Process for isolation and purification of high-quality protein

Abstract

The present disclosure relates to a process for isolation and purification of High-Quality Protein from a feed distiller's dried grains and mixed with solubles (DDGS). The process comprises the steps of: selecting an extraction solvent to maximize the protein yield and purity; extracting the DDGS with the selected solvent along with a buffer, a polar solvent, an alkaline solvent to obtain a extracted solid; processing the extracted solid by ultrasonication assisted solvent extraction method to obtain a High-Quality Protein. Further, the tannis were removed by enzymatic treatment.

Description

PROCESS FOR ISOLATION AND PURIFICATION OF HIGH-QUALITY PROTEINFIELD OF INVENTION

[0001] The present disclosure belongs to the technical field of biotechnology. More particularly, the present disclosure relates to a process for isolation and purification of High- Quality Protein.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] The Protein Supplements Market size is estimated at USD 27.99 billion in 2024 and is growing at a CAGR of 7.45% during the forecast period (2024-2029) [Protein Supplements - Market Share Analysis, Industry Trends & Statistics, Growth Forecasts (2024 - 2029), Market Research Report, 2024, 1-120; Protein Supplements Market Size (2024 - 2029), Mordor Intelligence] . Protein has become a very popular macronutrient, and it is not only limited to bodybuilders and athletes, but also consumed by public to meet their daily nutritional need and senior citizens consume it to maintain their muscle mass. The boost in sales and consumption of proteins encouraged manufacturers to develop processes for improved food and feed ingredients containing higher protein content, reduced gluten, higher soluble fiber content, etc. These nutritional supplements have provided health benefits in both humans and animals. In this regard, there are ongoing efforts in developing improved process technologies that should not add incremental risks to health. Therefore, physical purification processes that do not include the use of solvents or the addition of synthetic chemicals are preferred.

[0004] In order to achieve net-zero target and reduce reliance on fossil-fuels, Indian Government has accelerated the target of 20% ethanol blending in gasoline by 5 years from 2025 to 2030. To meet this goal, Government has allowed use of damaged and surplus food grains for ethanol production. In the grain-based ethanol production process, grains are crushed and mixed with water to create a slurry. This slurry is then heated to break down starch, followed by the addition of enzymes to further convert them into sugars. Finally, yeast is introduced to ferment these sugars into ethanol. After the ethanol is separated through distillation, the leftover solids - a rich mix of protein, oil, fiber, and minerals - are spun incentrifuges, dried, and recombined to create a valuable co-product called DDGS. The surge in fuel ethanol production in India has led to a growing supply of DDGS, prompting the need for continued market development to ensure its effective utilization. India's policy think tank, NITI Aayog has predicted a jump of 482-crore litre grain-based ethanol production by 2025, suggesting ample room for expansion in the grain-based distilleries. In grains-based distilleries, some by-products like CO2 & Dried Distillers Grains with Solubles (DDGS) are also generated which can also be utilized for production of value-added products, thus increasing the revenue potential of grain-based distilleries.

[0005] Rice (Oryza sativa) is the staple crop for more than half of the world’s population. It contains a significant amount of protein including four different fractions such as glutelin, albumin, globulin, and prolamin having solubility in alkali, water, salt, and alcohol, respectively. The dominant protein in rice is glutelin. Also, these proteins exhibit a richer amino acid profile, highly nutritious and possess several functional properties, like casein and soy protein isolates and are highly recommended for infants and the elderly due to their nutritional quality, digestibility, and hypo-allergenicity. Therefore, rice protein is considered an alternative source of protein over animal-based protein. Compared with many other cereal grains, rice protein is hypoallergic due to the absence of gluten, and therefore it is used to formulate food for infants and gluten-allergic people. Protein obtained from the rice bran is used in several food industries such as bread, breakfast cereals, protein supplements, beverages, and even meat and sausages. Furthermore, consumers having allergies and aversion to wheat or gluten are looking for a high-protein option that is wheat and gluten- free. Rice DDGS contains albumin, prolamin, globulin and glutelin.

[0006] However, the process technologies required to produce these improved ingredients should not add incremental risks to health, either real or perceived.

[0007] It is prudent to mention here that the rice has dense deposits of protein bodies (PBI and PBII). These PBs are known for their amino acid profile comprising essential amino acids like threonine, leucine, and phenylalanine along with sulfur-rich amino acids like methionine and cysteine.

[0008] The limitation of the use of rice protein is less than 2% solubility in water, that may be due to extensive aggregation and crosslinking through hydrophobic interaction and disulfide bonds result in the insoluble precipitate. While alkaline extraction effectively isolates rice proteins, it also elevates the sodium content in the final product. Therefore, post- alkaline protein extraction, it is necessary to purify the product, which is achieved byultrafiltration, a well-established membrane technology for protein concentration and purification.

[0009] Com-based Distillers Dried Grains with Solubles (DDGS) is a protein-rich coproduct derived from ethanol production. As com undergoes fermentation, starches convert to ethanol, leaving behind nutrient-dense components like protein, fiber, and essential amino acids in the DDGS. With a protein content typically between 25-35%, DDGS is widely used as a cost-effective feed ingredient, especially for livestock, such as cattle, swine, and poultry. Zein is a type of prolamin protein found in com, making up a major portion of com protein content. Known for its film-forming properties, zein is commonly used in biodegradable coatings for food products, pharmaceuticals, and even biodegradable plastics. While not a complete protein due to its limited essential amino acids, zein has unique applications in industries that leverage its water-insolubility and strong binding capabilities, making it valuable beyond traditional food and feed uses.

[0010] The fiber enriched fraction could be utilized for production of cellulosic ethanol, fiber oil, fiber gum, phytosterols, and oligosaccharides.

[0011] Tannins are water-soluble phenolic compounds with molecular weights typically ranging from 300 to 500. Known for their ability to precipitate gelatin, alkaloids, and proteins, tannins are considered to have antinutritional properties. Also, they are reported to form complexes with essential nutrients like proteins and certain minerals, hindering their absorption. Ingestion of tannic acid can lead to the hardening of the gastrointestinal mucosa, potentially reducing nutrient absorption.

[0012] Therefore, there remains a need in the art for methods of extracting protein from plant-based sources, such as cereal grains and byproducts thereof, wherein tannins and aflatoxins are effectively removed during the extraction process. Such improved methods are desirable to enhance protein purity, safety, and functionality for food and feed applications.OBJECTIVES OF THE INVENTION

[0013] The primary objective of the present disclosure is to provide a DDGS biofractionation process to separate oil, sugars, protein and fiber.

[0014] An object of the present disclosure is to provide a method of preparation of a human edible grade protein.

[0015] Another object of the present disclosure is to provide a process which uses a combination of steps involving mechanical reduction of DDGS to an optimize particle size, two step solvent extraction process for oil and protein recovery. The protein is furtherprecipitated at the isoelectric point and purified to human edible grade by subsequent membrane filtration and chromatographic steps.

[0016] Another object of the invention is drying the DDG or DDGS feed mixture to a specified moisture level.

[0017] Still another object of the present disclosure is importance of particle size for efficient extraction of oil and protein.

[0018] Still another object of the present disclosure is reduction of particle size to a specified median particle size < 500 pm.

[0019] Still another object of the present disclosure is to use the remaining sugars for ethanol production.

[0020] Further another object of the present disclosure is to provide a structured protein product and method of making such protein product.

[0021] Another object of the present disclosure is to provide a process for production of a gluten free product.

[0022] Still another object of the present invention is to provide a process for production of a gluten free protein that substantially free of anti-nutrient factors and chemicals of concern.

[0023] Yet another object of the present invention is to provide a process for production of a protein that offers several -fold advantages over the conventional.

[0024] Yet another object of the present invention to provide a process with significantly reduced tannin content.SUMMARY OF THE INVENTION

[0025] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0026] The present disclosure belongs to the technical field of biotechnology. More particularly, the present disclosure relates to an innovative process for isolation and purification of High-Quality Protein.

[0027] Accordingly, in one aspect, the present disclosure relates to a process for isolation and purification of High-Quality Protein from a feed distiller's dried grains and mixed with solubles (DDGS). The process comprises the steps of: selecting an extraction solvent to maximize the protein yield and purity; extracting the DDGS with the selected solvent alongwith a buffer, a polar solvent, an alkaline solvent to obtain a extracted solid; processing the extracted solid by ultrasonication assisted solvent extraction method, removing tannins by treatment with tannase and polyphenol oxidase (PPO / tyrosinase) to obtain a High-Quality Protein; and increasing the protein purity by membrane-based fractionation and / or chromatographic separation selected from ultrafdtration, diafdtration, nanofiltration, ionexchange chromatography, and / or size-exclusion chromatography..

[0028] In first aspect, the present invention relates to a method for isolation and purification of protein from a dried distillers grains with solubles (DDGS) comprising the steps of:(a) suspending the DDGS flour at 8-15% w / v in 25-65% v / v polar solvent with or without buffer followed by adjusting pH to 8.5-9.5;(b) applying ultrasound at 20-30 kHz with jacket cooling at 10-15 °C and separating solids to obtain a supernatant;(c) concentrating the supernatant by ultrafiltration and performing diafiltration to remove phenolics and to obtain retentate;(d) enzymatically treating the retentate with at least one enzyme at pH 5.0-7.0 to obtain tannin free solution; and(e) microfiltering and drying by spray-drying with outlet temperature not exceeding 60 °C or by freeze-drying to obtain the protein.

[0029] In another aspect of the present invention, the dried distillers grains with solubles is rice, wheat, maize, sorghum, or millets based DDGS.

[0030] In another aspect of the present invention, the polar solvent is selected from alcohol, alkaline water, l-ethyl-3-methylimidazolium chloride, l-hexyl-3-methylimidazolium chloride, l-butyl-3-methylimidazolium acetate, l-butyl-3-methylimidazolium hydroxide, N- butylpyridinium hydroxide, choline hydroxide and combination thereof.

[0031] In yet another aspect of the present invention, the at least one enzyme is selected from the group consisting of tannase, polyphenol oxidase (PPG), laccase and mixtures thereof.

[0032] In another aspect of the present invention, the method for isolation and purification of protein further comprising contacting the supernatant prior to enzymatic treatment with polyvinylpolypyrrolidone (PVPP) and passing the filtrate through a hydrophobic resin column followed by rinsing with water, to reduce phenolic content before ultrafiltration.

[0033] In another aspect of the present invention, the ultrasound is applied as pulsed ultrasonication with cycles of 10 seconds on and 20 seconds off for 8-12 minutes at 20-25 kHz, and vortex mixing is applied before and after sonication for at least 1 minute.

[0034] In another aspect of the present invention, the ultrafiltration is performed using metallic or ceramic membranes with a molecular weight cutoff is 1 kDa to 10 kDa for concentration to 2-5 x, followed by diafiltration with 2-3 diavolumes.

[0035] In another aspect of the present invention, the pulverized DDGS is pre-treated with high-power ultrasound at 10 kHz to 30 kHz and 900 W for about 5 minutes with intermittent mixing prior to suspension in polar solvent.

[0036] In another aspect of the present invention, the alcohol is methanol, ethanol, propanol, isopropyl alcohol or butanol.

[0037] In another aspect of the present invention, the dried distillers grains with solubles (DDGS) a D50 particle size of less than 500 micrometers (pm).

[0038] In another aspect of the present invention, the protein obtained by the present method has a purity of at least 82% by weight.

[0039] In yet another aspect, the present invention relates to a protein composition obtained by the method, characterized by: at least 82% protein by weight on a dry basis, NSI at least 88-90% at pH 7, tannic acid equivalent at or below 1-10 ppb by HPLC-UV with gallate calibration, free gallic acid at or below 0.2-0.5 ppm.

[0040] In yet another aspect, the present invention relates to a protein composition derived from distillers’ dried grains with solubles (DDGS) comprising at least 82% protein by weight on a dry basis, having a nitrogen solubility index (NSI) of at least 88% at pH 7, a tannic acid equivalent not exceeding 10 parts per billion as quantified by HPLC-UV using gallic acid calibration, and free gallic acid not exceeding 0.5 parts per million.

[0041] In another aspect of the present invention, the protein content is at least 85%, the NSI is at least 90% at pH 7, and the tannic acid equivalent is not more than 5 parts per billion as quantified by HPLC-UV using gallate calibration.

[0042] Various objects, features, aspects and advantages of the present disclosure will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like features.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawing(s) are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The diagrams are for illustration only, which thus is not a limitation of the present disclosure.

[0044] FIG. 1 illustrates raw DDGS and purified protein from DDGS.DETAILED DESCRIPTION OF THE INVENTION

[0045] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0046] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0047] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0048] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0049] The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0050] Unless the context requires otherwise, throughout the specification which follows, the word “comprise” and variations thereof, such as “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0051] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0052] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Furthermore, the ranges defined throughout the specification include the end values as well, i.e., a range of 1 to 10 implies that both 1 and 10 are included in the range. For the avoidance of doubt, the applicant shall be entitled to any equivalents according to applicable law.

[0053] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0054] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

[0055] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0056] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to asprocesses. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0057] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0058] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0059] The term “or”, as used herein, is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0060] Various terms are used herein to the extent a term used is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0061] The present disclosure belongs to the technical field of biotechnology. More particularly, the present disclosure relates to an innovative process for isolation and purification of High-Quality Protein.

[0062] An embodiment of the present disclosure provides a process for isolation and purification of High-Quality Protein from a feed distiller's dried grains and mixed with solubles (DDGS). The process comprises the steps of selecting an extraction solvent to maximize the protein yield and purity; extracting the DDGS by combination of the selected solvent along with a buffer, a polar solvent, an alkaline solvent to obtain a extracted solid; processing the extracted solid by ultrasonication assisted solvent extraction method to obtain a High-Quality Protein.

[0063] In an embodiment, the present invention relates to a method for isolation and purification of protein from a dried distillers grains with solubles (DDGS) comprising the steps of:(a) suspending the pulverized DDGS at 8-15% w / v in 25-65% v / v polar solvent with or without aqueous phosphate buffer followed by adjusting pH to 8.5-9.5;(b) applying ultrasound at 20-30 kHz with jacket cooling at 10-15 °C and separating solids to obtain a supernatant;(c) concentrating the supernatant by ultrafiltration and performing diafiltration to remove phenolics and to obtain retentate;(d) enzymatically treating the retentate with at least one enzyme at pH 5.0-7.0 to obtain tannin free solution; and(e) microfiltering and drying by spray-drying with outlet temperature not exceeding 60 °C or by freeze-drying to obtain the protein.

[0064] In another embodiment of the present invention, the dried distillers grains with solubles is rice, wheat, maize, sorghum, or millets based DDGS. Preferably, the DDGS is maize based DDGS.

[0065] In an embodiment, the extraction solvent is selected based on the nature of protein and solubility of protein. In some embodiments, the extraction solvent is selected from a group consisting of phosphate buffer, Osborne, alcohol, alkaline ethanol (10% to 95%), alkaline water (0.2-10 N NaOH), l-ethyl-3-methylimidazolium chloride, l-hexyl-3- methylimidazolium chloride, l-butyl-3-methylimidazolium acetate, l-butyl-3- methylimidazolium hydroxide, N-butylpyridinium hydroxide, choline hydroxide and combination thereof. In a preferred embodiment, the solvent used in the extraction process is a combination of solvents as given above.

[0066] In another embodiment of the present invention, the polar solvent is alcohol, 1-butyl- 3-methylimidazolium hydroxide or combination thereof.

[0067] In another embodiment of the present application, the polar solvent is ethanol, 1- butyl-3-methylimidazolium hydroxide or combination thereof. Preferably, the polar solvent is ethanol or mixture of ethanol and l-butyl-3-methylimidazolium hydroxide.

[0068] In an embodiment, the buffer is selected from a group consisting of phosphate buffer, tris buffer, citrate-phosphate buffer and combination thereof.

[0069] In another embodiment, the buffer is aqueous phosphate buffer.

[0070] In an embodiment, the polar solvent is selected from a group consisting of ethanol concentration (10% to 95%), l-butyl-3-methylimidazolium hydroxide and combination thereof.

[0071] In an embodiment, the alkaline solvent is selected from a group consisting of alkaline ethanol (10% to 95%), alkaline water (0.2-10 N NaOH), l-butyl-3-methylimidazolium hydroxide and combination thereof.

[0072] In another embodiment, the method for isolation and purification of protein further comprises contacting the supernatant prior to enzymatic treatment withpolyvinylpolypyrrolidone (PVPP) and passing the filtrate through a hydrophobic resin column followed by rinsing with water, to reduce phenolic content before ultrafiltration.

[0073] In another embodiment of the present invention, the supernatant contacting step uses insoluble PVPP at 0.1-3% w / v with a contact time of 2-30 minutes.

[0074] In another embodiment of the present invention, the hydrophobic resin is a styrene divinylbenzene porous adsorbent operated at linear velocities of 1-5 bed volumes per hour.

[0075] In an embodiment, the ultrasonication is carried out at a temperature in the range of 4 to 60°C for a period in the range of 30 sec to 30 min.

[0076] In an embodiment, the method of the present disclosure separates fats, proteins, oil and fibers.

[0077] In an embodiment, the method of the present disclosure significantly reduces tannin content.

[0078] In an embodiment, the method may also include milling process for reducing the particle size of the DDGS.

[0079] In an embodiment, the method may also include protein precipitation.

[0080] In an embodiment, the method may also include membrane filtration.

[0081] In an embodiment, the method also includes chromatography.

[0082] The present disclosure provides a combination of dried milling and air classifying based bio fractionation process of 1G Biorefinery byproduct i.e. dried distiller's grains (DDG) or distiller's dried grains and mixed with solubles (DDGS) into fibers, sugars, protein and oil, wherein the separation of these components will increase the value of these components thereby increasing the overall economic viability of the 1G biorefinery. Additionally, the disclosure provides a physical separation process for purifying a plantbased protein from cereal grains and byproducts of cereal grain processing operations, thereby does not add incremental risks to health, either real or perceived. Moreover, the present invention also focuses on production of gluten free protein. The method includes combination of the following steps: Milling and Air Classifying based separation to separate fats, proteins and fibers, a combination of ultrasonication and solvent extraction to separate oil and protein, followed by membrane filtration.

[0083] In another embodiment, the the present invention relates to a protein composition derived from distillers’ dried grains with solubles (DDGS) comprising at least 82% protein by weight on a dry basis, having a nitrogen solubility index (NSI) of at least 88% at pH 7, a tannic acid equivalent not exceeding 10 parts per billion as quantified by HPLC-UV using gallic acid calibration, and free gallic acid not exceeding 0.5 parts per million.

[0084] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person skilled in the art.EXAMPLES

[0085] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. The Pulverized DDGS is obtained from Archana Traders, Panipat, Haryana- 132103.Example 1: Purification of protein from DDGS by Solvent Extraction

[0086] Based on the nature of protein and solubility, it is desirable to select the extraction solvent to maximize the protein yield and purity. For the efficient protein extraction with minimal loss, extraction was carried out in solvent / combination of solvents including phosphate buffer, Osborne extraction method, ethanol concentration (10% to 95%), alkaline ethanol (10% to 95%), alkaline water (0.2-10 N NaOH), phosphate buffer, Osborne, alkaline ethanol (10% to 95%), alkaline water (0.2-10 N NaOH), l-ethyl-3-methylimidazolium chloride, l-hexyl-3-methylimidazolium chloride, l-butyl-3-methylimidazolium acetate, 1- butyl-3-methylimidazolium hydroxide, N-butylpyridinium hydroxide, choline hydroxide. To further reduce the total reaction time from 24 hours to less than 20 minutes, ultrasonication assisted solvent extraction method was optimized. After primary extraction and clarification, the DDGS protein solution was contacted with an enzyme complex comprising tannase, polyphenol oxidase (PPO / tyrosinase), and laccase under controlled conditions to reduce tannins and phenolic residues while preserving protein functionality. Fig. 1 shows the images of DDGS and isolated protein.Example 2:

[0087] Pulverized DDGS (D50 < 500 pm) was resuspended at 10% w / v in 20 mM phosphate buffer pH 7.0 with 30% v / v ethanol; pH was adjusted to 9.0 with 0.1 N NaOH. The slurry was vortexed for 1 min, sonicated for 9 min at 30 kHz in a 12 °C jacket, and vortexed for 1 min. The mixture was centrifuged for 10 min at 10,000 g, and the supernatant was collected. The supernatant obtained was adjusted to pH 6.0 using 0.1 N HC1 and concentrated 5x by 5 kDa ultrafiltration, followed by diafiltration with 2 diavolumes to remove low-molecularphenolics. The retentate was incubated with tannase (100 U / g protein), polyphenol oxidase (30 U / g protein) and laccase (15 U / g protein) for 20 min at pH 5-7 and 38 °C and then cooled to 30 °C. The treated solution was microfdtered at 0.2 pm, concentrated by 10 kDa UF, and spray-dried with an outlet temperature <60 °C. The protein powder (>85% protein) met tannic acid equivalent <5 ppb by HPUC-UV (gallate calibration), free gallic acid <0.5 ppm, and NSI >90% at pH 7.Example 3:

[0088] Pulverized DDGS (D50 < 500 pm) was suspended at 12% w / v in 20 mM phosphate buffer pH 7.2 containing 40% v / v ethanol and 0.03% w / v l-butyl-3-methylimidazolium hydroxide. The slurry was vortexed for 1 min, subjected to pulsed ultrasonication (10 s on / 20 s off cycles) for 12 min at 22 kHz in a 12 °C jacket, then vortexed for 1 min. The mixture was centrifuged for 10 min at 10,000 g to separate solids. The supernatant was neutralized to pH 6.0 using 0.1 N HC1 and contacted with PVPP (3 g / L) for 10 min under gentle stirring, then depth fdtered. The fdtrate was passed through a hydrophobic resin column at 3 BV / h for phenolic adsorption and rinsed with 1 BV water. The eluate was concentrated 5x and diafiltered with 2 diavolumes by 10 kDa ultrafiltration. Tannase (30 U / g protein) was added and incubated at pH 5.8, 30 °C for 12 min. The solution was cooled to 18 °C and diafiltered (3 diavolumes), then spray-dried with outlet temperature <60 °C. The product had >82% protein purity, <1 ppb tannic acid equivalent, residual ionic liquid <50 ppb, free gallic acid <0.2 ppm, and NSI >88% at pH 7.Example 4

[0089] Pulverized DDGS with 10% moisture. The pulverized DDGS was suspended at 10% w / v in 65% v / v ethanol adjusted to pH 9.0 with 0.4 N NaOH and was subjected to high- power ultrasound pretreatment at 20 kHz and 900 W for 5 min with intermittent mixing. The slurry was vortexed for 1 min, sonicated for 8 min at 25 kHz under 15 °C jacket, then vortexed for 1 min. The mixture was centrifuged at 10,000 g for 10 min. The supernatant was concentrated 4x by 3 kDa ultrafiltration and subjected to a two-stage counter-current diafiltration (total 3 diavolumes). The retentate was adjusted to pH 6.8 using 0.1 N HC1 and treated with laccase (3 U / g protein) at 15 °C with oxygen limited to 25% saturation for 8 min, then cooled and microfdtered at 0.2 pm. The protein concentrate was freeze-dried. Final protein content was >84%, tannic acid equivalent <10 ppb, free gallic acid <0.5 ppm, and NSI >90%.

[0090] The characteristics of purified protein are provided in the table below.ADVANTAGES OF THE INVENTION

[0091] The process of the present disclosure provides a high quality protein.

[0092] The process of the present disclosure provides a human edible grade protein.

[0093] The production of a protein by the method of the present disclosure offers several- fold advantages over the conventional.

Claims

We Claim:

1. A method for isolation and purification of protein from a dried distillers grains with solubles (DDGS) comprising the steps of:(a) suspending the pulverized DDGS at 8-15% w / v in 25-65% v / v polar solvent with or without buffer followed by adjusting pH to 8.5-9.5;(b) applying ultrasound at 20-30 kHz with jacket cooling at 10-15 °C and separating solids to obtain a supernatant;(c) concentrating the supernatant by ultrafiltration and performing diafiltration to remove phenolics and to obtain a retentate;(d) enzymatically treating the retentate with at least one enzyme at pH 5.0-7.0 to produce a tannin free solution; and(e) microfiltering and drying by spray-drying with outlet temperature not exceeding 60 °C or by freeze-drying to obtain the protein.

2. The method as claimed in claim 1, wherein the dried distillers grains with solubles is rice, wheat, maize, sorghum, or millets based DDGS.

3. The method as claimed in claim 1, wherein the polar solvent is selected from alcohol, alkaline water, l-ethyl-3-methylimidazolium chloride, l-hexyl-3-methylimidazolium chloride, l-butyl-3-methylimidazolium acetate, l-butyl-3-methylimidazolium hydroxide, N- butylpyridinium hydroxide, choline hydroxide and combination thereof; or the buffer is selected from a group consisting of phosphate buffer, tris buffer, citrate-phosphate buffer and combination thereof.

4. The method as claimed in claim 1, wherein the at least one enzyme is selected from the group consisting of tannase, polyphenol oxidase (PPG), laccase and mixtures thereof.

5. The method as claimed in claim 1, further comprising contacting the supernatant prior to enzymatic treatment with polyvinylpolypyrrolidone (PVPP) and passing the filtrate through a hydrophobic resin column followed by rinsing with water, to reduce phenolic content before ultrafiltration.

6. The method as claimed in claim 1, wherein ultrasound is applied as pulsed ultrasonication with cycles of 10 seconds on and 20 seconds off for 8-12 minutes at 20-25 kHz, and vortex mixing is applied before and after sonication for at least 1 minute.

7. The method as claimed in claim 1, wherein the ultrafiltration is performed using metallic or ceramic membranes with a molecular weight cutoff ranging from lkDa-10 kDa for concentration of supernatant to 2-5 x, followed by diafiltration with 2-3 diavolumes.

8. The method as claimed in claim 1, wherein the pulverized DDGS is pretreated with high- power ultrasound at 10 kHz to 30 kHz for about 5-10 minutes with intermittent mixing prior to suspension in polar solvent.

9. The method as claimed in claim 3, wherein the alcohol is methanol, ethanol, propanol, isopropyl alcohol or butanol.

10. The method as claimed in claim 1, wherein the dried distillers grains with solubles (DDGS) a D50 particle size of less than 500 micrometers (pm).

11. The method as claimed in claim 1, wherein the protein has a purity of at least 82% by weight.

12. A protein composition obtained by the method as claimed in any one of claims 1-10, characterized by: at least 82% protein by weight on a dry basis, NSI at least 88-90% at pH 7, tannic acid equivalent at or below 1-10 ppb by HPLC-UV with gallate calibration, free gallic acid at or below 0.2-0.5 ppm.

13. A protein composition derived from distillers’ dried grains with solubles (DDGS) comprising at least 82% protein by weight on a dry basis, having a nitrogen solubility index (NSI) of at least 88% at pH 7, a tannic acid equivalent not exceeding 10 parts per billion as quantified by HPLC-UV using gallic acid calibration, and free gallic acid not exceeding 0.5 parts per million.

14. The protein composition as claimed in claim 13, wherein the protein content is at least 85%, the NSI is at least 90% at pH 7, and the tannic acid equivalent is not more than 5 parts per billion as quantified by HPLC-UV using gallate calibration.

15. The method as claimed in claim 5, wherein the hydrophobic resin is a styrene-divinylbenzene porous adsorbent operated at linear velocities of 1-5 bed volumes per hour.

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