WO2022155730A1

WO2022155730A1 - Process for refining plant protein

Info

Publication number: WO2022155730A1
Application number: PCT/CA2022/050064
Authority: WO
Inventors: Thavaratnam Vasanthan
Original assignee: Thavaratnam Vasanthan
Priority date: 2021-01-19
Filing date: 2022-01-18
Publication date: 2022-07-28

Abstract

A process for producing a refined protein product from a de-hulled grain is provided. The process includes the steps of milling the de-hulled grain to produce a flour; hydrolyzing non-protein biomolecules in the flour to produce a hydrolyzed flour thereby reducing intermolecular interactions between the protein and non-protein biomolecules and among the non-protein biomolecules; removing fiber from the hydrolyzed flour to provide fiber-depleted flour; and removing starch from the fiber-depleted flour to produce the refined protein and starch products.

Description

Process for Refining Plant Protein

FIELD

[0001] The invention relates to processes for isolating proteins and other products from grains. Such proteins are used in food, pet food and animal and fish feed formulations for protein enrichment and functional properties and applications.

BACKGROUND

[0002] The plant-based protein refining industry is experiencing significant exponential global growth due to fast growing consumer interest in plant protein enriched foods with a majority of the growth in North America. This is primarily due to the desire among the general population, especially in the developed nations, for clean labels, ease of digestion, the need or desire to avoid allergens, compatibility with vegetarian and vegan lifestyles and concerns about the sustainability of animal protein production. In addition, the human health benefits of consuming plant proteins, and the negative health impacts of excessive red meat consumption as well as the benefits to the environment resulting from becoming less reliant on animal-based proteins has been highlighted in many media reports. In the recent past, in response to these consumers driven trends, a wide range of food products enriched in plant proteins is emerging from the food and beverage industry.

[0003] Seeds and grains from pulses, beans, cereals and oilseeds are a good source of protein and the content ranges between 10-45% (dry basis). Such grains provide a great opportunity to refine proteins for different food and industrial applications. Grains from soy and wheat are being used by the industry to refine proteins with target functionalities, while concerns over phytoestrogen content and GMO status of soy as well as gluten content of wheat are growing.

[0004] Proteins from other sources, especially those from non-GMO plant sources such as pulses/beans (field pea, faba bean, mung bean, chickpea, lentil, northern white bean, navy bean and black bean) are quickly gaining popularity. Protein concentrates from hemp, flax and rice are some other sources considered favorably by the supplement industries for applications related to human and pet nutrition. Plant proteins often lack one or more amino acids which are required to meet human dietary needs, and therefore, cereal-pulse complementary combinations and amino acid supplementation can help to overcome this shortcoming in vegetarian and vegan diets.

[0005] Protein concentrates and isolates processed from these plant sources are increasingly used in food formulations for protein enrichment as well as for their novel functional properties to manipulate the sensory and functional dynamics of food (such as texture, mouth-feel, gelling, emulsion stability and flavor profile). Ingredient technologies are being developed to address the lack of texture formation and negative “beany” flavor in raw pulse proteins that are considered challenges in food formulation. Meat analogs currently on the market are mainly based on soy protein concentrates or isolates that lack consumer desirability due to their GMO status, allergen concerns and off-flavors. Thus, pulse/bean proteins are quickly gaining popularity in the market (ex: pea protein) since they do not suffer from these drawbacks. The development and marketing of plant proteins from lentil and mung bean for use as egg replacements is also quickly growing.

[0006] There continues to be a need for improved processes of isolating plant based proteins from grain products.

SUMMARY

[0007] According to one embodiment of the technology, there is provided a process for producing a refined protein product from a de-hulled grain. The process includes: milling the de-hulled grain to produce a flour; hydrolyzing non-protein biomolecules in the flour to produce a hydrolyzed flour thereby reducing intermolecular interactions between proteins and the non-protein biomolecules and intermolecular interactions among non-protein biomolecules; removing fiber from the hydrolyzed flour to provide fiber-depleted flour; and removing starch from the fiber-depleted flour to produce the refined protein product.

[0008] The step of hydrolyzing the non-protein biomolecules may include addition of a solution comprising one or more non-protease hydrolases to the flour. The hydrolases may include at least one phytase and/or at least one carbohydrase. The carbohydrase may be xylanase. [0009] In some embodiments, the solution is prepared by mixing the hydrolases with water. The solution may include about 0.5 to about 2% (w/w, flour weight basis) of each of the non-protease hydrolases.

[0010] In some embodiments, the addition of the solution to the flour is performed while mixing the flour in a temperature-controlled blender or tank.

[0011] The step of removing the fiber from the flour may be performed by mixing the hydrolyzed flour with water or an aqueous ethanol solution to prepare a slurry, screening the slurry and recovering a filtrate containing a second fiber depleted flour fraction.

[0012] The step of screening the slurry may be performed with a pressure-fed or vibratory screening system.

[0013] In some embodiments, the process further comprises recovering a solid retentate fraction from the screening step by centrifuging, and subsequently drying the solid retentate fraction to produce a fiber concentrate. The process may further include recovering a solid portion of the filtrate by centrifuging the filtrate and subsequently drying the solid portion to produce a fiber-depleted flour with higher protein content than the hydrolyzed flour.

[0014] In some embodiments, the aqueous ethanol solution is between about 40% to about 95% ethanol.

[0015] In some embodiments, the process further comprises recovering and recycling aqueous ethanol obtained from the centrifuging and/or drying steps.

[0016] The step of removing starch from the fiber-depleted flour may include processing the fiber-depleted flour in an air classifier, thereby producing a first protein concentrate or isolate and a first starch concentrate or isolate.

[0017] In some embodiments, the process further comprises milling the fiber-depleted flour prior to processing the fiber-depleted flour in the air classifier. The step of milling the fiber-depleted flour may be performed by pin-milling. [0018] In some embodiments, the process further comprises milling the first starch concentrate or isolate to produce a milled starch concentrate and processing the milled starch concentrate in the air classifier, thereby providing a second starch concentrate or isolate and a second protein concentrate or isolate. In these embodiments, the step of milling the starch concentrate may be performed by pin-milling.

[0019] In some embodiments, the step of milling the dehulled grain may be performed by hammer milling, pin milling, or roller milling.

[0020] The technology described herein provides a protein concentrate product produced by embodiments of the process described herein, which has greater than 60% (w/w), greater than 70% (w/w), or greater than 80% (w/w) protein content.

[0021] The technology described herein provides a starch concentrate product produced by embodiments of the process described herein, having greater than 80% (w/w) or greater than 90% (w/w) starch content.

[0022] In some embodiments, the protein isolate or concentrate product has less than 0.6% (w/w) of total mass of a group of oligosaccharides comprising raffinose, stachyose and verbascose.

[0023] In some embodiments, the starch isolate or concentrate product has less than 0.6% (w/w) of total mass of a group of oligosaccharides comprising raffinose, stachyose and verbascose.

[0024] In some embodiments, the fiber concentrate product produced by embodiments of the process described herein has less than 0.6% (w/w) of total mass of a group of oligosaccharides comprising raffinose, stachyose and verbascose.

[0025] In some embodiments of the process, the raw material de-hulled grain is from a legume grain. In other embodiments, the raw material de-hulled grain is from field pea, faba bean, mung bean or chickpea. In other embodiments, the raw material de-hulled grain is from a cereal grain. In other embodiments, the raw material de-hulled grain is from corn, wheat, buckwheat, barley, rye, triticale, oat, sorghum or millet. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Various objects, features and advantages of the invention will be apparent from the following description of particular embodiments of the invention, in context of conventional processes, as illustrated in the accompanying drawings. Emphasis is placed upon illustrating the principles of various embodiments of the invention.

Figure 1 is an illustration of the cotyledon from yellow field pea (a legume/pulse grain) showing the close associations between protein bodies, starch, dietary fibre, phytates, minerals, and phenolic acids.

Figure 2 is a process flow diagram for a process of refining protein products from pulse grains by pin-milling and air-classification (PM-AC) technology.

Figure 3 is a process flow diagram for a process for refining protein products from pulse grains by conventional aqueous-alkali extraction technology.

Figure 4 is another process flow diagram for another process for refining protein products from pulse grains by conventional aqueous-salt extraction technology.

Figure 5 is a flow diagram for one embodiment of an inventive process for refining plant protein from pulse grains.

DETAILED DESCRIPTION

Introduction and Rationale

[0027] Challenges in Refining Plant Proteins from Grains - Technologies currently available for refining plant proteins are not cost efficient. The existing protein manufacturing capacity is inadequate to meet demand and most of the manufacturing technologies are complicated by having too many steps or unit operations, as well as being expensive to assemble and costly to operate as a result of excessive water usage, water recycling and drying of final products. In addition, these technologies are not effective at producing good protein yields. Typical protein recovery from such processes can be as low as 55% of the total protein in the parent material (de-hulled whole grains). [0028] A variety of dry and wet processing technologies for refining plant proteins have been developed and currently are being used by the grain processing industry. Dry processing technologies such as “milling and sieving” or “milling and air-classification” are relatively robust and cost efficient, but result in low purity protein concentrates (less than 58%, dry basis) with inferior functional properties due to contaminants such as dietary fiber, ash and minerals in the protein concentrates. As used herein, the term “dry processing” refers to the use of processing steps which do not include the use of water or other solvents. Wet processing technologies yield protein isolates with greater purity (greater than 80%, dry basis) and better functional properties (if proteins remain undenatured by the conditions applied during processing). As used herein, the term “wet processing” refers to the use of processing steps which include the use of water or other solvents such as ethanol. However, there are several challenges that increase the cost of production and consequently limit the wider usage of refined protein isolates in food and industries. As used herein, the noun “isolate” refers to a product of relatively higher purity than a “concentrate.” As used herein, the noun “concentrate” refers to a product of a relatively lower purity than an “isolate.”

[0029] The shortcomings in the wet processing technologies are primarily attributed to a lack of robustness and poor protein recovery due to fiber hydration and consequent requirement for high volume of waters at commercial scale; a lack of process cost efficiency due to multiple processing steps such as high shear mixing, centrifugation, membrane filtration and spray drying involving high volume water usage; a large capital cost for equipment setup; alkaline and acid chemical usage to improve protein recovery that alters protein functionality and prevents “clean label” applications that have less environmental impact; inferior quality of refined protein isolates due to partial or complete denaturation of proteins and loss of functionality as well as altered sensory properties (flavor, color, etc.) attributed to the impact of heat or alkaline and chemical usage during processing; and the presence of significant amounts of sodium (due to alkaline and acid chemical use) in the final product (protein isolate). Thus, improvements in processes for isolating plant proteins which address these challenges are needed.

[0030] Legume pulse/bean grains such as yellow peas, faba bean, mung bean, and chick pea, are rich sources of nutritive and functional proteins (23-30%, dry weight basis). Although albumins (water soluble) and globulins (salt-water soluble) are the two dominant (>70%, w/w) types of protein in pulse grains, they exist as seed organelles known as protein bodies, which are used to sustain growth during germination. General relative volumes of protein bodies in a cotyledon cell are illustrated in Figure 1. The proportions between albumins and globulins differ among different sources. Furthermore, these proteins exist in the cotyledon of the pulse grain in tight association with other grain components such as dietary fiber, starch, phytic acids and phytates, ash/minerals, phenolics, and other compounds, as illustrated in Figure 1. The phytates, although a minor component of pulse grains (approximately 2%, dry basis), mostly exist in association with dietary fibre, protein and minerals. Phytates can chelate minerals and ash which include divalent and monovalent cations and can form cross-linkages with grain components such as dietary fibre and protein molecules. These associations present challenges in refining pulse protein to its highest purity. The composition as well as the extent of associations among grain components differ with plant source. Therefore, one single protein refining approach or technology cannot be used to quantitatively and cost efficiently concentrate or isolate proteins from different pulse grains. Therefore, an improved new technological approach is required to address the abovementioned innate molecular associations between grain biomolecules. The inventor has recognized that a range of non-protease hydrolase enzymes such as phytases and carbohydrases are now commercially available and can be used to disentangle these associations between grain components.

[0031] Problems with Dry Processing Technologies - Pin-milling (i.e. fine grinding) and air-classification (PM-AC) are conventional dry processing technologies (Figure 2) that are used in processes to produce protein concentrates from de-hulled/hull free pulse grains (also called as pulse groats or whole grains) such as field pea and faba beans. These protein concentrates are obtained at purity levels that range between 50-58% and at 33- 35% yield based on the whole grain flour weight. As used herein the term “whole grain” refers to the hull-free kernels of various pulse grains such as pea, faba, mung bean and chickpea. However, the challenges attributed to this technology (PM-AC) have been overlooked for years and must be addressed. The fine grinding of the raw whole pulse grains (hull-free) by pin milling is unavoidably required in order to break and pulverize the grain tissue to discretely separate grain components one from each other at micron scales. Thus, during air-classification, the components such as protein (low density) and starch (high density) separate into different fractions/streams. However, it is important to note that the finely ground fiber co-concentrates with protein during air-classification, which not only dilutes the protein concentration, but also negatively impact the functionality of protein concentrates. An analysis performed by the inventor has indicated that the total dietary fiber content of the pulse protein concentrates produced by PM-AC technology usually ranges between 19-25%, dry basis. Thus, the inventor arrived at the recognition that upstream removal of dietary fiber and minerals from whole grain pulse flour, prior to PM- AC, would yield better protein products.

[0032] Problems with Wet Processing Technologies - Conventional wet/water- based technologies such as those illustrated in process flow diagrams of Figures 3 and 4 for refining pulse proteins generally involve addition of relatively harsh chemical compounds in order to maximize protein recovery. In these technologies, pulse seeds/grains are initially wet-ground in water adjusted to higher pH levels >8, by adding chemicals/alkaline salts such as sodium hydroxide (NaOH), sodium carbonate (Na₂CC>3), and/or calcium hydroxide (Figure 3). Aqueous alkaline media can quantitatively solubilize proteins. The solubilized protein is subsequently separated by decanter centrifugation into the alkaline water (i.e. supernatant) and then recovered by iso-electric precipitation, usually at pH between 3.5 to 5, by adding mineral acids such as hydrochloric acid (HCI). This chemical processing is not only unsuitable for producing “clean label” proteins, but also not environmentally friendly due to the large amount of water used (the requirement for multiple washing steps to remove chemicals/salts raises sustainability concerns), the high cost of drying of purified protein and a very significant effluent treatment cost (i.e. water recycling). Furthermore, at the end of the processing, the protein slurry must be neutralized prior to drying (usually spray drying). This neutralization step results in significant amounts of sodium or calcium salts being added to the final product, and requires washing with water.

[0033] Water or salt water-based extraction technologies, which are considered as clean label processing, (Figure 4) are also used for pulse protein refining because >70% of the pulse or bean proteins belong to the Osborne protein types albumin (water soluble proteins) and globulin (salt soluble proteins). Once solubilized and separated into a salt solution, the quantitative recovery of protein from the solution is achieved by isoelectric precipitation or the removal of salt by membrane filtration/dialysis and subsequent spray drying of the protein slurry (Figure 4). Laboratory and pilot trials on protein isolate production technologies, comparing alkali versus salt protocols (simulating commercial processing conditions), from whole field pea and faba bean flours, resulted in a protein recovery efficiency (original grain weight basis) as low as 55%. Although salt-based refining is preferred by the industry due to its “clean label” nature, improving the cost efficiency of this technology is important to ensure sustainability of this process.

[0034] Whole grain pulse/beans (de-hulled) are commonly used as raw materials in water based processing technologies, such as the processes shown in Figures 3 and 4, production of protein isolates. Since whole grain pulses are composed of 25-30% protein and 70-75% non-protein components, a significant amount of non-protein material is unnecessarily carried through all the unit operations of wet processing technology. This leads to excessive water requirement for preparing a slurry of the raw-material, an increased requirement for equipment with a greater capital cost to handle bulk quantities, and a greater energy cost at each unit operation due to bulk mixing, centrifugation, dialysis, product drying and effluent handling. This unnecessarily increases the cost of production and compromises the cost efficiency of the process. Therefore, an improved approach for protein refining is warranted.

[0035] One of the inventor’s earlier laboratory and pilot-scale studies investigated using protein concentrates (produced by pin milling and air classification of whole grain pulse flour) as raw material for water based refining of pulse protein isolates through aqueous alkali technology (Figure 3) or aqueous salt water technology (Figure 4). It was found that this approach to protein refining was technically difficult as a result of the viscosity/thickness of the slurry attributed to the high content of fine particulates of dietary fibre (>19%) present as contaminants in the protein concentrate and the large hydration/water binding capacity of the same fine fiber. The viscosity significantly increases over the processing time course. High viscosity impairs the efficiency of each downstream unit operation which includes mixing, screening, and other steps. Furthermore, the thick slurry demands a high degree of dilution with water, resulting in excessive water usage and requires high centrifugation speeds (large g-forces) that cannot be reliably achieved using commercial decanter centrifuges. Even with high dilution it is not feasible to produce protein isolate (>80% purity) at larger scale, because significant amounts of fine fiber particulates co-concentrate with the protein during centrifugation. Thus, reaching concentrations greater than 80% purity was impossible.

[0036] The inventor of the present technology has recognized the aforementioned shortcomings and difficulties in pulse protein refining by conventional processes, and developed a new processing approach. On embodiment of this new approach is illustrated in Figure 5. This process refines protein products (concentrates and isolates) from pulse grains with upstream removal of dietary fibre and minerals from whole grain pulse flour using a combination of hydrolysis of non-proteinaceous biomolecules and aqueous ethanol washing steps to generate a fibre depleted flour (FDF), followed by the removal of starch from the fiber depleted flour by pin-milling and air classification.

[0037] Production of Protein Isolate from Grains - Results of application of the new technology for production of protein isolates from pulse grains will now be described with respect to a flow diagram shown in Figure 5 and Tables 1-4 which provide protein refinement data for processing of yellow field peas, faba beans, mung beans and chickpeas. Other grains suitable for such processing may include, but are not limited to: legume grains, cereal grains, corn, wheat, buckwheat, barley, rye, triticale, oat, sorghum and millet.

[0038] Protein recovery percentages presented in Tables 1-4 are adjusted proportionally for the total material loss encountered during processing. In the tables below, the data represent means of triplicate determinations. “FDF” refers to fiber-depleted flour. “PC” refers to protein concentrate. “PM-AC” refers to pin-milling and air classification. The protein refinement data sets generated for yellow field peas, faba beans, mung beans and chickpeas are shown in Tables 1-4.

[0039] In Figure 5 and the ensuing description of related examples, emphasis is placed on highlighting various aspects of the technology. A number of possible alternative features are introduced during the course of the description of the general process outlined in Figure 5. It is to be understood that, according to the knowledge and judgment of persons skilled in the art, such alternative features may be substituted in various combinations to arrive at different embodiments. [0040] In Figure 5, the starting material for the process is de-hulled pulse grain which may be produced by a conventional dehulling step using a machine known as a huller. The dehulled pulse grain is subjected to milling using a milling technique such as hammer milling, fluidized particle milling, pin milling, or roller milling.

[0041] Application of Roller Milling - In the current example, the dry milling of the de-hulled pulse grain was performed using a roller mill, which comminutes materials without too much damage to the fibers. A typical roller mill is a type of mill consists of two rotating steel rollers with corrugated or smooth surface. The rollers are placed parallel to each other with a small clearance. The substance to be homogenized/milled is fed into the clearance space between the rollers while rotated at low to medium speeds.

[0042] Application of Hammer Milling - In alternative embodiments of the inventive process, the dry milling of the de-hulled grain may be performed using a hammer mill. A hammer mill is essentially a steel drum containing a vertical or horizontal rotating shaft or drum on which hammers are mounted. The hammers are free to swing on the ends of the cross, or fixed to the central rotor. The rotor is spun at a high speed inside the drum while material is fed into a feed hopper. The material is impacted by the hammer bars and is thereby shredded and expelled through screens in the drum of a selected size. The hammer mill can be used as a primary, secondary, or tertiary crusher.

[0043] Fluidized Particle Milling - As another alternative to roller milling, a dry milling technique known as “fluidized particle milling” could be included in embodiments of the process. Fluidized particle milling provides reduced particle sizes while loosening the associations among grain components (starch, protein, fiber, etc.), without extensively reducing the dietary fiber into undesirable finer particles. In fluidized particle milling, the pulverizing action of a rotor mill is supplied by a rotor which spins at high speed. This rotor is supported by heavy duty bearings which are located at either end of the shaft. This provides the stability necessary for greater material loading while also extending bearing life. The bearings are out of the grinding chamber and are protected from contamination. The rotor includes top and bottom sections. The bottom section includes a fan which provides air flow for the grinding system. In addition, the fan helps to accelerate and distribute the feed material prior to the material entering the grinding chamber. The top section is the grinding part of the rotor mill. It consists of a number of rows containing grinding plates which accelerate the air causing it to react with the grooved lining of the rotor mill. This interaction creates miniature pockets of rotating air at very high velocities. This air stream causes the particles to collide with each other and disintegrate while the heat caused by the size reduction is instantly absorbed by the rapidly moving air stream. An optional dynamic air classifier can be added. Finely ground material will pass through the classifier blades to collection while larger particles will be flung outward by centrifugal force into an adjustable recycle port for regrinding. The classifier speed may be changed to control the size particles that are rejected.

[0044] Fine milling of a wide variety of materials can be accomplished by adjusting the grinding plates, the style of grinding plates, and air flow to permit the fine milling of a wide variety of materials at high production rates without the temperature rise normally associated with the grinding of fine powders. Many heat sensitive materials can be milled without cryogenic processing with a separate variable speed drive. Rotor mills can be constructed in carbon or stainless steel. Interiors can be furnished with hardened material for extended life, for grinding abrasive materials.

[0045] Application of Pin Milling - In alternative embodiments, the dry milling of the dehulled grain is performed using a pin mill, which comminutes materials by the action of pins that repeatedly move past each other, to break up substances through repeated impact. A typical pin mill is a type of vertical shaft impactor mill and consists of two rotating discs with pins embedded on one face. The discs are arrayed parallel to each other so that the pins of one disk face those of the other. The substance to be homogenized is fed into the space between the disks and either one or both disks are rotated at high speeds.

[0046] Treatment of the Parent Flour with Hydrolases - In the present example, the parent flour was treated with the non-protease hydrolases phytase and xylanase. A phytase (phytic acid is myo-inositol hexakisphosphate phosphohydrolase) is any type of phosphatase enzyme that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) - an indigestible, organic form of phosphorus that is found in many plant tissues, especially in grains and oil seeds. Xylanase is any member of a class of enzymes that degrades the linear polysaccharide xylan into xylose or branched arabinoxylan into oligosaccharides, thus breaking down hemicellulose, one of the major components of plant cell walls. In the present example, phytase and xylanase (each enzyme 0.5%, w/w, flour dry matter basis) were dissolved in water (enzyme : water, 1 :100 w/w), and the water-enzyme mixture was gradually sprayed onto the flour (the ratio between flour : water-enzyme mixture was 1 :1 , w/v) while thoroughly blending flour in a jacketed ribbon blender to provide temperature control of the flour. A ribbon blender includes a U-shaped horizontal trough and a ribbon agitator formed of a set of inner and outer helical agitators, wherein the outer ribbon moves materials in one direction and the inner ribbon moves the materials in the opposite direction. In alternative embodiments, an alternative blender such as a paddle blender is used instead of a ribbon blender. A paddle blender includes a U-shaped horizontal trough and a fabricated paddle agitator. The paddle agitator includes multiple paddles positioned to move materials in opposing lateral directions and radially. The paddle design is normally applied where friable materials are being blended and when batches as small as 15% of the total capacity are going to be mixed in one blender. It is predicted that using an enzyme solution having a range of about 0.5% to about 2.0% (w/w) of each enzyme will be effective at generating sufficient hydrolase activity to free the desirable proteins from non-proteinaceous cellular components. In alternative embodiments, the ratio between flour : water-enzyme mixture can be greater than 1 :1 (w/v) to form an aqueous slurry in order to enhance hydrolase activity. The mixing of the slurry can be performed in a jacketed tank to provide temperature control of the flour.

[0047] The mixture was then allowed to incubate at 50°C for 3 hours under continued mixing. The resulting mixture is herein designated as “hydrolyzed flour.” It is to be understood that in this context, the term “hydrolyzed” refers to treatment with the objective generally hydrolyzing biomolecules other than proteins to aid in release of proteins from other cellular components. The term should not be construed to mean that the flour is completely hydrolyzed into monomers of all biological polymers.

[0048] While the present example included phytase and xylanase, it is soundly predicted that addition of one or more additional non-protease hydrolases may also be useful to include in the same step in alternative embodiments. As used herein the term “nonprotease hydrolases” refers to any hydrolase which does not catalyze hydrolysis of protein. Such non-protease hydrolases may include, but are not limited to carbohydrase, lipase, mannanase, glucanase, cellulase, exoglucanase, laccase, amylase, galactosidase, galacturonase, and glucosidase. It is believed that these additional nonprotease hydrolases may also help to free proteins from other non-proteinaceous biomolecules, thereby contributing to the objective of enhancing refinement of protein.

[0049] Wet Processing with Ethanol Solution - At the end of the incubation period, the hydrolyzed flour mixture was emptied into a stainless steel processing tank containing 95% ethanol. The volume of ethanol is 2-fold greater than of the amount of water used to mix/slurry the enzymes. The resulting slurry of flour was homogenized thoroughly for 10 mins, and then a volume of water was added to the tank in order to adjust the ethanol concentration to 50% (v/v). The homogenization was continued until a smooth slurry was achieved. The slurry of flour was then subjected to screening through a 75 pm screen using a pressure-fed screening system. Pressure screens are used primarily to separate unwanted debris or contaminants from the desirable fibers in papermaking pulp and are also useful for the inventive process described herein. A typical pressure screen consists of a vertical cylindrical housing that contains a perforated cylindrical screen basket. The solids remaining on the screen, referred to as “retentate” were collected, re-slurried with 50% ethanol (1 : 1 (w/v), based on the starting flour weight), homogenized and rescreened to ensure satisfactory filtration efficiency. The “retentate” was then collected and dried. This sample, predominately containing fibre, is referred to as “fibre concentrate.” At the second screening step, use of an inline grinder as a homogenizer may further improve fiber separation efficiency. The filtrates from both screenings were pooled and decanter centrifuged in order to recover the solids which are significantly depleted in dietary fibre, but enriched in starch and protein. The solids were subsequently dried to moisture levels below ~8%, w/w. The used ethanol can be efficiently recovered and recycled. The dried “fiber-depleted flour” (FDF) was then pin-milled (Hosokawa 250 CW Pin-mill; door pin speed, 5000 rpm and house pin speed, 10000 rpm) into fine powder and air-classified (Hosokawa ATP 200 air-classifier; classifier wheel speed 5000 rpm) into two fractions namely starch concentrate (SC) and protein concentrate, (PC). The starch concentrate from the first air-classification was re-milled and re-air classified to obtain two new fractions as shown in Figure 5.

[0050] Application of Air Classification - Certain embodiments of the process described herein employ air classification to remove starch from fiber-depleted flour. An air classifier is an industrial machine which separates materials by a combination of size, shape, and density. An air classifier operates with injection of the material stream to be sorted into a chamber which contains a column of rising air in cyclonic motion. Inside the separation chamber, air drag on the objects supplies an upward force which counteracts the force of gravity and lifts the material to be sorted up into the air. Due to the dependence of air drag on object size and shape, the objects in the moving air column are sorted vertically and can be separated in this manner. Air classifiers are commonly employed in many different types of industrial processes where a large volume of mixed materials with differing physical characteristics need to be separated quickly and efficiently. Repeated pin-milling and air-classification improves recovery of target components such as protein. An airclassification facility usually has three pieces of equipment connected in series i.e. a flour feeder connected to an air-classifier that is connected to a pneumatic flour collection system with a blower fan. The main component of an air-classifier unit is a chamber that contains an “air-classifier wheel” (cylindrical in shape with vanes on the surface placed at an angle) that can rotate at a very high speed. The blower fan of the pneumatic flour collection unit pulls air into the air-classifier through an inlet. The air stream goes up in a cyclonic fashion, and flows through the vanes of the fast rotating cylindrical wheel and the flour collection unit, and finally exits through the blower fan. Flour is usually injected into the airstream closer to the air inlet of the air-classifier. When the air stream carrying the flour particulates hits the fast rotating vane surfaces, separation of flour particulates into light-out and heavy-out fractions is achieved. The composition of these fractions usually varies in relation to the flour. The flour feed rate, speed of the rotating air-classifier wheel (centrifugal/centripetal force) and the air-flow speed/rate through the vanes (drag force) helps to achieve the separation of finely milled flour particulates based on the differences in their density, mass and projected area in the direction of air flow. Pin-milling and airclassification, as a dry process, is relatively energy efficient and has a lower capital cost when compared to wet processes.

[0051] Analysis of Protein Refinement - Protein refinement was investigated for four different grains: yellow field peas, faba beans, mung beans and chickpeas. Results are listed in Tables 1 to 4 below. Table 1 : Refinement of Protein from Yellow Field Peas

Abbreviations: FDF - fiber depleted flour; PC - protein concentrate; PM- AC - pin-milling and air classification. [0052] In experiments using yellow field peas, four different flour washing steps were investigated (data in Table 1). It was found that ethanol washing of the hydrolyzed flour to produce sample FDF4 provides the best protein recovery (86.6%) as well as minimal ash, fat and total dietary fibre. Subsequent pin milling and air classification of FDF4 to yield PC9 provides a total protein recovery of 54.6%, similar to pin-milling and air classification of parent flour, but with significant reduction in moisture, ash, fat and TDF. The subsequent step 8 of Figure 5 may be performed to recover additional protein from the starch concentrate generated in step 7. The second pool of protein concentrate generated in step 8 (PC10) has significantly lower levels of ash and TDF. The fiber concentrates recovered from the slurry screening in step 4 had TDF contents that ranged from 45-53% (w/w), whereas the starch concentrates/isolates recovered from the air-classification steps-7 and 8 had starch content that ranged from 80-92% (w/w).

Table 2: Refinement of Protein from Faba Beans

Abbreviations: FDF - fiber depleted flour; PC - protein concentrate; PM- AC - pin-milling and air classification. [0053] In a set of experiments using faba beans as the protein source (data in Table 2), ethanol washing (steps 1 to 6) of Figure 5 combined with air classification in step 7, and subsequent steps to step 8 were performed, as well as processing of the parent flour by air classification, represented by steps 1 and 7, as well as steps 1 , 7 and 8. The best individual recovery of protein was obtained for sample PC9 (48.6%). Furthermore, ash, fat and total dietary fiber (TDF) were reduced significantly in sample PC9 relative to percentages of these components in samples PC1 and PC2. The second air classification step performed on the FDF4 sample led to additional recovery of protein with similar percentages of ash, fat and TDF in sample PC10. Therefore, the processes including steps 1 to 7 and including steps 1 to 8 are established as more effective than conventional air classification alone in refining protein from parent flour of faba beans. The fiber concentrate recovered from the slurry screening in step 4 had a TDF content of 54% (w/w), whereas the starch concentrates/isolates recovered from the air-classification steps-7 and 8 had starch contents that ranged from 80-91 % (w/w).

Table 3: Refinement of Protein from Mung Beans

Abbreviations: FDF - fiber depleted flour; PC - protein concentrate; PM- AC - pin-milling and air classification. [0054] In a set of experiments using mung beans as the protein source (data in Table 3), ethanol washing (steps 1 to 6) of Figure 5 combined with air classification in step 7, and subsequent steps to step 8 were performed, as well as processing of the parent flour by air classification, represented by steps 1 and 7, as well as steps 1 , 7 and 8. The best individual recovery of protein was obtained for sample PC1 (57.8%). However, sample PC9 had good protein recovery as well (50.8%) in addition to having ash, fat and total dietary fiber (TDF) reduced significantly relative to percentages of these components in samples PC1 and PC2. The second air classification step performed on the FDF4 sample led to additional recovery of protein with similar percentages of ash, fat and TDF in sample PC10. Therefore, the processes including steps 1 to 7 and including steps 1 to 8 are established as more effective than conventional air classification alone in refining protein from parent flour of mung beans. The fiber concentrate recovered from the slurry screening of step 4 had a TDF content of 50% (w/w), whereas the starch concentrates/isolates recovered from the air-classification steps 7 and 8 had starch contents that ranged from 80-93% (w/w).

Table 4: Refinement of Protein from Chickpeas

Abbreviations: FDF - fiber depleted flour; PC - protein concentrate; PM-AC - pin-milling and air classification. * Processing was not feasible due to high fat content and viscosity (stickiness) of the parent flour

[0055] In a set of experiments using chickpeas as the protein source (data in Table 4), ethanol washing (steps 1 to 6) of Figure 5 combined with air classification in step 7, and subsequent steps to step 8 were performed, as well as processing of the parent flour by air classification, represented by steps 1 and 7, as well as steps 1, 7 and 8. The best individual recovery of protein was obtained for sample PC9 (56%) in addition to having ash, fat and total dietary fiber (TDF) reduced significantly. The second air classification step performed on the FDF4 sample led to additional recovery of protein with similar percentages of ash and fat and an improved reduction of TDF in sample PC10. Air classification of the parent flour of chickpeas was deemed to be not feasible. Therefore, the processes including steps 1 to 7 and including steps 1 to 8 succeeded in protein refinement whereas performing conventional air classification alone parent flour of chickpeas was deemed a failure. The fiber concentrate recovered from the slurry screening of step 4 had a TDF content of 52% (w/w), whereas the starch concentrates/isolates recovered from the air-classification steps had starch contents that ranged from 80-91% (w/w).

[0056] Certain carbohydrates are not efficiently digested by humans and undergo fermentation which produces gases that can cause flatulence. Examples of such carbohydrates include, but are not limited to raffinose, stachyose and verbascose. These carbohydrates were measured in all four parent whole grain pulse flours of the present investigation, and found to be in the range between 2-6%, w/w. The process outlined in Figure 5 significantly reduces this content to <0.6%, w/w in all protein and starch concentrates and isolates generated. This is a desirable outcome in producing a more easily digested protein product.

[0057] An in house sensory testing with 9 subjects were performed on all the protein and starch concentrates and isolates. The testing indicated that all the protein and starch concentrates and isolates were brighter in appearance and generally bland tasting whereas other protein concentrates and isolates tend to have unpleasant taste. This is another desirable outcome in producing a protein product without an unpleasant taste. Equivalents and Scope

[0058] Other than described herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and current rate, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0059] Any patent, publication, internet site, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0061] While the technology been particularly shown and described with references to embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the technology encompassed by the appended claims.

[0062] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.

[0063] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed. Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the technology, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where the term “about” is used, it is understood to reflect +/- 10% of the recited value. In addition, it is to be understood that any particular embodiment of the present technology that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein

Claims

1. A process for producing a refined protein product from a de-hulled grain, the process comprising: milling the de-hulled grain to produce a flour; hydrolyzing non-protein biomolecules in the flour to produce a hydrolyzed flour thereby reducing intermolecular interactions between proteins and the non-protein biomolecules and intermolecular interactions among non-protein biomolecules; removing fiber from the hydrolyzed flour to provide fiber-depleted flour; and removing starch from the fiber-depleted flour to produce the refined protein product.

2. The process of claim 1, wherein the step of hydrolyzing the non-protein biomolecules comprises addition of a solution comprising one or more non-protease hydrolases to the flour.

3. The process of claim 2, wherein the hydrolases include at least one phytase.

4. The process of claim 2 or 3, wherein the hydrolases include at least one carbohydrase.

5. The process of claim 4, wherein the carbohydrase is xylanase.

6. The process of any one of claims 2 to 5, wherein the solution is prepared by mixing the hydrolases with water.

7. The process of any one of claims 2 to 6, wherein the solution comprises about 0.5 to about 2% (w/w, flour weight basis) of each of the non-protease hydrolases.

8. The process of any one of claims 2 to 7, wherein the addition of the solution to the flour is performed while mixing the flour in a temperature-controlled blender or tank.

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9. The process of claim 1, wherein the step of removing the fiber from the flour is performed by mixing the hydrolyzed flour with water or an aqueous ethanol solution to prepare a slurry, screening the slurry and recovering a filtrate containing a second fiber depleted flour fraction.

10. The process of claim 9, wherein the step of screening the slurry is performed with a vibratory or pressure-fed screening system.

11. The process of claim 9 or 10, further comprising recovering a solid retentate fraction from the screening step by centrifuging, and subsequently drying the solid retentate fraction to produce a fiber concentrate.

12. The process of claim 10, further comprising recovering a solid portion of the filtrate by centrifuging the filtrate and subsequently drying the solid portion to produce a fiber- depleted flour with higher protein content than the hydrolyzed flour.

13. The process of claim 10, wherein the aqueous ethanol solution is between about 40% to about 95% ethanol.

14. The process of any one of claims 11 to 13, further comprising recovering and recycling aqueous ethanol obtained from the centrifuging and/or drying steps.

15. The process of any one of claims 1 to 14, wherein the step of removing starch from the fiber-depleted flour comprises processing the fiber-depleted flour in an air classifier, thereby producing a first protein concentrate or isolate and a first starch concentrate or isolate.

16. The process of claim 15, further comprising milling the fiber-depleted flour prior to processing the fiber-depleted flour in the air classifier.

17. The process of claim 16, wherein the step of milling the fiber-depleted flour is performed by pin-milling.

18. The process of any one of claims 15 to 17, further comprising milling the first starch concentrate or isolate to produce a milled starch concentrate and processing the milled starch concentrate in the air classifier, thereby providing a second starch concentrate or isolate and a second protein concentrate or isolate.

19. The process of claim 18, wherein the step of milling the starch concentrate is performed by pin-milling.

20. The process of any one of claims 1 to 19, wherein the step of milling the dehulled grain is performed by hammer milling, pin milling, or roller milling.

21. A protein concentrate product produced by the process of any one of claims 15 to 19, having greater than 60% (w/w) protein content.

22. A protein concentrate product produced by the process of any one of claims 15 to 19, having greater than 70% (w/w) protein content.

23. A protein isolate product produced by the process of any one of claims 15 to 19, having greater than 80% (w/w) protein content.

24. A starch concentrate product produced by the process of any one of claims 15 to 19, having greater than 80% (w/w) starch content.

25. A starch isolate product produced by the process of any one of claims 15 to 19, having greater than 90% (w/w) starch content.

26. A protein isolate or concentrate product of any one of claims 21 to 23 having less than 0.6% (w/w) of total mass of a group of oligosaccharides comprising raffinose, stachyose and verbascose.

27. A starch isolate or concentrate product of claim 24 or 25 having less than 0.6% (w/w) of total mass of a group of oligosaccharides comprising raffinose, stachyose and verbascose.

28. A fiber concentrate product produced by the process of claim 11 having less than 0.6% (w/w) of total mass of a group of oligosaccharides comprising raffinose, stachyose and verbascose. to 20, wherein the raw material de-hulled grain to 20, wherein the raw material de-hulled grainr chickpea. to 20, wherein the raw material de-hulled grain to 20, wherein the raw material de-hulled graine, triticale, oat, sorghum or millet.

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