverage may comprise the protein isolate at from 0.1 % to 30%, more preferably from 0.5 to 20%, even more preferably from 1 to 10%, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10% of dry matter by weight of the food or beverage product.

[0118] In preferred embodiments, the beverage, preferably a carbonated beverage, comprises the protein isolate in an amount of from 0.5 to 20% of dry matter by weight of the beverage. The beverage may be a “soft” drink, e.g. an energy drink, a sport hydration drink, a flavoured water beverage, a ready-to-drink (RTD) beverage such as RTD coffee, cola or fruit-flavoured carbonated beverage, or a ready-to-mix (RTM) beverage. The beverage may be an alcoholic beverage, which may be a fermented alcoholic beverage, such as a beer or cider, or a distilled alcoholic beverage. The beverage may additionally be non-alcoholic or low alcohol beverage, such as a no- 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, for example 0%ABV. The carbonated beverage may be provided in a bottle, can, keg or any other suitable container.

[0119] The food or beverage may further comprise additives commonly found 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, and fumaric acid; colours; flavourants, such as raspberry, strawberry, mixed berry, 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 described by the following non-limiting examples.

[0120] EXAMPLES

[0121] Sample A is a protein isolate prepared from BSG by the general method described in WO 2021 / 028509 A1. Sample B is a protein isolate prepared by the general method described in WO 2021 / 028509 A1 with the additional step that the permeate from the microfiltration step is bleached using hydrogen peroxide (35% w / w) in an amount of 4 mol / kg protein at 90°C for 100 minutes, nanofiltered and the nanofiltration retentate spray dried. Sample C was prepared according to Example 3 below.

[0122] Optimisation of acid solubilisation conditions

[0123] An investigation into the stability of the isolates having lower solubility at acidic pH at various temperatures and pHs was carried out in order to determine the conditions under which the step of incubating should be performed. Sample A was combined with water to a concentration of 3.5% by weight and incubated with stirring under the conditions shown in Table 1 .

[0124] Table 1

[0125] Samples were removed at 0 mins, 30 mins, 3 h, 6 h, 8 h, 14 h and 24 h and centrifuged (15 minutes at 4,000 rpm). The supernatant was analysed for lightness (L*) using the Cl ELAB method (CR-5 Konica Minolta) and haze (ISO 7027 turbidity method). The results are shown in Figures 3a-3f.

[0126] Incubation at 75°C resulted in a darker supernatant and higher haze than at 4°C or 20°C, which may be the result of increased solubility of unstable proteins at higher temperature, which then precipitated once the sample returned to ambient temperature. Incubation at lower temperature resulted in higher haze than at 20°C. It is theorised that the lower temperatures caused unstable proteins to precipitate. At 20°C, the haze was low and stable and similar haze and lightness results were achieved for each pH value at this temperature. This indicates that these conditions are favourable for obtaining an isolate having high solubility at low pH.

[0127] General method for producing acid-soluble protein powder from BSG

[0128] Protein isolates were prepared according to the following general method using brewer’s spent grain comprising spent barley and either spent corn or spent rice.

[0129] The incoming grains were received into a jacketed, mixed tank with water to make 10.5:1 water to dry weight ratio. The resulting slurry was heated to 55°C and treated with a glucoamylase enzyme (Novozymes® glucoamylase) for 45 minutes to hydrolyse the starch. The pH was then raised to 9 using alkali and maintained for 45 minutes.

[0130] The mixture was then treated with a food-grade protease enzyme (Novozymes® alkaline protease) for 20 to 60 minutes at 60°C to hydrolyse the protein component. Thereafter, the enzymes were deactivated by heating the mixture to 80°C and holding for up to 25 minutes.

[0131] The solids were separated from the liquid protein stream by decanting centrifuges. The liquid protein stream was fed into a microfiltration system (0.1 pm membranes; 70 to 80°C; suitable membranes available from Pall Corporation).

[0132] The permeate from the microfiltration was processed in a nanofiltration system (MW CO of c. 1000 Da; suitable membranes available from MICRODYN-NADIR).

[0133] Optionally, the output retentate was bleached using hydrogen peroxide (35% w / w) in an amount of 4 mol / kg protein at 90°C for 100 minutes. The bleached retentate was further processed in a nanofiltration system (MWCO of c. 1000 Da; suitable membranes available from MICRODYN-NADIR).

[0134] The output retentate from the nanofiltration system was adjusted to pH 3.5 and incubated at a protein content of 10% by weight for 1 hour at 15-20°C. Bulk solids were removed from the resulting suspension using a disk-stack centrifuge or decanter (suitable decanters include those marketed by GEA). Optionally, the filtrate from the bulk solid removal was pasteurised at 77°C for 90 seconds. Second filtration process

[0135] The present inventors have discovered that the long term solubility of protein isolate at acidic pH can be improved by performing a second / polishing filtration at low temperatures after the incubation process.

[0136] In Examples 1 -6, the filtrate from the bulk solid removal underwent a second filtration according to the conditions set out in the 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).

[0137] In Examples 7-17, the filtrate from the bulk solid removal underwent a second filtration according to the conditions set out in the 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 to 17, the filtrate from the bulk solid removal was pasteurised at 77°C for 90 seconds.

[0138] The output filtrate of secondary filtration was spray dried to a powdered product.

[0139] Stability Evaluation

[0140] Evaluation of the stability of the products of Table 3 was performed by mixing the powdered product with de-ionised water at a concentration of 2.5% protein concentration by weight at pH 3.5 and 4 and visually inspecting the mixture after the specified time.

[0141] Evaluation of the stability of the products of Tables 4 and 5 were performed by mixing the powdered product with de-ionised water at a concentration of 2.5% protein concentration by weight at pH 3.5 and 4 and visually inspecting the mixture after 3-4 weeks of storage. The symbols used to describe the stability of the product are defined in Table 2 below. Table 2

[0142] Table 3

[0143] Table 4

[0144] Table 5

[0145] Compositional analysis

[0146] A sample prepared according to the general method above including the bleaching process (Example 3, Sample C) and a commercial pea protein product adapted for low pH applications analysed and the results are shown Table 6 below.

[0147] Table 6

[0148] Solubility analysis

[0149] The solubility of Samples A, B and C were measured as a function of pH and protein concentration according to the following method. A suspension of the sample was prepared at the desired protein concentration at 20°C and the pH adjusted to the desired value using HCI (1 M) or NaOH (1 M). The samples were centrifuged at 4,000 rpm for 10 minutes (Eurolabs centrifuge). A control sample was prepared at the desired protein concentrations with no pH adjustment (Native). The protein content of the Native sample and the supernatants of the test samples were measured using the Kjeldahl method, conversion factor 6.25. Solubility was calculated according to the below equation:

[0150] Protein content sample

[0151] Solubility (%) = x 100

[0152] Protein content Native

[0153] The solubility of Samples A-C is shown in Table 7 below and Figures 1a and the solubility of Sample C at 2%, 5%, 10%, 15% and 20% protein concentration is shown in Table 8 and Figure 1 b. Table 7

[0154] Table 8

[0155] Sample C shows high solubility (>95%) across the pH range at 2 and 5% protein content. The solubility remains high (90% or above) at 10% protein concentration by weight at pH 3-8. Lower solubility is observed at protein concentrations of 15% and 20% by weight.

[0156] In Table 9 below and Figure 1 d the solubility of the product of Example 13 is shown at 2%, 5%, and 10% protein concentration as measured using the Kjeldahl method, with a conversion factor of 6.25.

[0157] Table 9

[0158] The product of Example 13 shows high solubility across pH 2 to pH 6, even at protein concentrations of 5% and 10%.

[0159] Haze analysis and CIELAB (or L*a*b*) colour measurement

[0160] Cl ELAB measurements were taken using a Konica Minolta Bench-top Spectrophotometer CM-5. Liquid samples were prepared at the protein concentration by weight and pH recited 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 10 mm - 0.1 mm thickness front / 0.23 mm thickness side) and measured in transmittance mode. The protein concentration described herein is 5% by weight unless otherwise stated. Solid samples were measured in reflectance mode. Haze analysis was performed using a Hach® 2100Q IS Portable Turbidimeter according to the ISO 7027 turbidity method at the protein concentration by weight and pH recited in Table 9 and 10 below.

[0161] The samples of Examples 2, 3, 5 and 14 contained no visible sediment, whereas the samples of Sample B contained sediment which began to settle shortly after preparation. The samples of Sample B were therefore centrifuged and the haze and Cl ELAB values shown in Tables 9 and 10 are those of the resulting supernatant.

[0162] Table 9

[0163] * Measurements performed on supernatant after centrifugation

[0164] Table 10

[0165] Measurements performed on supernatant after centrifugation

[0166] Molecular weight characterisation

[0167] Molecular weight analysis was performed using a Thermofisher Vanquish high performance liquid chromatography (HPLC) according to the following method. A sample (200 pL) was injected onto a series of two columns (Superdex® 75 GL (molar mass range 70,000-3,000 Da) and Superdex® 30 GL (molar mass 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 method was run at room temperature for between 90 and 160 mins. Ultraviolet detection was performed at 218 nm.

Claims

CLAIMS:

1. A protein isolate isolated from a grain material, preferably brewer’s spent grain, wherein the protein isolate has: a protein content of at least 60% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25; and, a solubility of at least 90%, preferably at least 95%, in water at a pH of below 7 and a protein concentration of 2% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25.

2. The protein isolate according to claim 1 , wherein the protein isolate has a haze value, as measured by the ISO 7027 turbidity method, of less than 65 FNU, preferably less than 40 FNU, more preferably from 5 FNU to 35 FNU, 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; or, wherein the protein isolate has a haze value of from 2 FNU to 20 FNU, preferably from 4 FNU to 17 FNU, more preferably from 5 FNU to 15 FNU, 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.

3. The protein isolate according to claim 1 or 2, wherein the protein isolate has an L* score of at least 60, preferably from 75 to 100, more preferably from 85 to 98, as measured by the Cl ELAB method.

4. The protein isolate according to any one of the preceding claims, wherein the protein content is from 65 to 95% of dry matter by weight.

5. The protein isolate according to any one of the preceding claims, wherein the solubility of the protein isolate in water is at least 97%, preferably at least 98%, at a pH of from 2 to 5 and a protein concentration of 2% or 2.5% of dry matter by weight, as determined by the Kjeldahl method (conversion factor 6.25).

6. The protein isolate according to any one of the preceding claims, wherein the solubility of the protein isolate in water is at least 70% at a pH of from 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.

7. The protein isolate according to any one of the preceding claims, wherein the solubility in water is at least 80%, preferably 85%, at a pH of from 2 to 6 and a protein concentration of up to 10% of dry matter by weight, as determined by the Kjeldahl method using a conversion factor of 6.25.

8. The protein isolate according to any one of the preceding claims, wherein the solubility in water of the protein isolate may be at least 5% higher, preferably from 10 to 20%, than a protein isolate produced without a process for increasing the solubility of the protein isolate in water at a pH below 7.

9. The protein isolate according to any one of the preceding claims, wherein the protein isolate has an L* score of at least 70, preferably from 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.

10. The protein isolate according to any one of the preceding claims, wherein the percentage of protein in the protein isolate having a molecular weight of from 3,000 to 30,000 Da, is at least 45%, preferably from 50 to 75%, by weight of the protein isolate.

11. The protein isolate according to any one of the preceding claims, 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 from 2 to 12% by weight.

12. The protein isolate according to any one of the preceding claims, 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 from 1 to 5% by weight.

13. The protein isolate according to any one of the preceding claims, wherein the protein isolate remains soluble following 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.

14. A process for producing a protein isolate from a grain material, preferably brewer’s spent grain, 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 from 2 to 5, preferably at a temperature of from 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.

15. The process according to claim 14, wherein incubating the liquid protein stream is carried out at a pH of from 2 to 5, preferably from 3 to 4, more preferably about 3.5.

16. The process according to claim 14 or 15, wherein incubating the liquid protein stream is carried out at a temperature of from 10 to 30°C, more preferably from 15 to 25°C.

17. The process according to any one of claims 14 to 16, wherein the concentration of the proteinaceous material during the step of incubating is up to 30% solids by weight, preferably from 1 to 20% solids by weight, more preferably from 5 to 15% solids by weight.

18. The process according to any one of claims 14 to 17, wherein the step of incubating is carried out at a pH of from 3 to 4 and a temperature of from 15 to 25°C for from 45 to 90 minutes.

19. The process according to any one of claims 14 to 18, wherein removing solids from the liquid protein stream comprises centrifugation or decantation.

20. The process according to any one of claims 14 to 19, wherein the second filtration process is performed and comprises subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 4 to 70°C, preferably from 5 to 25°C.

21. The process according to claim 20, wherein the ceramic membrane has a pore size of from 0.1 pm to 1.4 pm, preferably from 0.14 to 0.45 pm, more preferably from 0.1 to 0.3 pm, optionally from 0.1 to 0.2 pm.

22. The process according to any one of claims 14 to 21 , wherein the second filtration process is performed and comprises subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 5 to 25°C, wherein the ceramic membrane has a pore size of from 0.1 to 0.3 pm.

23. The process according to any one of claims 20 to 22, wherein the microfiltration is carried out at a pressure of from 0.5 to 2 bar, more preferably about 1 bar.

24. The process according to any one of claims 14 to 23, wherein: the step of incubating is carried out at a pH of from 3 to 4 and a temperature of from 15 to 25°C; the second filtration process is performed and comprises subjecting the liquid protein stream to microfiltration carried out using a ceramic membrane at a temperature of from 5 to 25°C, or from 4 to 20°C, wherein the ceramic membrane has a pore size of from 0.1 to 0.3 pm; and, wherein the second filtration optionally further comprises a step of filtration with activated carbon.

25. The process according to any one of claims 14 to 19, wherein the second filtration process is performed and comprises subjecting the liquid protein stream to filtration, preferably using a cellulose filter, at below 4°C, preferably from -2 to 2°C.

26. The process according to claim 25, wherein the filtration is carried out with one or more stabilisation agents, preferably selected from the group consisting of diatomaceous earth, perlite, activated carbon, polyvinylpolypyrolidone, silica gel, colloidal silica, and tannic acid.

27. The process according to claim 26, wherein the filtration comprises mixing the liquid protein stream with diatomaceous earth before filtration, preferably at a concentration of from 1 g / l to 10 g / l, more preferably from 3 to 9 g / L.

28. The process according to claim 26 or 27, wherein the filtration further comprises mixing the liquid protein stream with tannic acid before filtration, preferably at a concentration of from 1 to 20 g / hL, more preferably from 10 to 15 g / hL.

29. The process according to any one of claims 14 to 19 or 25 to 28, wherein: the step of incubating is carried out at a pH of from 3 to 4, a temperature of from 15 to 25°C for from 45 to 90 minutes; the second filtration process is performed and comprises subjecting the liquid protein stream to filtration, preferably using a cellulose filter, at from -2 to 2°C with diatomaceous earth; and, wherein the second filtration optionally further comprises a step of filtration with activated carbon.

30. The process according to any one of claims 14 to 29, wherein the second filtration is performed and comprises a step of filtration with a stabilisation agent, preferably activated carbon.

31. The process according to any preceding claim, wherein processing the liquid protein stream to produce the protein isolate comprises evaporation 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.

32. The process according to any preceding claim, 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.

33. A protein isolate obtained or obtainable by the process according to any one of claims 14 to 32.

34. A food or beverage comprising the protein isolate according to any one of claims 1 to 13 or according to claim 33.

35. The food or beverage according to claim 34 comprising the protein isolate in an amount of up to 50% of dry matter by weight of the food or beverage product, preferablyfrom 0.1% to 30%, more preferably from 0.5 to 20% of dry matter by weight of the food or beverage product.

36. The food or beverage according to claim 34 or 35, wherein the food or beverage is a beverage, preferably a carbonated beverage, comprising the protein isolate in an amount of from 0.5 to 20% of dry matter by weight of the beverage.

37. The beverage according to claim 36, wherein the beverage has a pH of from 2 to 6, preferably from 3 to 5.

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