A process for producing millet protein concentrate

Abstract

The present invention relates to a process for producing a millet protein concentrate from millet grains The process comprises preparing an aqueous slurry of milled millet, gelatinizing the slurry by heating, enzymatically liquefying the gelatinized slurry to hydrolyze starch, and separating a first solid biomass. The first solid biomass is then subjected to a critical alkaline washing step at a pH of about 10 and a temperature of about 45 °C, followed by separation, neutralization, and drying to obtain a protein concentrate. The alkaline wash selectively removes non-protein components while preserving protein integrity, resulting in enhanced protein purity and recovery. The process avoids the use of organic solvents and harsh chemicals and is scalable for industrial application. The millet protein concentrate obtained exhibits high protein content and retains significant dietary fiber, making it suitable for use in food products, nutritional supplements, and plant-based formulations.

Description

[0001] A PROCESS FOR PRODUCING MILLET PROTEIN CONCENTRATE TECHNICAL FIELD

[0002] The present disclosure relates to food and nutraceutical processing. More particularly, it relates to an enzyme-alkali hybrid process for producing a high-purity millet protein concentrate from millet grains.

[0003] BACKGROUND OF THE INVENTION

[0004] Millets are increasingly recognized as sustainable, gluten-free sources of plant protein. Proso millet and foxtail millet typically contain about 11-18% protein, but efficient recovery of this protein in concentrated form remains challenging. Conventional extraction techniques rely on organic solvents such as ethanol or isopropanol, strong acids or alkalis, or high salt concentrations. These approaches often result in low protein recovery, degradation of functional properties, increased processing cost, and environmental burden.

[0005] Park and Bean (2003) disclose production of sorghum protein concentrate (-60%) using sodium borate buffer with reducing agents such as sodium metabisulfite or glutathione, while Bean et al. (2006) report that 70% ethanol extraction yields very low protein recovery (<4%), although recovery can exceed 70% with sodium metabisulfite; however, these disclosures are silent on achieving high protein purity. Mohamed et al. (2009) describe alkaline extraction of foxtail millet proteins with high purity (-80%) but do not disclose yield or protein recovery, which are critical for commercial viability. US4254022A reports recovery of >80% protein from proso millet using aqueous isopropyl alcohol, sodium hydroxide, and dilute mineral acid, but alcohol removal increases process cost. CN114106083 A discloses millet protein concentrates with high purity (81.8-94.6%) using n-hexane defatting, though the use of harsh organic solvents may adversely affect protein quality and process sustainability.Enzyme-based starch hydrolysis has been explored as a milder alternative; however, previously reported processes either require high enzyme dosages, extreme temperatures, or solvent-based washing steps, and do not simultaneously achieve high protein purity, yield, and scalability.

[0006] Accordingly, there exists a need for a commercially viable, scalable, and environmentally benign process that selectively removes starch and non-protein components from millet flour while preserving protein quality and achieving high protein purity. The present disclosure addresses this need through a specifically optimized alkaline washing step following enzymatic liquefaction.

[0007] OBJECTS OF THE INVENTION

[0008] An object of the present application is to provide an efficient process for producing millet protein concentrate with high protein purity;

[0009] Another object of the present application is to enhance protein recovery from proso millet;

[0010] Another object of the present application is to preserve nutritional and functional properties of millet proteins; and

[0011] Yet, another object of the present application is to provide a scalable and industrially feasible process.

[0012] SUMMARY OF THE INVENTION

[0013] The present disclosure provides a process for producing millet protein concentrate comprising:

[0014] (a) preparing an aqueous slurry of milled millet;

[0015] (b) gelatinizing the slurry by heating;(c) enzymatically liquefying the gelatinized slurry using an amylolytic enzyme to hydrolyze starch to form a liquefied slurry;

[0016] (d) separating a first solid biomass and a first liquid fraction from the liquefied slurry;

[0017] (e) subjecting the first solid biomass to an alkaline wash at a predetermined pH and a predetermined temperature for a predetermined time, thereby selectively removing non-protein components and forming a second liquid fraction and a protein-rich solid fraction;

[0018] (f) separating a protein-rich solid fraction from a second liquid fraction;

[0019] (g) neutralizing and drying the protein-rich solid fraction to obtain a millet protein concentrate.

[0020] The inventiveness of the process resides primarily in step (e), wherein the alkaline wash selectively solubilizes residual carbohydrates and non-protein components without denaturing the protein, resulting in unexpectedly high protein purity and recovery.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 illustrates a flow diagram of the process for producing millet protein concentrate, according to the present disclosure.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] In the description that follows, several terms are used, and the following definitions are provided to facilitate understanding of various aspects of the disclosure. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including”, but not limited to, and the word “comprises” has a corresponding meaning. The present application now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the applications are indicated. Indeed, the application may be embodied in manydifferent forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present application is described fully herein with non-limiting embodiments and exemplary experimentation.

[0025] The invention will now be described in detail with reference to preferred and non-limiting embodiments. Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by a person skilled in the art. The present application describes an improved process for producing millet protein concentrate mainly involving enzymatic hydrolysis treatment and alkali treatment. The process conditions lead to an increase in the purity and yield of the protein concentrate while maintaining its nutritional properties and minimizing the need for harsh chemicals, thereby improving the overall quality of the final protein concentrate. Moreover, the process reduces the reliance on harsh chemicals, improves the protein yield and purity, and produces a protein concentrate that retains desirable nutritional properties.

[0026] In one aspect, the process of producing millet protein concentrate from millet grains comprises the steps (a) preparing an aqueous slurry of milled millet; (b) gelatinizing the aqueous slurry by heating to a temperature sufficient to gelatinize starch; (c) liquefying the gelatinized slurry by enzymatic treatment to hydrolyze starch to form a liquefied slurry; (d) separating a first solid biomass and first liquid fraction from the liquefied slurry;(e) subjecting the first solid biomass to an alkaline wash at a predetermined pH and a predetermined temperature for a predetermined time, thereby selectively removing non-protein components and forming a second liquid fraction and a protein-rich solid fraction; (f) separating the protein-rich solid fraction from the second liquid fraction; and (g) neutralizing and drying the protein-rich solid fraction to obtain a millet protein concentrate.The process includes extracting protein concentrates from millet sources, wherein the process concentrates the protein within the raw material and is isolated by removing non-protein components while abstaining from overuse of harsh chemicals, salts, organic solvents, and alcohols.

[0027] Referring to FIG. 1, the process for extracting protein concentrates from millet will be described in detail.

[0028] Raw Material Preparation (102)

[0029] The millet grains used in the process is selected from proso millet, pearl millet, foxtail millet, sorghum, or combinations thereof. In some embodiments, the millet grain is a mixture of proso-millet and foxtail millet. In another embodiment, the millet grain is proso-millet. In another embodiment, the millet grain is foxtail-millet. The Proso millet was sourced from local market of Theni Thaniyam, 1092, Periyakulam road, Near mini bus stand, Theni, Tamil Nadu, India

[0030] The millet grains are cleaned and optionally dehulled. In some embodiments, the millet grains are thoroughly cleaned and dehulled, if necessary, to remove impurities. Dehulling is performed to remove the outer husk, which is nonedible and can interfere with protein extraction. Dehulling can be achieved mechanically using abrasive or friction-based dehulling apparatus known in the art.

[0031] The grains are subjected to size reduction to obtain a millet flour, preferably having a particle size of less than about 250 pm. In some embodiments, the millet grains are subjected to a size reduction process using a milling process, wherein the millet grains are milled into millet flour.

[0032] Typically, the millet grain milling process is performed using conventional milling equipment until the particle size is reduced to at least 250 pm. In an embodiment, the milled millet powder is sieved using a 250 pm sieve to ensure that only fine particlesare used for the protein concentrate extraction. Typically, the particle size of the millet flour is selected in the range of 1-250 pm.

[0033] Slurry Preparation (104)

[0034] The millet flour is dispersed in water in a flour-to-water ratio ranging from about 1 : 5 to about 1:15, to form an aqueous slurry. In some embodiments, the aqueous slurry of the millet grain is prepared by mixing the millet flour with water in a ratio ranging from about 1 : 10 to 1:15. Typically, the ratio of millet flour to water is 1:15.

[0035] In some embodiments, the water is at least one selected from including but not limited to RO water, distilled water, double distilled water and deionized water.

[0036] The aqueous slurry is subjected to homogenization under controlled stirring which leads to enhance the extraction of soluble proteins.

[0037] In non-limiting examples, 30 kg of milled millet powder is mixed with RO water (water filtered using reverse osmosis) until a homogeneous slurry is formed. Preferably, the aqueous slurry is continuously stirred for 10-15 minutes to ensure uniformity.

[0038] In one embodiment, the pH of the aqueous slurry is adjusted in the range of 5-7 using an acid medium. The pH adjustment prepares the aqueous slurry for an enzymatic hydrolysis step and ensures optimal conditions for enzyme activity. In an exemplary embodiment, the pH of the aqueous slurry is adjusted to 6 by adding 5N hydrochloric acid (HC1) while stirring.

[0039] Gelatinization (106)

[0040] The slurry is heated to about 80-90°C to gelatinize the starch. In some embodiments, the temperature of the aqueous slurry is increased until the aqueous slurry is converted into a gelatinized slurry. Typically, the temperature of the aqueous slurry israised to 85 °C using a suitable heating medium known in the art. The gelatinized slurry is characterized by a thick and viscous texture.

[0041] Enzymatic Liquefaction (108)

[0042] The gelatinized slurry is liquified by enzymatic treatment to hydrolyze starch to form a first liquid fraction. In one embodiment liquified gelatinized slurry is the first liquid fraction.

[0043] In some embodiments, the gelatinized slurry of the millet grain is subjected to cooling until the temperature of the gelatinized slurry reaches 70 °C before the enzymatic treatment. The starch component in the gelatinized slurry gets hydrolyzed into smaller fragments including but not limited to dextrin.

[0044] In some embodiments, the enzymatic treatment includes enzymatic hydrolysis using alpha-amylase enzyme. Examples of suitable enzyme used in the industry and may be obtained from Novozymes, for example, BAN480L.

[0045] In some embodiments, the dosage of alpha-amylase enzyme is at least selected but not limited to 0.1% - 0.4% with respect to the total mass of millet grain feed. The dosage of alpha-amylase enzymes may be selected from about 0.1%, 0.2% 0.3% or 0.4%, or any intermediate value.

[0046] In some embodiments, the enzymatic starch hydrolysis is preferably carried out at the natural pH of the gelatinized slurry. The pH may be selected from about 5 to about 7 (for example 5, 5.5, 6, 6.5, 7 or 7.5, or any intermediate value). In an exemplified embodiment, the pH of the hydrolysis step is 6. In some embodiments, enzymatic hydrolysis is preferably carried out at a temperature of from about 60 °C to about 80 °C (for example 60, 61, 62, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 °C, or any intermediate temperature). In some embodiments, the enzymatic hydrolysis is preferably carried out for a period in the range of 80-100 minutes, preferably at least about 80 minutes, and up to about 90 minutes, preferably about 90minutes. For example, the enzymatic hydrolysis may be carried out for a period of 80, 85, 90, 95 or 100 minutes, or any intermediate period.

[0047] The enzymatic hydrolysis is preferably carried out until at least about 50% by weight, preferably at least about 90% by weight, more preferably at least about 95% by weight, of the initial starch content, has been hydrolyzed to sugars (i.e. to dextrin, glucose and / or to other water-soluble saccharides, including di saccharides and other short-chain oligosaccharides).

[0048] In another embodiment, during hydrolysis step, the enzyme breaks down starch molecules into smaller dextrin and sugars, making it easier to separate the protein fraction from non-protein components.

[0049] First Solid-Liquid Separation (110)

[0050] The first liquid fraction is subjected to solid-liquid separation. In some embodiments, a first solid biomass is separated from the first liquid fraction.

[0051] In some embodiments, the solids from the first liquid fraction are separated to obtain the first solid biomass using a suitable separation process known in the art. In some embodiments, the separation process includes but is not limited to centrifugation and / or micro, ultra or nanofiltration, to separate the first solid biomass from the first liquid fraction.

[0052] Typically, the solids from the first liquid fraction are separated to obtain the first solid biomass using centrifugation. The centrifugation of the first liquid fraction results into first solid biomass in the residual phase and solubilized starch fraction in the supernatant phase. The residue contains valuable protein fractions and is retained for further processing.

[0053] Residue Resuspension (112)In some embodiments, the first solid biomass is resuspended in water followed by separation of the first solid biomass.

[0054] Alkaline Washing Step (114)

[0055] The first solid biomass is subjected to an alkaline wash. In some embodiments, the first solid biomass is suspended in an alkaline medium at predetermined temperature and time to obtain second liquid fraction. More particularly, the residual solid biomass obtained from the centrifugation separation process is resuspended in an alkaline medium. Typically, the pH of the alkaline medium from about 8 to about 12 (for example (8, 8.5, 9, 9.5, 10, 10.5, 11, or 11.5 or 12 any intermediate value). In a preferred embodiment, the pH of the alkaline medium for resuspending the first solid biomass is adjusted to 10 using 5M NaOH. In some embodiments, the suspended first solid biomass in the alkaline medium is kept for incubation for a predetermined temperature and time.

[0056] In some embodiments, the alkaline wash of the residual solids in the first solid biomass is carried out at a temperature of about 30 °C to about 60 °C (for example 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 °C, or any intermediate temperature).

[0057] In some embodiments, the alkaline wash of the residual solids in the first solid biomass is preferably carried out for a period of at least selected including but not limited to 10-90 minutes, preferably at least about 60 minutes, and up to about 70 minutes, preferably about 60 minutes. For example, the alkaline wash may be carried out for a period of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 minutes, or any intermediate period.

[0058] In some embodiments, the first solid biomass is suspended in an alkaline medium under constant stirring during incubation for a predetermined temperature and time.In a non-limiting example, the residue is then washed with RO water to remove residual sugars and starch fragments resulting from enzymatic hydrolysis. The alkaline wash step, involving the resuspension of solid residue at pH 10 using NaOH at a 45 °C temperature with constant stirring, is a critical phase in the production of millet protein concentrate. This alkaline wash step greatly enhances protein extraction by breaking down protein-starch complexes and improving the overall yield and purity of the final protein product. In another exemplary embodiment, the solid residue is resuspended in 75 liters of reverse osmosis (RO) water. The pH of the suspension is adjusted to 10 using 5M sodium hydroxide (NaOH), and the suspension is incubated at 45°C for 1 hour with constant stirring.

[0059] The alkaline wash step selectively solubilizes residual carbohydrates and disrupts protein-starch complexes while maintaining protein integrity. Experimental data demonstrate that washing at pH 10 and 45 °C provides a marked increase in protein purity compared to lower pH values or the absence of alkaline washing, without excessive protein loss. The alkaline treatment further solubilizes the carbohydrates which include but are not limited to dextrin and other water-soluble saccharides, thereby leading to efficient extraction of protein in the insoluble fraction.

[0060] In some embodiments, during the suspension of first solid biomass in an alkaline medium at predetermined temperature and time to obtain second liquid fraction step, the parameters such as pH, temperature and time are optimized to enhance protein purity and recovery. The pH of the slurry is adjusted to a slightly alkaline range i.e., 10 to promote the solubilization of carbohydrates while ensuring maximum enzymatic activity. Sodium hydroxide is commonly used in extraction due to its strong alkalinity, which promotes the solubilization of carbohydrates. The addition of NaOH results in an increase in pH, which causes the carbohydrate to separate.

[0061] Centrifugation and separation of residue (116, 118)In some embodiments, the solids from the second liquid fraction are separated to obtain a protein concentrate fraction using a suitable separation process known in the art. In some embodiments, the separation process step includes but is not limited to centrifugation and / or micro, ultra or nanofiltration, to separate the first solid biomass form from the first liquid fraction.

[0062] Neutralization and Drying (120)

[0063] The protein-rich solid fraction is neutralized to about pH 7 using a food-grade acid and subsequently dried using freeze drying, spray drying, or equivalent techniques to obtain a millet protein concentrate powder.

[0064] Typically, the solids from the second liquid fraction are separated out to obtain a protein concentrate fraction using centrifugation. The parameters and the operating conditions of the centrifugation process are optimized to effectively separate the protein concentrate in the residual phase. The centrifugation of the second liquid fraction results in the separation of the protein concentrate fraction in the residual phase and solubilized starch fraction in the supernatant phase. More preferably, the residual phase primarily includes high solid protein concentrates.

[0065] In some embodiments, the pH of the protein concentrate fraction is neutralized to obtain a neutralized protein concentrate. Typically, pH of the protein concentrate fraction is adjusted to 7 using 5N HC1.

[0066] In some embodiments, the neutralized protein concentrate is subjected to at least one drying process selected from including but not limited to, drum drying, spin flash drying, fluidized bed drying, dried to a moisture of less than 10% to obtain a millet protein concentrate. The drying process involves atomizing the slurry into a fine mist and rapidly drying it in a stream of hot air, producing a fine, protein-rich powder. Overall, the process for producing millet protein concentrate is optimized by systematically adjusting extraction conditions i.e., using controlled temperature,controlled pH for alkaline extraction, and optimized enzyme dosage in enzymatic hydrolysis enhances protein purity and yield. This produces high-quality millet protein concentrate.

[0067] In one embodiment, the millet protein concentrate has a protein recovery of at least 80%.

[0068] In some embodiments, the obtained millet protein concentrate from the process described above is characterized by high-protein and high-fiber concentrate. Typically, the protein recovery is at least 80 wt.%

[0069] In another aspect, the present application relates to a high-purity protein concentrate suitable for use in various food applications, including nutritional supplements, plantbased protein formulations, beverages and other food ingredients.

[0070] The millet protein concentrate obtained has a protein purity in the range of about 50 to 65 wt% on a dry basis. In one embodiment, the millet protein concentrate has a protein purity of about 50 to 60 wt% on a dry basis. In another embodiment, the millet protein concentrate has a protein purity of about 55 to 65 wt% on a dry basis. In some embodiments, the millet protein concentrate comprises total dietary fiber in the range of 15 to 25 wt% on a dry basis. In an embodiment, the millet protein concentrate has total dietary fiber of about 22 wt% on a dry basis.

[0071] In some embodiments, the millet protein concentrate obtained from the above process is a high-purity, protein ingredient suitable for use in food products, dietary supplements, and other applications. The protein concentrate retains essential amino acids and exhibits high purity, and improved digestibility as compared to conventional millet protein products.

[0072] Furthermore, in one embodiment, the production of high-quality protein concentrates from proso and foxtail millets through the alkali-enzyme hybrid process providesvarious advantages. Instead of relying on harsh chemicals, mild alkaline conditions are used to extract protein efficiently. In terms of functional properties, millet proteins exhibit favorable emulsifying, water-holding, and foaming capacities, making them suitable for a variety of food applications. Hence, the extraction of the protein concentrates from proso and foxtail millets can address consumer demands for nutritious and plant-based options.

[0073] Additionally, in one embodiment, the protein concentrates from proso and foxtail millets are also beneficial from a nutritional standpoint. These millets are naturally high in essential amino acids, including methionine, which is often limited to other plant-based proteins like soy.

[0074] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known / conventional processes and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[0075] Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

[0076] While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples and drawings and are described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims.EXAMPLES

[0077] The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

[0078] Example 1: Effect of Enzyme Concentration on Protein Enrichment

[0079] Proso millet flour having a protein content of about 14.41% and a moisture content of about 11.01% was used as the raw material. 60g of the flour was dispersed in 600 mL of distilled water and mixed at room temperature for about 10 minutes to obtain an aqueous slurry. The pH of the slurry was adjusted to about 6 using IN hydrochloric acid. The slurry was heated to about 85 °C to gelatinize starch, followed by cooling to about 70 °C.

[0080] An alpha-amylase enzyme was added at varying concentrations ranging from 0.05% to 0.50% (w / w, based on millet solids). The slurry was incubated at about 70 °C for about 90 minutes to effect enzymatic liquefaction. The liquefied slurry was centrifuged at about 3000 ref for about 10 minutes to separate a first liquid fraction and a first solid biomass.

[0081] The first solid biomass was resuspended in an equal amount of distilled water, and the pH was adjusted to about 10 using IN sodium hydroxide. The suspension was mixed for about 45 minutes at room temperature to carry out an alkaline wash. The mixture was centrifuged again at about 3000 ref for about 10 minutes to recover a protein-rich solid fraction. The pH was neutralized to about 7 using IN hydrochloric acid, and the product was freeze-dried to obtain a millet protein concentrate.

[0082] The results obtained are shown in Table 1

[0083] TableEnzyme concentration Solids yield (%) Protein purity (%)

[0084] (% w / w)

[0085] 0.05 25.1 39.7

[0086] 0.10 23.0 44.6

[0087] 0.15 24.4 42.5

[0088] 0.20 22.2 48.2

[0089] 0.25 20.9 49.7

[0090] 0.30 20.3 48.9

[0091] 0.35 20.7 50.6

[0092] 0.40 18.9 52.6

[0093] 0.50 19.2 51.3

[0094] Example 2: Effect of Enzymatic Liquefaction Time

[0095] Proso millet flour was processed as described in Example 1, using a fixed alphaamylase concentration of about 0.4% (w / w). The enzymatic liquefaction time was varied at 45, 90, and 180 minutes while maintaining the incubation temperature at about 70 °C.

[0096] After liquefaction, the slurry was centrifuged to obtain a first solid biomass. The first solid biomass was subjected to an alkaline wash at pH 10 for about 45 minutes, followed by centrifugation, neutralization to pH 7, and freeze-drying as described in Example 1.The results obtained are shown in Table 2.

[0097] Table 2

[0098] Enzyme concentration Liquefaction time Solids yield Protein purity (%) (min) (%) (%)

[0099] 0.4 45 29.3 33.25

[0100] 0.4 90 18.9 52.6

[0101] 0.4 180 20.9 49.71

[0102] Example 3: Effect of pH in the Alkaline Wash Step

[0103] Proso millet flour was processed as described in Example 1, using an alpha-amylase concentration of about 0.4% and a liquefaction time of about 90 minutes. The first solid biomass obtained after centrifugation was resuspended in water and subjected to alkaline washing at different pH values (8, 9, 10, and 11), adjusted using sodium hydroxide. Each alkaline wash was carried out for about 45 minutes at room temperature.

[0104] After alkaline washing, the mixtures were centrifuged, neutralized to pH 7, and freeze-dried to obtain protein concentrate powder which were analyzed for protein purity.

[0105] The results of solid yield and protein purity obtained are shown in Table 3

[0106] Table 3Alkaline wash pH Solids yield (%) Protein purity (%)

[0107] No washing 50.8 33.5

[0108] 8 22.7 45.7

[0109] 9 21.2 49.2

[0110] 10 18.9 52.6

[0111] 11 16.5 51.8

[0112] Example 4: Scale-Up of Millet Protein Concentrate Production

[0113] The process was scaled up using 60 kg of proso millet flour having a protein content of about 13.55%. The flour was dispersed in 600 L of water to form an extraction slurry. Gelatinization was carried out at about 85 °C, followed by enzymatic liquefaction using about 0.4% alpha-amylase at about 70 °C for about 90 minutes. The slurry was subjected to decantation to obtain a first solid biomass. The first solid biomass was resuspended in water, and an alkaline wash was carried out at pH 10 and about 45 °C for about 45 minutes. The protein-rich solid fraction obtained after separation was neutralized, pasteurized and spray-dried to obtain a millet protein concentrate.

[0114] The product exhibited a protein purity of about 56.42 wt% (dry basis) with a protein recovery of about 85%, confirming the scalability and industrial applicability of the process.

[0115] The above experimental data demonstrate that enzymatic liquefaction alone is insufficient to achieve high protein purity. As shown in Example 3, omission of thealkaline wash results in protein purity of only about 33.5%. Introduction of an alkaline wash leads to a substantial increase in protein purity, with a marked and unexpected improvement observed at pH 10.

[0116] The results further indicate that pH 10 provides an optimal balance between selective solubilization of non-protein components and preservation of protein integrity. Increasing the pH beyond 10 does not result in a proportional improvement in protein purity, indicating a non-linear and non-obvious technical effect.

[0117] Examples 1 and 2 show that appropriate enzymatic liquefaction enhances the effectiveness of the subsequent alkaline wash by reducing starch content and enabling efficient separation of protein. Example 4 confirms that the same technical effect is reproducible at an industrial scale.

[0118] Collectively, the examples establish that the alkaline wash step carried out at about pH 10 and about 45 °C constitutes a critical and enabling feature of the invention, resulting in synergistic and unexpected enhancement of protein purity and recovery.

[0119] Example 5: Dietary Fiber Characterization of Millet Protein Concentrate The millet protein concentrate obtained according to the process described in Example 4, including enzymatic liquefaction followed by the alkaline wash at about pH 10 and about 45 °C, was further analyzed for dietary fibre content.

[0120] Soluble dietary fibre and insoluble dietary fibre were determined in accordance with AO AC Official Method 991.43 (21st Edition, 2019), and total dietary fibre was determined in accordance with AOAC Official Method 985.29 (21st Edition, 2019). The results obtained are shown below:

[0121] Table 4Parameter Content (g / 100 g)

[0122] Insoluble dietary fibre 20.81

[0123] Soluble dietary fibre 1.43

[0124] T otal dietary fibre 22.57

[0125] The dietary fibre analysis demonstrates that the millet protein concentrate retains a significant proportion of insoluble dietary fibre while maintaining high protein purity. This indicates that the alkaline wash at about pH 10 and about 45 °C selectively removes non-protein carbohydrates such as solubilized starch and dextrin, without indiscriminately degrading or removing structurally bound dietary fibre.

[0126] The coexistence of elevated protein content together with substantial dietary fibre content is advantageous for nutritional and functional food applications. Importantly, this result further confirms that the alkaline wash step does not merely act as a harsh extraction treatment, but operates in a controlled and selective manner, thereby preserving desirable nutritional components.

[0127] The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognizethat the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

[0128] While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims

We Claim:

1. A process for producing a millet protein concentrate, the process comprising the steps:(a) preparing an aqueous slurry of milled millet;(b) gelatinizing the aqueous slurry by heating to a temperature sufficient to gelatinize starch;(c) liquefying the gelatinized slurry by enzymatic treatment to hydrolyze starch to form a first liquid fraction;(d) separating a first solid biomass from the first liquid fraction;(e) subjecting the first solid biomass to an alkaline wash at a predetermined pH and a predetermined temperature for a predetermined time, thereby selectively removing non-protein components and forming a second liquid fraction and a protein-rich solid fraction;(f) separating the protein-rich solid fraction from the second liquid fraction; and(g) neutralizing and drying the protein-rich solid fraction to obtain a millet protein concentrate.

2. The process as claimed in claim 1 , wherein the alkaline wash is carried out at a pH in the range of 8 to 12.

3. The process as claimed in claim 1 or 2, wherein the alkaline wash is carried out at a temperature in the range of 30 °C to 60 °C.

4. The process as claimed in any one of claims 1 to 3, wherein the alkaline wash is carried out for 10 to 90 minutes.

5. The process as claimed in any one of claims 1 to 4, wherein the alkaline wash employs sodium hydroxide as the alkali.

6. The process as claimed in any one of claims 1 to 5, wherein the enzymatic liquefaction is performed using an alpha-amylase enzyme derived from Bacillus species.

7. The process as claimed in claim 6, wherein the enzyme is used at a concentration of 0.1 to 0.4 wt%, based on millet solids.

8. The process as claimed in any one of claims 6 to 7, wherein enzymatic liquefaction is conducted at a temperature in the range of 60-80 °C, for a time in the range of 80-100 min.

9. The process as claimed in claim 1, wherein the solids separation in step (d) and step (f) is performed using centrifugation at about 3000 ref for about 10 min.

10. The process as claimed in any one of claims 1 to 9, wherein the millet grains comprise proso millet, foxtail millet, or a mixture thereof.

11. The process as claimed in any one of claims 1 to 10, wherein the aqueous extraction slurry comprises millet flour and water in a ratio of 1 : 5 to 1:

15.

12. The process as claimed in any one of claims 1 to 11, wherein the millet protein concentrate obtained has a protein purity in the range of about 50 to 65 wt% on a dry basis.

13. The process as claimed in claim 12, wherein the millet protein concentrate has a protein recovery of at least 80%.

14. A millet protein concentrate, obtainable by the process as claimed in any one of claims 1 to 13.

15. The millet protein concentrate as claimed in claim 1 to 14, wherein the concentrate comprises total dietary fiber in the range of 15 to 25 wt% on a dry basis.

16. The millet protein concentrate as claimed in claim 1 to 14 for use in food products, nutritional supplements, plant-based protein formulations, or beverages.

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