Natural edestin protein isolate and its use as a texturizing ingredient

Abstract

Processes and products that solve problems related to isolating hemp protein, preparing raw material inputs, and processing raw material inputs to produce superior hemp protein meat and dairy analogs. The compositions and processes include processes for isolating hemp kernel protein, pasteurizing, forming liquid solutions, forming gels, texturizing, and producing meat and dairy analogs. The processes of the present disclosure result in structured protein foods or meat analogs with superior properties when compared to existing products or similar products produced using known technology.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 124,973, filed December 14, 2020, which is incorporated herein by reference.

[0002] The present disclosure relates to protein isolation and plant-based meat and dairy analogs, more particularly to plant-based products having the texture, appearance, and taste of meat or dairy products. The present disclosure also relates to compositions and methods for preparing liquid, gel, or solid products for use in producing meat and dairy analogs. [Background technology]

[0003] Hemp-based meat or dairy analogs produced using only hemp kernels as a protein source are not known to be commercially available or described in the food industry or food science literature. For food use, hemp protein is considered inferior to soy and pea proteins, particularly with respect to the properties required for the production of meat and dairy analogs. Meat and dairy analogs, also referred to herein as structured protein foods, require proteins capable of forming a strong gel matrix, and hemp protein has not been found to have a strong ability in that regard.

[0004] According to Wang, "the emulsifying and gel-forming properties of hemp proteins have generally been found to be inferior to soy proteins" (Wang et al., 2019). Malomo showed that salt micellization isolation of hemp proteins can improve their gel-forming ability, but Shen revealed that this complex, costly and time-consuming protein isolation method can have a negative effect on the protein structure, and chemical cross-linkers may be required to obtain sufficient gel-forming ability in hemp proteins (Shen et al., 2021;Malomo et al, 2014;Wang et al., 2019).

[0005] As indicated by Wang, currently soy protein is preferred over hemp protein for the production of meat analogues. "Currently, soy protein is mainly used to mimic animal proteins due to its favorable gelling properties and the resulting interlaced fibrous matrix" (Schreuders et al., 2019). The latter fiberization of soy occurs at a typical temperature of 130 °C (266 °F). For example, IMPOSSIBLE FOODS uses soy protein in its IMPOSSIBLE BURGER. However, BEYOND MEAT, IMPOSSIBLE FOODS' largest competitor, uses yellow pea protein in its BEYOND BURGER, mainly due to health and nutrition-related concerns about soy products. However, yellow pea "has much lower gelling ability than soy protein" and "the heat-induced gel of soy protein isolate (SPI) is stronger than that of pea protein isolate (PPI)" (Schreuders et al., 2019).

[0006] Soybean and pea proteins are known to have limitations in taste, texture, and phytochemicals as protein sources for meat and dairy analogue production, but no plant-based alternative protein has yet been found to be a successful replacement for meat analogues. However, hemp protein has been investigated as a potential replacement for soybean protein in meat analogues. Recently, Zahari reported that hemp protein has not yet been used in meat analogue production, even though it has been recognized for its excellent nutritional and functional properties. "Previous studies have shown that hemp seed protein, especially, has high protein quality and functionality. However, there have been no studies using hemp seed protein as a raw material for meat analogue production" (Zahari et al, 2020).

[0007] Zahari went on to demonstrate that hemp protein concentrate (HPC) can be used in combination with soy protein isolate (SPI) to produce meat analogs by traditional extrusion, but not as the sole protein source. The study concluded, "HPC may therefore be a promising novel ingredient for inclusion in extruded products, and the study shows that the resulting meat analogs give textures comparable to SPI alone and can replace up to 60% of soy protein with hemp protein" (Zahari et al, 2020). The study showed that using high concentrations of hemp protein in the formulation unacceptably reduced the hardness and chewiness of the meat analogs. Thus, Zahari, Wang and Shen teach against the use of hemp protein as the sole protein source in meat analog production.

[0008] Despite the need for new and improved plant protein sources to meet the growing demands of the plant-based food industry, hemp protein has yet to achieve significant market share in food manufacturing. Despite the nutritional and environmental benefits of hemp, soy and pea proteins continue to dominate the plant-based food market. Soy and pea proteins have benefited from decades of research and widespread commercial availability for use in the manufacture of meat analogs and other food products, which have significantly improved ingredient and product quality and provided cost benefits of scale.

[0009] Improvements in soy and pea based meat analogs have been made through extensive research and development at all stages of meat and dairy analog production. Meat analog production generally involves four steps. The first step involves protein isolation from the selected plant material. The second step involves combining the isolated protein with water and oil to form a matrix for thermal gelling or extrusion. The third step involves thermal gelling or extrusion of the raw materials to set and texture the protein. The final step involves the use of binding and water binding agents, such as carrageenan, cellulose fibers, starch, gluten, or flour, to form the meat analog, which is then cooked to mimic products such as burgers, filets, chicken strips, and pulled pork.

[0010] The first step in meat analogue production involves protein isolation from plant material. Traditionally, soybean and pea proteins are used as plant materials for protein isolation. However, hemp grain protein has excellent digestibility and desirable essential amino acid composition and is considered as a possible protein source for meat analogues (Tang, Ten, Wang, & Yang, 2006;Wang, Tang, Yang, & Gao, 2008;Russo and Reggiani, 2015a;Callaway, 2004;House et al., 2010;Docimo et al., 2014;Zahari et al., 2020). A recent proteomic characterization of hemp grain concluded that it is an underdeveloped non-legume plant and a protein-rich cereal (Aiello et al., 2016).

[0011] Although the nutritional potential of hemp proteins is high, the nutritional value of plant proteins, as measured by amino acid composition and digestibility, is influenced by many factors. Amino acid composition may be influenced by genotypic variability or agricultural conditions such as soil fertility and post-harvest treatments (e.g., dehulling) that alter the ratio of grain components. Protein digestibility may be influenced by the structure of the protein and the presence of anti-nutritional compounds in the plant material or formed during alkaline or high temperature treatments (Sarwar, 1997). However, Aiello found that anti-nutritional factors such as condensed tannins, phytic acid, and trypsin inhibitors are present in low concentrations in hemp grains (Aiello et al., 2016).

[0012] The functional characteristics of hemp protein have hindered its use as a food protein source. Hemp protein concentrate is commercially available as a result of hemp seed oil production. After hemp seeds are crushed and pressed to obtain profitable oil, they become a protein-rich seed cake. The seed cake is green in color, rich in fiber, and represents about 40% protein concentrate. Unfortunately, seed cake has a very grassy, ​​earthy flavor that is not tolerated in most foods. By crushing and dry sieving the cake, the protein content can be increased to about 50%. Recognizing the nutritional value of this protein-rich source, many researchers have isolated hemp protein and used it as a starting material to improve the taste and functional qualities of hemp protein. Tang found that hemp protein isolate (HPI) from seed cake was inferior to SPI for use in the creation of plant-based foods (Tang et al., 2006). Tang showed that HPI is considered to have poor emulsifying and water-retaining properties when compared to soy protein isolates due to the poor water solubility of hemp globulins. (Tang et al., 2006;Hadnadev et al. 2020). According to Tang, "The data suggest that HPI can be used as a valuable nutritional source for infants and children, but has poor functional properties when compared to SPI. The poor functional properties of HPI are mainly attributed to the formation of disulfide covalent bonds between individual proteins due to the high free sulfhydryl content from sulfur-containing amino acids and subsequent aggregation at neutral or acidic pH." (Tang et al., 2006). Furthermore, "Differential scanning calorimetry (DSC) analysis showed that HPI has only one endothermic peak with a denaturation temperature (T(d)) of approximately 95.0 °C, which is attributed to the edestin component." (Tang et al., 2006).

[0013] Despite the apparently inferior functional aspects of hemp proteins, their superior nutritional value has sparked continued interest in the use of hemp proteins in food production. To this end, individual proteins have been isolated from hemp and further studied for their potential functional properties. Furthermore, researchers have investigated whether the functionality of hemp proteins can be improved by different extraction and isolation methods. "The value and uses of hemp proteins in food are closely related to the structure and functional properties of the proteins" (Wang et al., 2019).

[0014] To investigate the nutritional and functional properties of individual hemp kernel proteins, researchers employed methods to extract and separate the two major proteins present in hemp kernels. Hemp kernel proteins are primarily composed of the proteins edestin and albumin. Edestin, a globulin, accounts for approximately 60%-80% of the total protein content (Odani & Odani 1998; Tang et al., 2006), while albumin, a globular protein but not a globulin, accounts for the difference. Edestin and albumin have different amino acid compositions and functional characteristics.

[0015] Malamo studied the nutritional differences between edestin and albumin in hemp kernels and concluded that the edestin fraction of hemp protein is nutritionally superior, containing more sulfur-containing amino acids (methionine and cysteine), aromatic amino acids (AAA), branched-chain amino acids, and hydrophobic amino acids (Malomo and Aluko 2015). Malamo separated edestin from albumin and measured the nutritional and functional characteristics of each. These characteristics include water solubility, amino acid content, and digestibility.

[0016] Malomo reported that the albumin fraction is soluble in water, whereas the edestin fraction is soluble in salt solutions. Extracted edestin has very low solubility in water at neutral or acidic pH and dissolves only at high ionic strength or alkaline pH (Malomo & Aluko, 2015). "Many of the functions of proteins, such as surface active properties, are correlated with the solubility of the protein" (Jackman & Yada, 1989;Malomo & Aluko, 2015). In hemp kernels, the solubility and foaming ability of albumin was found to be higher than that of edestin, although edestin has better emulsion forming ability (Malomo & Aluko, 2015).

[0017] Research has shown that edestin may only be found in hemp kernels, but edestin-like proteins may be present in kernels from the family including pumpkin and squash (Vickery, 1940). Thus, the present disclosure and its uses may relate to edestin and edestin-like proteins that may have similar or identical properties to edestin. Vickery disclosed that a potential substitute for edestin may be found in plants of the Cucurbitaceae family, including pumpkin seeds. Hirohata investigated the globulins of 38 varieties and species of eight genera in this family, noting the considerable similarity of globulins from closely related species (Vickery, 1940;hirotata, 1932). Vickery suggested that globulins of the Cucurbitaceae family may contain edestin-like proteins that meet the requirements of a nutritional substitute for hemp kernel edestin (Vickery 1940).

[0018] Edestin was first isolated and analyzed by Thomas Osborne (Osborne, 1892). In its complete native form, edestin is composed of six identical subunits, each of which is composed of an acidic (AS) subunit and a basic (BS) subunit linked by a single disulfide bond (Farinon 2020; Patel, Cudney, & McPherson 1994). Recently, it has been shown that edestin can exist in multiple forms within a single variety of hemp (Docimo et al., 2014). For example, in one variety of Cannabis Sativa, seven genes code for edestin globulin, which result in divergent forms of two edestin types. Within a given strain of hemp, one type of edestin is virtually identical to each other, while the second type of edestin is substantially different from the first. Ponzoni identified a type 3 edestin gene, CsEde3, which shows approximately 65% ​​and 58% sequence homology when compared to the genomic forms CsEde1 and CsEde2, respectively (Ponzoni, Brambilla, & Galasso, 2018). The amino acid composition can differ significantly between the two types of edestin, making one form more nutritional (Docimo et al., 2014).

[0019] Edestin itself has a large particle mass of 309,000, but when denatured, it depolymerizes to 51,000 in concentrated urea solution [Burk & Greenberg, 1930] and 17,000 in dilute hydrochloric acid [Adair & Adair, 1934]. These units are approximately 1 / 6 and 1 / 18 the size of the native molecule, respectively. In the native state, they have a particular polypeptide pattern, held together in part, probably by some form of chemical bond (e.g., S-S bonds), but mainly by lateral attractions between adjacent CO and NH groups, and by interactions between free acid and basic groups in the side chains. As can be seen from the following analytical data, the number of these latter groups is high: glutamic acid, 19-2%; aspartic acid, 10-2% [Jones & Moeller, 1928]; arginine, 17-76% [Vickery, 1940]; lysine, 2-4%; histidine, 2-03% [Tristram, 1939]; amide-N, 1-73% [Bailey, 1937, 2]. Taking into account the amidated COOH groups, these correspond to a total of 670 charged groups per 309,000 molecules. This spatial arrangement of charges results in a certain charge symmetry on which the stability of the molecule ultimately depends, which, within definite limits of pH, is capable of some variation, as reflected in the change in the dipole moment. Beyond these limits, the ionization of acid or basic groups is further inhibited, which leads to attractions and repulsions within the molecule, which distort and ultimately destroy the inherent polypeptide conformation, especially in the absence of small mobile ions. (Bailey, 1940).

[0020] Thus, edestin as referred to in this disclosure may incorporate all forms of edestin, currently known or currently unknown, that have similar or identical properties as the edestin disclosed for the purposes of this disclosure.

[0021] Edestin is rapidly degraded to edestane under mildly acidic conditions. Edestane, an intermediate product derived from edestin, occurs during the denaturation of edestin and was first identified by Osborne (Osborne, 1901; 1902). Edestane is formed when edestin comes into contact with dilute acid. Edestane liberates a sulfhydryl group (Bailey, 1942). Bailey demonstrated that edestin can be rapidly converted to edestane under acidic conditions in less than 20 minutes (Bailey, 1942). This study showed that the liberation of the sulfhydryl group occurs simultaneously with the conversion of edestin to edestane. Bailey also reported a reduced nitrogen content in edestane compared to edestin, which can be explained by the reduction of tryptophan in edestin. Edestin in its undenatured native state has different functional properties than denatured or partially denatured edestin or edestane.

[0022] Conventional techniques for isolating hemp proteins or separating edestin from albumin may cause structural changes in the protein, some of which may be irreversible. Various protein extraction and isolation techniques and conditions (pH, presence or absence of monovalent and multivalent salts, ionic strength of the medium used for protein extraction, time, temperature, etc.) can affect the functional properties of the protein (Hadnadev et al., 2018). These changes can have adverse effects on the function of the protein (Hadnadev et al., 2018;Shen et al., 2021). These adverse effects may include changes in digestibility, protein-oil interactions, taste, solubility, and emulsification and gel-forming ability (Shen et al., 2021). Therefore, it is very important to maintain the native structure of the protein as much as possible, especially when extracting and isolating edestin for food use.

[0023] A number of techniques have been used to separate hemp proteins and edestin. These techniques include the use of high temperatures, alkaline or acidic conditions on aqueous or solvent slurries, isoelectric precipitation, isoelectric focusing, micellization, ultrafiltration, and mechanical processes such as squeezing, grinding or sieving the kernel or dehulled kernel, or grinding the kernel and sieving the kernel slurry. Any of these techniques can alter the structure of the protein and reduce its function.

[0024] High temperatures generated by mechanical processes can adversely affect protein function. For example, grinding grains to produce flour can generate high temperatures that can alter the structure of proteins. These temperatures can potentially cause denaturation of edestin and binding or aggregation between edestin, albumin or fiber, thereby preventing their independent isolation.

[0025] Dry milling of grains generates temperatures of at least 80°C to 100°C, which can potentially denature edestin. Mohammad found that the heat and mechanical forces generated during milling can denature globular proteins (Mohommad, 2015). Mohommad showed that the bulk properties of globular proteins can be altered by mechanical stresses applied during milling.

[0026] Therefore, the high temperatures caused by dry milling to produce flour may alter the structure of hemp proteins. Farinon calculated the denaturation temperature of hemp grain protein (edestin) to be 92°C (Farinon et al., 2020). Furthermore, Malamo showed that heat treatment, as well as changes in pH, may alter the secondary structure of hemp grain albumin and edestin (Malomo and Aluko, 2015). High temperatures may cause proteins to unfold, thereby exposing hydrophobic groups and favoring protein-protein interactions over protein-water interactions.

[0027] By using chemical means of protein extraction, heating during extraction can be avoided or minimized, but many chemical extraction methods require first mechanical particle size reduction. Solvent extraction is a common method of isolating proteins from plant material and involves the use of a liquid solvent to which the protein-containing material is added. The solvent may be water, alcohol, acetone, hexane, or other liquid solvent. Solvent extraction can be combined with mechanical or other extraction means that first disrupt the plant material to release the protein. Solvent extraction may involve the use of a solvent that disrupts the plant's cell walls or fibrous material, thereby releasing the protein.

[0028] Some solvents used in protein extraction have the drawback of denaturing proteins. Moreover, these solvents can be toxic and are not suitable for consumption even in small amounts. Furthermore, the solvents generally require long extraction times, laborious procedures, and can leave residues in the food that can be difficult to dispose of safely. Hexane is an example of this type of solvent. Many solvents cannot be used in the production of certified organic foods, according to the United States Department of Agriculture (USDA) organic food labeling guidelines.

[0029] An alternative process for protein extraction that does not require a solvent is aqueous extraction, which involves adding crushed or pressed plant material to water followed by separation of proteins based on their solubility in the aqueous fraction or in a fat-containing fraction when the fat in the plant material is separated from the water. Aqueous extraction may be followed by isoelectric precipitation or isoelectric focusing or salt extraction to isolate proteins.

[0030] Alkaline extraction is a common technique in which highly basic solvents disrupt the cellular structure, thereby releasing proteins from the cells. However, this process can result in damage to proteins, such as racemization of amino acids, formation of lysinoalanine, reduced digestibility, and loss of essential amino acids (Moure et al., 2006). According to Xu, under alkaline conditions, polyphenols found in many plant materials, including hemp kernels, can oxidize and then react with proteins, resulting in a dark green or brown color in the extracted protein solution (Xu and Diosady, 2002).

[0031] When used during the extraction of hemp kernel proteins, the pH of the alkaline extraction is generally raised to 9 or 10, higher than that of legume protein extraction (pH 8), because native hemp kernel proteins are tightly packed and may be tightly integrated with other components, such as phenolic compounds (Wang and Xiong, 2019). Generally, the alkaline extraction is followed by precipitation of the target hemp proteins at isoelectric point, and after several washing steps, the induced color often cannot be removed from the protein isolate.

[0032] Generally, water extraction or alkaline extraction is followed by isoelectric precipitation or salt extraction to isolate proteins.Isoelectric precipitation may be used to extract soluble proteins after alkaline extraction or solvent extraction, and involves adjusting the pH until a charge equilibrium between the target protein and the solvent is reached, thereby precipitating the protein from the solution.Isoelectric precipitation requires a change in pH, which may alter the structure of the protein, thereby adversely affecting the function of the protein.

[0033] Regarding isoelectric precipitation of edestin, Bailey disclosed that the isoelectric zone of edestin is pH 5.5 (Bailey, 1942). In this process, albumin can be largely removed during precipitation of edestin at isoelectric point (Papalamprou et al., 2009). This result can be attributed to the high solubility of hemp albumin at pH 5.0 (>75%) compared to hemp globulin (<10%) (Malomo & Aluko, 2015). One advantage of isoelectric precipitation over other protein isolation methods is that a higher water binding capacity has been found for protein isolates obtained by isoelectric precipitation compared to the same isolate obtained by micellar extraction (Krause et al., 2002). However, a disadvantage of isoelectric precipitation during edestin isolation is the lower solubility of the protein compared to edestin isolated by salt extraction, suggesting that the protein is no longer in its native state (Hadnadev, 2018).

[0034] Compared to alkaline extraction and isoelectric precipitation, salt extraction may involve micellization, is a gentler extraction procedure, and does not result in oxidation, polymerization, and co-extraction of polyphenols with proteins. Salt extraction involves "salting in" a group of proteins, followed by "salting out" the target protein. "Salting in" refers to the effect of increasing the ionic strength of a solution, which increases the solubility of solutes, such as proteins. This effect tends to be observed when the ionic strength is low. "Salting out" involves further increasing the salt concentration, which reduces the solvating power of the salt ions due to the presence of salt ions, thus reducing the solubility of the target protein, resulting in precipitation.

[0035] One method of salt extraction includes a micellization step, as described in US Patent No. 6,005,076 to Murray. In salt extraction using micellization, the protein is first solubilized with a salt solution having a certain ionic strength. The saline solvent is then diluted with the concentrated protein solution to reduce the ionic strength below a certain level, thereby forming discrete protein particles in the aqueous phase, at least partially in the form of protein micelles. The protein micelles then settle to form a mass of target protein isolate. The protein isolate can then be separated from the supernatant.

[0036] Salt-based micellization extraction as disclosed by Murray has the advantage of producing protein isolates with higher solubility compared to those obtained by isoelectric precipitation (Karaca et al., 2011; Krause et al., 2002; Paredes-Lopez and Ordorica-Falomir 1986). In addition to improved solubility, protein isolates obtained by micellization techniques had higher interfacial activity compared to isoelectric precipitation. Furthermore, according to Krause and Papalamprou, micellization extraction resulted in protein isolates with better preservation of the native protein structure compared to isoelectrically precipitated proteins (Krause et al., 2002; Papalamprou et al. (2009). In general, isoelectric precipitation will denature the extracted proteins to some extent, which may cause hydrophobic interactions between protein molecules and lead to the formation of insoluble protein aggregates. Although salt extraction and micellization may be the least damaging known methods to isolate hemp proteins, the addition of salt during isolation will adversely affect protein structure and function. "The addition of NaCl also has a different effect on the gel structure. Specifically, increasing NaCl concentration (up to 300 mM) promotes intensive protein-protein interactions and aggregation, leading to the formation of HMI [hemp protein micellized isolate] gel structures with larger pore sizes." (Shen et al., 2021).

[0037] Salt extraction was the first method used to isolate edestin from hemp kernels (Osborne, 1892). This method was further developed by Malomo, who utilized micellization techniques to extract edestin (Malomo and Aluko, 2015). As Malomo demonstrates, during salt-based micellization extraction, after removing the salt in a dialysis step, albumin remains in the supernatant, while globulins precipitate and can be collected by centrifugation. At Malomo, globulin isolates were produced by salt extraction of hemp kernel flour followed by dialysis against water in dialysis tubing.

[0038] Dialysis of the salt extract of hemp kernel flour resulted in the precipitation of water-insoluble globulins in micellar form while albumin remained in solution (Malomo and Aluko, 2015). The precipitate was then collected and freeze-dried. Comparing the albumin and globulin fractions of hemp protein, albumin had significantly higher protein solubility and foaming ability than globulin, while no difference in emulsion forming ability was observed between the two protein fractions. Salt extraction and micellization entails high labor, time, material, equipment, and waste disposal costs and is not currently considered commercially viable for protein extraction for use in food products.

[0039] Ultrafiltration is another method that can be used to produce protein isolates with improved functional properties compared to other conventional protein extraction techniques. For example, when compared to alkaline extraction, protein isolates obtained by ultrafiltration generally have better emulsification properties. However, one of the drawbacks of ultrafiltration is the clogging of the membrane due to precipitates formed in the final product, which can lead to higher extraction costs.

[0040] Newer protein extraction methods include ultrasonic-assisted extraction, enzyme-assisted protein extraction, and electrical protein extraction. These methods have drawbacks such as high cost, low yield, protein degradation, and protein impurities. Therefore, traditional extraction methods such as salt extraction, alkaline extraction, and isoelectric precipitation are still the mainstream methods for extracting protein from plant materials such as hemp kernels.

[0041] With regard to published methods of extracting and isolating hemp using the methods described herein above, an example of aqueous protein extraction of hemp grain followed by isoelectric precipitation is disclosed in U.S. Patent No. 10,555,542 to Crank. Crank first discloses grinding hemp grain using any suitable means including grinding using a hammer mill, roller mill, or screw type mill. Grinding by these processes is a high energy process, generally resulting in high temperatures of about 140°F to 150°F. As is known in the art of peanut butter manufacturing, certain high temperatures are required to form a paste from a solid, so these high temperatures are necessary to obtain a paste. These temperatures may cause undesirable interactions between the protein components of the grain material depending on the end use of the product. In Crank, grinding produces a paste or a fine flour (fine flour if the kernel is first pressed to remove the oil), and water can be added to the ground material in a ratio of about 4 to about 16 parts by weight for each portion of plant material. Crank discloses adjusting the pH to about 7.5 by adding a base such as calcium hydroxide to facilitate extraction of the protein.

[0042] The resulting solution is then centrifuged to separate the fat fraction from the aqueous fraction or reduced-fat extract. The reduced-fat extract can be used as a reduced-fat plant milk or further processed to produce a protein concentrate or protein isolate. In Crank, the proteins in the reduced-fat extract were concentrated by precipitation and separated to produce a plant protein concentrate or isolate from partially defatted plant material. Crank discloses that the proteins in the reduced-fat extract can be precipitated by adding an acid, such as citric acid, to the isoelectric point of the protein. Crank does not disclose that edestin and albumin can be separated using aqueous extraction alone. Additionally, Crank mentions in the application that hemp seeds may be a source of isolated protein for food use according to the Crank process, and that hemp seeds contain edestin, but Crank does not disclose the purification and isolation of edestin. Crank discloses that the fiber and protein-containing portions of the hemp protein are discarded after centrifugation.

[0043] Beran's Czechoslovak Patent No. 33,545 discloses a method for extracting edestin from hemp kernels to produce protein for human consumption. Beran discloses in the background section of the patent that hemp proteins are often produced as spray-dried hemp protein isolates, which often utilize high heat and can cause denaturation of the protein. According to Beran, spray drying can require temperatures of 150°C to 250°C; temperatures at which hemp proteins can denature. Beran discloses that "thermal denaturation of proteins adversely affects solubility and dispersibility, foaming properties and emulsifying properties."

[0044] In order to avoid the thermal denaturation during the preparation of edestin caused by spray drying, Beran discloses a method that includes first crushing or squeezing hemp kernels to remove oil, followed by water extraction and either isoelectric precipitation or salt extraction to purify edestin. The preferred method of particle size reduction used in Beran appears to be dry milling. According to the patent, the milled powder is then added to water at a concentration of 5:1 water to powder. Beran then discloses shaking the solution to produce an albumin-containing water fraction and a precipitate fraction.

[0045] Beran discloses that the precipitate contains edestin, but discloses further steps to isolate edestin for use in foods. These steps include extraction of the protein by isoelectric precipitation, salt extraction, or ultrafiltration. Beran does not disclose the level of purity of edestin in the precipitate prior to the additional steps to isolate edestin, but the need for such additional steps indicates that the purity of edestin in the precipitate is not sufficient for the stated purpose of Beran's patent, which is to use edestin to "increase the protein content of high protein foods and smoothie protein beverages." In conclusion, Beran discloses that "due to its emulsifying properties and beneficial effects on the organoleptic properties of the final product, this product can be used in these foods."

[0046] Both Crank and Beran generally disclose the use of crushed or pressed hemp kernels (to remove oil) and then crushed to use hemp flour as the starting material for protein extraction. As a result, the hemp flour has been subjected to a dry-grinding or milling and oil extraction process that affects the structure and function of the protein. Additionally, both Crank and Beran disclose the isolation of edestin by at least isoelectric precipitation, which causes a structural change in the protein and, as a result, reduces its function.

[0047] During the process of extracting protein from grains for use in plant-based meat, oil may also be extracted from the grains. In some cases, oil extraction is the primary objective, as plant-based oils have food and cosmetic value. Common methods for extracting oil from grains, nuts, and seeds include expression-based extraction methods, such as cold pressing and expeller pressing, as well as solvent extraction.

[0048] Kernel pressing to extract oil involves mechanically compressing the plant material to force the oil out of the solid. Solvent extraction involves placing the plant material in a liquid to extract the oil. In some cases, pressing and solvent extraction may be combined. Oil recovery from the extruder pressing process may be relatively inefficient, leaving a fairly high percentage of fat in the cake. Thus, the press cake may be further extracted using oil solubilizing solvents. Commercial cakes and flours produced by pressing or pressing and solvent processes are believed to have reduced protein functionality.

[0049] Conventionally produced hemp kernel oil may have a green color, which may be due to the destruction of the protoplasts or chloroplasts during extraction. Hemp kernels may contain chlorophyll-containing substances that release chlorophyll when destroyed. Compared to other types of kernels, hemp kernels contain more of these substances, which is why hemp oil extracted using conventional methods tends to have a green color.

[0050] According to Leonard et al., "unrefined hemp seed oil has a dark green color, which is due to the presence of chlorophyll" (Leonard, et al., 2019). Additionally, the presence of chlorophyll in the oil can lead to fat oxidation and off-flavors. Soe's U.S. Patent No. 9,493,749 discloses that "vegetable oils derived from oilseeds, such as soybean oil, palm oil, rapeseed (canola) oil, cottonseed oil, and peanut oil, typically contain some chlorophyll. However, the presence of high levels of chlorophyll pigments in vegetable oils is generally undesirable, as chlorophyll imparts an undesirable green color and can cause oxidation of the oil during storage, leading to oil spoilage."

[0051] Various methods have been used to remove chlorophyll from vegetable oils. These methods include chemical bleaching and ultrasonic bleaching. Chlorophyll may be removed during many stages of the oil production process, such as grain milling, oil extraction, degumming, caustic treatment, and bleaching. However, the bleaching step is usually the most critical to reduce chlorophyll residues to acceptable levels. During bleaching, the oil is typically heated and passed through an adsorbent to remove chlorophyll and other color-bearing compounds that affect the appearance and / or stability of the finished oil. The adsorbent used in the bleaching step is typically clay.

[0052] Conventional methods for removing chlorophyll from hemp oil are expensive and can cause waste disposal problems. In addition, methods for removing chlorophyll from hemp oil after the chloroplasts are destroyed can cause the oil to oxidize due to temporary exposure to chlorophyll. Therefore, there is a need for improved methods for extracting oil from hemp kernels.

[0053] In producing meat and dairy analogs, after obtaining the protein isolate and preferred oil source, step 2 is to combine the isolated protein with water and possibly oil to form a material for curing or extrusion. After the protein is isolated from the hemp kernel or other plant product, it must be combined with the other ingredients of the meat or dairy analog to form the final meat analog. The three basic ingredients for meat analog production are protein, water, and fat. These ingredients can be combined in various concentrations and processed in various ways to form the meat and dairy analogs.

[0054] Considering that meat analogs require gelation, or structuring to provide a chewy, meat-like texture, proteins typically combine with water and possibly oil to form a gel that can be hardened by heat to create texture. With regard to forming hemp protein isolate gels using these ingredients, or just protein and water, studies have shown that hemp protein does not have good gel-forming properties. As disclosed above, Wang, Shen, and Zahari teach that hemp does not have good gel-forming capabilities, making it a poor candidate for use as a primary protein in meat or dairy analogs (Wang et al., 2019; Shen et al., 2021; Zahari et al., 2020). For example, Wang showed that the combination of hemp protein isolate and water alone does not form a desirable gel upon heating. Wang also showed that a mixture of hemp protein, water, and oil does not form a desirable gel upon heating, even when oil is added to Wang's protein and water mixture and heated.

[0055] In the production of meat analogs, extrusion typically involves a third step of heat-setting the protein, water, and oil, which texturizes the product and forms a more meat-like material. To form textured meat, an extruder is used to form textured vegetable protein (TVP). TVP is typically a soy-based product, although other plant proteins, such as pea, can be used alone or in combination with soy. To produce TVP, the plant-based ingredients are fed into an extruder for texturization. Traditionally, dried plant protein is fed into the extruder, and water, starch, and sometimes fat are added to the protein through separate inputs as the protein is conveyed through the extruder. After extrusion, the extruder output may undergo marinating, coating, and / or cooling steps.

[0056] A common problem with conventional plant-based meat substitute products, including TVP and HMMA, is the non-dispersible texture and rubbery mouthfeel compared to meat. This texture and mouthfeel of conventional meat analogs is due in part to the lack of fat, oil, or a combination thereof incorporated into the molecular structure of the protein peptide chains or "fibers." Animal-derived meats have fat molecules incorporated between the muscle fibers that make up the majority of animal meat products. This fat is released during chewing, providing the consumer with positive and continuous sensory feedback regarding taste and mouthfeel as chewing continues. The sensory feedback obtained when chewing current conventional meat analogs is not equivalent to that obtained with meat, in part because of the lack of fat between the peptide layers of the protein. In conventional meat analogs, fat is added after the protein is fully denatured, so it may surround cooked protein pieces of significant size, but is not incorporated within the peptide layers of the protein itself.

[0057] Soy and pea proteins, commonly used to produce TVP and HMMA fibers, may only retain about 10% of their weight in fat. Meat muscle fibers typically have anywhere from 5 to about 50% of their weight in fat incorporated within the protein fiber, depending on the source of the meat. Thus, with conventional soy and pea meat analogs, much of the fat added to the product during extrusion remains on the outside of the fiber, creating a greasy, unappealing product that does not release the fat in a controlled, juicy manner when chewed. As a result, conventional meat analogs appeal primarily to a limited number of committed vegan or vegetarian consumers, and not to the majority of meat-eating consumers.

[0058] Different extrusion methods may produce different meat analog textures. Extrusion has been developed over decades to create meat analogs that are more similar to meat. Extrusion and preparation of meat analogs for extrusion involves complex chemical changes and processes within the protein components of the extrusion mixture. During extrusion, the structure of the protein isolates changes significantly, causing the proteins to be partially or totally denatured or unfolded, rearranged, cross-linked with other protein molecules, or chemically bonded to other components of the extrusion mixture. Extruders cause these changes through changes in temperature and pressure, as well as the application of shear forces exerted by the screw as the mixture moves through the machine. The final texture, taste, and mouthfeel of meat analogs produced by extrusion are determined by the different types of chemical bonds formed between the components of the extrusion mixture before, during, and after extrusion.

[0059] Regarding early development of extrusion processes for producing textured meat analogs, US Patent No. 6,319,539 to Shemer et al. disclosed mixing protein with a majority of water and potentially fat, and heating, gelling, and forming the resulting paste in an extruder. Shemer discloses that during transfer to the extrusion device, the paste is heated, conveyed at a predetermined speed, and then extruded through an orifice. The resulting food product has a fibrous texture with substantially aligned axial fibers. However, problems with this process are that the flow rate is limited and it can only be performed with certain raw materials, particularly gluten, thus limiting the variety of products. Gluten is a known allergen, which has also limited the use of soy-based TVP in the past.

[0060] An additional disadvantage of the Shemer process and other earlier extruder processes is that the cooked product expands as it is conveyed from the extruder due to the release of water vapor as the hot product cools. The water vapor causes disruption of aligned protein fibers, which is undesirable for an acceptable texture of the meat analog.

[0061] To solve this problem, WO 2003 / 007729 to Bouvier et al. discloses a twin-screw rotor extruder, as opposed to a single-screw device, that has an elongated cooling chamber, allowing the ingredients to be mixed and extruded at a controlled temperature so that steam does not disrupt the alignment of the final product protein fibers. In addition to addressing the cooling and steam issues, the '729 application also recognized problems in existing technology with incorporating desired amounts of fats and oils into extruded products using conventional raw material blends.

[0062] To achieve the desired fat content, the '729 application disclosed a novel extrusion mixture containing a fat component mixed with lecithin or caseinate, protein, fiber, starch, and water. The mixture was kneaded to obtain a paste that was heated in the extruder to gel. However, the inclusion of large amounts of carbohydrates, such as starch, in the meat analog is undesirable due to taste and nutritional concerns.

[0063] To solve the problem of introducing fat into meat analogs without adding starch and other related problems associated with introducing oil into the extruder, Giezen et al., WO 2012 / 158023, discloses an extrusion process for converting vegetable protein compositions, such as soy protein, into a fibrous, meat-like structure. Giezen discloses that an extrusion outlet temperature higher than the boiling point of water results in an open product structure that can be infused with oil to reach a desired fat content. Giezen's problems include adding additional post-extrusion process steps and the end product being perceived by consumers as too oily and too fatty.

[0064] A commonly recognized problem in the art of meat analog extrusion is that a high amount of oil in the extrusion mixture prevents obtaining a product with the texture of animal meat. In conventional meat analog extrusion, the presence of oil reduces the high mechanical shear forces in the extruder that create the ideal meat analog fiber structure. Thus, when using conventional processes, the addition of an optimal amount of oil results in a meat analog with a suboptimal fiber structure and texture.

[0065] To overcome the problem of higher oil content resulting in suboptimal texture in textured meat analogs, Trottet et al., U.S. Patent Application No. 20180064137, developed the addition of oil separately from other raw materials during extrusion. The process involves feeding an extruder barrel with 40-70% water and 15-35% vegetable protein by weight, followed by injection of 2-15% oil by weight into the extruder barrel at a point downstream of the feeder. According to this disclosure, the downstream location for injecting the liquid oil is preferably within the latter half of the full length of the extruder barrel. Ostensibly, this configuration creates high shear forces in the first half of the extruder to promote fiber formation, allowing oil to be added downstream without disrupting fiber formation.

[0066] Although the Trottet process results in an improved product compared to the prior art, Trottet does not incorporate significant amounts of oil into the core of the protein fiber. Without the incorporation of oil into the fiber, the resulting product is perceived by the consumer as greasy and the release of fat during mastication is not controlled. This unsatisfactory result is because people are accustomed to eating animal meats that have a large amount of fat incorporated into the muscle fiber. Animal meat protein fibers contain up to about 50% fat by weight, but this varies depending on the source of the meat. In the Trottet process, only fat is incorporated into the fiber in an amount of about 10% of the weight of the vegetable protein fiber. This problem with the Trottet process is caused by both the type of protein used as the raw material for extrusion, which Trottet discloses as soybean and wheat, and the method by which the oil is added to the protein fiber.

[0067] In another patent application addressing the fat content of meat analogs, Pibarot's patent application WO2020 / 208104 was filed in 2020. In the application, entitled "Meat analogs and meat analog extrusion devices and methods," Pibarot acknowledges the problem of mimicking the fat content of animal meats, which contain fatty tissue within and outside of a protein matrix. Pibarot suggests that this complex structure may allow the appearance of the meat, as well as the texture and juiciness of the meat, to be manipulated.

[0068] To solve this problem, Pibarot discloses injecting fat into the extrusion mixture as it cools in the die. In Pibarot, gaps are created between the protein fibers of the extrusion mixture during extrusion. As the heated and sheared product is conveyed through the cooling die, fat is injected into these gaps, depositing fat between the protein fibers. Pibarot claims that this process gives the product a marbled appearance similar to lean meat and improves the texture and flavor of the product. Pibarot discloses using this process with soy, pea, and other traditional plant protein sources. However, Pibarot does not teach how to incorporate fat into the molecular structure of the protein fibers.

[0069] In summary, the Shemer process can only be used with a limited number of ingredients and the lack of temperature and cooling control resulted in an inferior product. Bouvier solved the cooling problem of Shemer, but blended large amounts of starch with the raw extruded material to achieve the desired fat content, which resulted in undesirable taste and nutritional value. Giezen solved the starch problem of the Bouvier process by adding fat after extrusion, but this required an additional step and resulted in an oily, unpalatable product. Trottet improved on both Giezen and Bouvier by introducing oil at a later stage of extrusion, but Trottet still suffers from the problem of low incorporation of oil into the protein fibers of the meat analog. Pibarot discloses injecting fat into the meat analog upon cooling, which introduces fat between the protein fibers, but does not produce a final product with fat incorporated into the protein fibers.

[0070] Hemp grain is highly valuable as both a protein source and an oil source. Although various methods exist for producing food products from hemp grain, it is clear that there is a need for more effective, efficient, cleaner, and lower-cost methods of extracting protein and oil from non-oxidized hemp grain with clean, pleasant tasting flavors to produce hemp proteins with excellent purity, gelling function, nutritional value, digestibility, and flavor, and oxidatively stable oils with clean flavors and bright colors suitable for cooking and cosmetics. Furthermore, there is a continuing need for processes and raw materials that can be used to create a variety of meat analogs with the appearance, taste, texture, juiciness, and chewiness of a variety of animal meats and dairy products. More specifically, there is a need for meat analogs with an optimal amount of oil or saturated fat in the final product, with the appearance, texture, and taste of meat, and with the oil or saturated fat incorporated into the protein fiber at a level that approximates the oil or saturated fat of animal meat or dairy sources. Summary of the Invention

[0071] The present disclosure solves the problems of the prior art with respect to isolating hemp protein, preparing raw material inputs, and processing raw material inputs to produce superior plant-based meat and dairy analogs. The compositions and processes of the present disclosure include processes for isolating hemp kernel protein, pasteurizing, liquid solution, gel formation, texturizing, and producing meat and dairy analogs. The processes of the present disclosure result in structured protein foods or meat analogs with superior properties when compared to existing products or similar products produced using known technology.

[0072] The preparation of meat or dairy analogs by the process of the present disclosure can be divided into three major steps. The first step involves the extraction, or isolation, of the protein from the hemp kernel. The second step involves combining the isolated protein with water and oil to form a feedstock for thermal gelling or extrusion. The third step involves thermal gelling or extrusion of the feedstock to set or texture the meat analog. The final meat analog may then be cooked to mimic meat or dairy products such as chicken, fish, and cheese.

[0073] With respect to the first step, isolation of hemp proteins, the process of the present disclosure incorporates, with modifications, known grain processing methods disclosed in U.S. Patent No. 7,678,403 to Mitchell ("Mitchell" or "'403 patent"). The '403 patent is incorporated herein by reference in its entirety. The Mitchell process discloses aqueous wet milling at low temperatures and sieving of the resulting product. In the present disclosure, the aqueous wet milling may be performed while maintaining the temperature of the slurry preferably between 33°F and 38°F. At high temperatures, especially above 42°F, microbial growth is a concern.

[0074] In some embodiments, milling can be performed using whole or dehulled hemp grains (also called dehulled hemp grains). Depending on whether whole or dehulled hemp grains are used, the color of the final meat or dairy analog may differ. Using whole hemp grains results in a darker, more beef-like color, whereas using dehulled hemp grains results in a whiter, more chicken or fish-like color. The use of some whole and some dehulled hemp grains, in one embodiment, where the whole hemp grains are used at a concentration of about 20-30% by weight relative to the amount of dehulled hemp grains, results in a beef-like color in the final product. In one embodiment, the husk previously removed by the dehulling of the hemp grains can be reintroduced into the dehulled hemp grains to add color, where in one embodiment, the husk can be added to the dehulled grains in an amount of about 10-15% by weight relative to the dehulled hemp grains to achieve a beef-like color.

[0075] After aqueous wet milling, Mitchell in the '403 patent teaches sieving at a different mesh size than the present disclosure, with sieving at a mesh size of 170-200 being preferred in the present disclosure. In the '403 patent, Mitchell, when discussing sieving rice grains for milk production, discloses a mesh size of 150 or less, which is suitable for certain grains but not for removing chloroplasts from hemp grains. In the present disclosure, it has been surprisingly discovered that a mesh size of 170-200, preferably or between about 160-200, will prevent chloroplasts or chlorophyll-containing particles from passing through the filter, while allowing sufficient protein particles to pass through the filter without significantly reducing protein or nutrient yield.

[0076] Processing hemp grain according to the modified Mitchell process results in an insoluble protein-containing precipitated by-product. This protein-containing material was discovered by Mitchell to have unique and valuable properties and is particularly well suited for the production of meat and dairy analogs. This hemp grain protein-containing material has not been previously published. Upon further investigation, Mitchell determined that the material is primarily composed of edestin and, importantly, is substantially free of albumin, the other major protein component of hemp grain. The processing parameters of the Mitchell process appear to maintain the edestin substantially in its native state. Due to the high concentration of substantially native edestin, this material is hereafter referred to as native edestin protein isolate (NEPI). NEPI is approximately 80% protein-based and also contains oil, fiber, carbohydrates, and ash.

[0077] After grinding and sieving, the NEPI can be separated from the albumin oil-water emulsion (AOAE) by centrifugation and decanting. The albumin oil-water emulsion can optionally be further processed to produce hemp oil and albumin. The NEPI extracted according to the present disclosure can be used in a variety of different plant-based food products that replicate meat or dairy products. The NEPI can optionally be combined with oil to form protein hydrosols and protein-fat hydrosols, which can be processed to produce evaporated or spray-dried products. The hydrosols can be used to produce plant-based meat analogs.

[0078] The present disclosure is based on methods and materials for producing plant-based products that more closely replicate meat products, including the texture, juiciness, fibrousness, and texture uniformity of animal meat. Described herein is a process for producing meat analogs, including protein unfolding or denaturation, selecting proteins based on their properties and fat-retaining ability. Additionally, the processes described herein include a method of preparing an extrusion mixture or input, which may be a pre-extrusion protein-fat hydrosol that incorporates water and fat with the selected protein in such a manner that the water and fat form a liquid matrix, and the protein-fat hydrosol may also be referred to as a protein-fat hydrosol that includes protein. In some embodiments, the liquid matrix may have additional ingredients, but the protein-fat hydrosol may have no ingredients other than the protein-fat and water. Additionally, the processes described herein include a method of extruding or otherwise heating a liquid matrix, which may also be referred to herein as an extrusion input or extrusion mixture, and the liquid matrix may be a fat protein hydrosol. The process of extruding the liquid matrix includes feeding the liquid matrix to a pump at a first end of an extrusion chamber. The liquid matrix is ​​fed to an extruder, the extruder being set to parameters appropriate for the liquid matrix.

[0079] The present disclosure relates to compositions containing edestin or edestin-like proteins, and methods for isolating edestin from hemp grain.As disclosed herein, edestin can be isolated from hemp grain or other grains and seeds containing edestin or edestin-like proteins.In one embodiment, hemp grain is wet milled during aqueous protein extraction, which results in an edestin-containing fraction and an albumin oil-aqueous emulsion.

[0080] The present disclosure, in one aspect, utilizes a method of aqueous wet milling to separate fats stored within hemp grains without breaking down the chloroplasts and releasing chlorophyll into the oil. Once the seeds are milled by this process, the resulting milled product is sieved through various sizes of mesh. Sieving between about 170 mesh and 200, or in some embodiments between 160 and 200 mesh, or in some embodiments between 200 and 270 mesh removes husks, chloroplasts, and fiber. More preferably, mesh sizes between 160 and 200 can be used. In one preferred embodiment, a mesh size of 170 can be used. A mesh size of 150 has openings that are too large, allowing undesirable materials, such as fiber and chlorophyll-containing materials, to enter the filtrate. Surprisingly, most of the protein-containing particles pass through a mesh of this size, while the chlorophyll-containing particles remain larger than the pore openings of the 170 mesh. According to the method of the present invention, chloroplasts, protoplasts, or other chlorophyll-containing particles are separated from the hemp oil and protein-containing fractions by sieving through different sized meshes, resulting in a final oil product that is pale yellow in color.

[0081] In the process of the present disclosure, an insoluble fraction containing NEPI and albumin oil-water emulsion may be present in the filtrate after sieving. The AOAE may be decanted after centrifugation. The insoluble fraction and pellet-containing portion may be washed to remove residual oil. In some embodiments, two cold water washes may be performed.

[0082] In some embodiments, the AOAE can be cooled to between about 33°F and 38°F, preferably 35°F, until the albumin begins to separate and precipitate from the oil in the emulsion, which in some embodiments can be assisted by centrifugation. According to the process of the present disclosure, the albumin binds strongly to the hemp kernel oil, thereby improving the separation of the oil and albumin from the insoluble edestin fraction, i.e., the NEPI. This process can separate the albumin from the hemp kernel oil. Gel electrophoresis shows that this process can remove substantially all of the albumin from the NEPI, leaving primarily edestin in the NEPI. The AOAE can be removed from the NEPI by centrifugation and decanting, leaving the NEPI as a solid material that can be washed to remove residual material.

[0083] In one embodiment, the NEPI can then be heated to a temperature of about 145°F for about 30 minutes to pasteurize the product. In some jurisdictions, 145°F may be the legal lower limit for pasteurization. Here, the temperature should be maintained at about 145°F, or between 145°F and 155°F, to prevent granulation that occurs at higher temperatures observed in this disclosure. With NEPI, granulation can occur at temperatures much lower than denaturation of edestin, for example, about 158°F; therefore, it is very important to pasteurize at a temperature lower than those that those skilled in the art would typically use for pasteurization. Those skilled in the art conventionally pasteurize protein isolates at temperatures that cause significant granulation in this disclosure in order to process the product quickly. After pasteurization is complete, the NEPI is spray dried or stored as a concentrate for use in meat and dairy analogs. Spray drying should be performed at a lower temperature, preferably about 145°F to 155°F, to prevent granulation or aggregation of the protein.

[0084] In some embodiments, particularly for commercial applications, the NEPI comes off the production line into a tank heated to 145° F., and the product is incubated at this temperature for 30 minutes before being sent to a cooling tank and cooled to about 35° F. After cooling, the NEPI is shipped for spray drying, freezing, freeze drying, or vacuum microwave drying, as appropriate, for subsequent use in the manufacture of meat and dairy analogs, or structured protein foods.

[0085] For the production of meat analogs, the pasteurized product can be prepared by first hydrating the NEPI if dry, or by maintaining the proper degree of hydration if not. In one embodiment, the amount of water can be about 3 parts water to 1 part NEPI. Before adding to the NEPI, the water may be preheated, preferably to about 135°F, to form a protein hydrosol before hardening. Salt should not be added during this process as it may destroy the hydrosol structure of the protein. Salt may be added just before or after hardening, but not before hardening. In some embodiments, the protein hydration and disentanglement can be carried out at 100°F to 135°F, or in some embodiments, between 100°F to 155°F; alternatively, in other embodiments, the protein hydrosol formation may be carried out at a lower temperature, but that temperature must be higher than a lower temperature that cannot disentangle the protein. Preferably, the temperature during the hydration and protein preparation steps should remain as close as possible to 145°F, which is considered the minimum temperature for pasteurization, without reaching a temperature that would result in granulation of the product.

[0086] Once the protein is hydrated, in one embodiment, oil can be added and mixed with the protein hydrosol to form the protein-fat hydrosol. Oil should not be added until the NEPI is sufficiently hydrated so that the protein hydrosol has a smooth appearance. Adding oil before hydration and protein preparation may result in granulation. Furthermore, according to the process of the present disclosure, the oil should be added before the material hardens, where hardening means when protein bonds form to create a more solid gel product, when protein aggregation occurs, and when proteins aggregate at high temperatures where protein denaturation typically occurs. In the present disclosure, there is no free oil during the hardening process, and all oil is incorporated into the emulsion or protein structure before the product is hardened in an extruder or other heat hardening means. In traditional extrusion, free oil is present in the material that is partially or fully hardened in the extruder. Therefore, in the present disclosure, it is important to add water to fully hydrate and disintegrate the NEPI before adding oil to ensure that there is no free oil during extrusion or hardening. This protein-fat hydrosol can then be heated or extruded to form a meat analog. In conventional extrusion, for example using soy or pea proteins, oil is added to the protein material after hardening begins at high temperatures in the extruder, for lubrication rather than oil incorporation.

[0087] Once the protein hydrosol is sufficiently hydrated, oil can be added to form the protein-fat hydrosol. The oil is preferably preheated, and the temperature of the oil can be preferably between about 130°F and 135°F. In other embodiments, the oil can be preheated between 100°F and 135°F, or between 100°F and 155°F, while in other embodiments, the oil can be added at a lower temperature, but the oil should not be added at a low temperature that will destroy the structure of the protein hydrosol and prevent the oil from being incorporated into the protein hydrosol to form the protein-fat hydrosol. The material can also be set in a retort system, although retort may not be able to produce a fiberized product as extrusion does.

[0088] The texture of the retorted NEPI meat analog was surprisingly good, with texture properties such as firmness and chewiness far superior to commercially available hemp protein concentrates and isolates under the same conditions. The process of the present disclosure unexpectedly resulted in the thermal gelation and extrusion of high-quality fiberized meat analogs produced using only hemp as a protein source. Due to the nature of traditionally used meat and dairy analog proteins such as soy and hemp, traditional meat and dairy analogs are unable to replicate textured meat fillets that resemble animal meat products, such as chicken breast. The unexpectedly advantageous properties and results obtained from the use of NEPI and the process using it described in the present disclosure have produced a much better structured meat analog using only hemp protein as a protein source when compared to other commercially available products. At present, hemp protein is only known to be used in combination with soy or other plant proteins to produce meat analogs.

[0089] In one aspect, this document features a meat analog extruded input, or liquid matrix, that can have a protein to fat ratio ranging from about 4:1 to 0.5:1.

[0090] In one aspect, this document features a process where water is added to the protein isolate in ratios disclosed herein below or water is maintained in the protein isolate at a specific ratio, and after the water is added to or maintained in the protein isolate, fat is added to the protein and water mixture in an approximately constant ratio of water to fat and protein to fat.

[0091] In one aspect, this document features a product having a moisture content target of between 35% and 75% by weight.

[0092] In any of the methods or compositions described herein, the isolated plant proteins in the liquid matrix can include seed oil proteins such as edestin, albumins, globulins, or mixtures thereof.

[0093] In any of the methods or compositions described herein, the isolated protein can first be isolated from all other plant proteins within the plant.

[0094] In any of the methods or compositions described herein, the isolated protein used may be one that is isolated in its native, or non-denatured, state; native can mean fully native, substantially native, partially native, or otherwise identified as substantially native by conventional methods of detecting protein structure, or as would be understood by one of skill in the art.

[0095] In any of the methods or compositions described herein, the protein isolated from seed proteins preferably has a cysteine ​​content higher than that typically found in soy or casein.

[0096] In any of the methods or compositions described herein, the liquid matrix may include flavorings, starch, fiber, or other carbohydrate sources.

[0097] In some embodiments, the meat and dairy analogs provided herein may be free of animal products, wheat gluten, soy protein, or pea protein.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. All references to percentages are by weight.

[0099] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description, drawings, and examples, and from the claims. [Brief description of the drawings]

[0100] [Figure 1] FIG. 1 is a flow diagram showing a process for producing native edestin protein isolate or NEPI according to the present disclosure. [Diagram 2] FIG. 1 is a flow diagram illustrating a process for producing pasteurized NEPI according to the present disclosure. [Diagram 3] FIG. 1 is a flow diagram showing a process for spray drying NEPI according to the present disclosure. [Figure 4] FIG. 1 is a flow diagram illustrating a process for making colored NEPI according to the present disclosure. [Diagram 5] FIG. 1 is a flow diagram illustrating a process for extracting hemp oil from hemp grain according to the present disclosure. [Figure 6] FIG. 1 is a flow diagram illustrating a process for forming a hydrosol according to the present disclosure. [Figure 7] 1 is a flow diagram illustrating a process for producing retort meat and dairy analogs according to the present disclosure; [Figure 8] 1 is a flow diagram illustrating an extrusion process for NEPI according to the present disclosure; [Figure 9] 1 is an SDS-PAGE electrophoresis gel under non-reducing conditions of NEPI proteins and hemp proteins from commercial hemp protein concentrates and isolates according to the present disclosure; [Figure 10] 1 is an SDS-PAGE electrophoresis gel under reducing conditions of NEPI proteins and hemp proteins from commercial hemp protein concentrates and isolates according to the present disclosure; [Figure 11A] SDS-PAGE electrophoresis gels under reducing conditions of hemp flour and hemp protein isolate from prior art publications; [Figure 11B]FIG. 2 is an SDS-PAGE electrophoresis gel of hemp proteins under non-reducing and reducing conditions of hemp protein isolates from prior art publications. [Figure 12A] 1 is a differential scanning calorimetry thermogram of NEPI 250 dehulled hemp grain spray dried powder. [Figure 12B] 1 is a differential scanning calorimetry thermogram of NEPI 250 whole hemp grain concentrate (slurry). [Figure 13A] FIG. 1 is a differential scanning calorimetry thermogram of VICTORY HEMP dehulled hemp grain spray dried powder. [Figure 13B] 1 is a differential scanning calorimetry thermogram of NUTIVA hemp powder. [Figure 14] 14A is a photograph of a cross section of boiled chicken breast meat. B is a magnified photograph of the cross section of the boiled chicken breast meat of FIG. 14A; C is a magnified photograph of the cross section of the boiled chicken breast meat of FIG. 14B. [Figure 15] A is a photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain concentrate; B is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp concentrate of FIG. 15A; C is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain concentrate of FIG. 15B according to the present disclosure. [Figure 16] A is a photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain powder; B is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp powder of FIG. 16A; C is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain powder of FIG. 16B according to the present disclosure. [Figure 17] A is a photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder; B is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp powder of FIG. 17A; C is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder of FIG. 17B according to the present disclosure. [Figure 18]A is a photograph of a cross section of a retort meat analog using HEMPLAND dehulled hemp grain powder; B is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp powder of FIG. 18A; C is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder of FIG. 18B according to the present disclosure. [Figure 19] 1 is a photograph of extruded NEPI from dehulled flour and boiled chicken breast pieces showing similarity in texture and fiber according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. All references to percentages are by weight. Details of one or more embodiments of the present invention are described in the accompanying drawings and the description below. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims.

[0102] In general, the present disclosure provides methods and materials for producing plant-based meat or dairy analogs, also referred to herein as structured protein foods, from hemp grain proteins. In any of the methods or compositions described herein, and in some embodiments, the extracted protein-containing product can be separated from other hemp grain proteins. In any of the methods or compositions described herein, edestin can be substantially isolated from some or all of the other proteins in hemp grain. In any of the methods or compositions described herein, the protein isolated from grain proteins preferably has a cysteine ​​content higher than the cysteine ​​content typically found in soybeans or casein.

[0103] The plant protein used according to the present disclosure may be an isolated plant protein. For the purposes of this disclosure, a "native" protein is a protein that may have the same tertiary and quaternary structure as in living, active cells. In some embodiments, a "native" protein may be substantially natural. In any of the methods or compositions described herein, the isolated protein may be isolated in a near-natural, substantially natural, or non-denatured state. In any of the methods or compositions described herein, the isolated protein used may be isolated in a natural state or in a non-denatured state; where native may mean fully natural, substantially natural, partially natural, or otherwise identified as substantially natural by conventional methods of detecting protein structure, or natural as understood by those skilled in the art. Changes and disruption of subunit structure and tertiary structure may occur due to changes in temperature (typically above 41° C.) or contact with aqueous acid or alkali, oxidizing or reducing agents, or organic solvents. Disruption of quaternary structure may render the protein biologically inactive or biologically inactive in living cells. However, the tertiary structure of the released subunits, with their specific shape created by hydrogen bonds, van der Waals forces, and disulfide bonds, may be functionally active and exhibit functions similar to those found in living cells, one example of this is the lock and key function of enzymes due to the tertiary shape of the protein.

[0104] Thus, for the purposes of this disclosure, if the quaternary or tertiary structure is substantially maintained after extraction as in living cells, they may be considered "native" proteins. This disclosure has found that certain oil grain globular proteins, which can be considered native in the sense that their tertiary structure has not been denatured by changes in temperature (typically above 41°C), or aqueous acid or alkali solutions, oxidizing or reducing agents, or organic solvents, have unique and superior functional properties.

[0105] Traditional plant protein extraction processes are known to destroy the quaternary and tertiary structures of proteins. In some cases, this destruction can result in the loss or reduction of the function of the quaternary or tertiary structure. The tertiary structure can be denatured by the destruction of functional bonds and forces, such as hydrogen bonds, van der Waals forces, or disulfide bonds, all of which cooperate to form the tertiary structure of a particular protein. The change in the protein's environment and the denaturation mode of the tertiary structure can change the tertiary structure or shape, bonds, forces, and linkages of the protein.

[0106] As used herein, the term "isolated plant protein" refers to plant proteins, which may include proteins such as edestin, glutelin, albumin, legumin, vicilin, convicilin, glycinin, and the like, and protein isolates from any seed or bean, including soybean, pea, lentil, and the like, or combinations thereof, or plant protein fractions (e.g., 7S fractions) that have been separated from other components of the source material (e.g., other animal, plant, fungal, algal, or bacterial proteins), and the protein or protein fractions include, but are not limited to, edestin, glutelin, albumin, legumin, vicilin, convicilin, glycinin, and the like. , meaning that the raw material is at least 2% (e.g., at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) free, by dry weight, from other components of the raw material. For example, isolated native globular proteins having a high cysteine ​​content can be used, alone or in combination with one or more other proteins (e.g., albumin), or from any other protein source, such as soy, pea, whey, etc.

[0107] In any of the methods or compositions described herein, the fat may be a non-animal fat, an animal fat, or a mixture of non-animal fat and animal fat. The fat can be algae oil, fungal oil, corn oil, olive oil, soybean oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil, linseed oil, palm oil, palm kernel oil, coconut oil, ahi oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, borage oil, blackcurrant oil, sea-buckhorn oil, macadamia oil, saw palmetto oil, conjugated linoleic acid, arachidonic acid-rich oil, docosahexaenoic acid (DHA)-enriched oil, eicosapentaenoic acid (EPA)-enriched oil, palm stearic acid, sea-buckhorn berry oil, macadamia oil, saw palmetto oil, or rice bran oil; or margarine or other hydrogenated fat.In some embodiments, for example, the fat is algae oil. The fat can contain flavorings and / or isolated plant proteins (e.g., conglycinin protein). The fat or oil composition of the liquid matrix can be tailored to preferentially match the saturated and unsaturated composition of the analog's target source material.

[0108] Thus, in some embodiments, the isolated protein can be a protein such as a natural edestin isolated from hemp grain or other grains that may have edestin or edestin-like proteins. In some embodiments, the proteins can be separated based on their molecular weight, for example, by size exclusion chromatography, ultrafiltration through a membrane, or density centrifugation. In some embodiments, the proteins can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Proteins can also be separated based on their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactant, detergent, or solvent extraction, including water extraction. Proteins can also be separated by their affinity for another molecule, for example, using hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography can also include using antibodies with specific binding affinity for heme-containing proteins, nickel nitroloacetic acid (NTA) for His-tagged recombinant proteins, lectins that bind to sugar moieties on glycoproteins, or other molecules that specifically bind to proteins. In some embodiments, the plant-based meats described herein are substantially or entirely composed of ingredients derived from non-animal sources, such as plant, fungal, or microbial-based sources. In some embodiments, the plant-based meat or plant-based dairy products may include one or more animal-based products. For example, meat replicas may be made by combining plant-based and animal-based sources.

[0109] Definition: Hemp seeds (HS) are generally defined herein as viable seeds that are typically used for further propagation and planting. HS may or may not be food grade based on washing practices and agricultural preservation practices of the seeds.

[0110] Whole grain hemp grain (WHG) is generally defined herein as hemp grain including both germinated and pasteurized hemp grain.

[0111] Viable hemp kernel (VHG) is generally defined herein as a viable hemp seed that has been further cleaned of all dust and foreign matter and is suitable for food grade and has the core and husk completely intact.

[0112] Pasteurized hemp grain (PHG) is generally defined herein as hemp grain that has been treated with heat or irradiation to destroy the germination ability of the seeds.

[0113] Defatted hemp kernel cake (DHGC) is generally defined herein as the dry solid residue resulting from the non-aqueous removal of oil from hemp kernels.

[0114] Hemp kernel oil (HGO) is generally defined herein as a raw, green oil obtained from the non-aqueous extraction of hemp kernels.

[0115] Hemp kernel oil sludge (HGOS) is generally defined herein as a crude oil sludge slurry obtained from the non-aqueous extraction of oil from hemp kernels.

[0116] Hulled hemp kernel (HHG) is generally defined herein as the equivalent of a hemp heart or hemp nut; the hemp kernel with the outer shell removed.

[0117] Defatted dehulled hemp kernel cake (DHHGC) is generally defined herein as the dry solid residue resulting from the non-aqueous removal of oil from dehulled hemp kernels.

[0118] Hulled hemp kernel oil (HHGO) is generally defined herein as a yellow oil obtained from the non-aqueous extraction of dehulled hemp kernels.

[0119] Hemp protein isolate (HPI) is generally defined herein as an isolate of albumin, edestin or aggregates thereof.

[0120] Albumin oil-in-water emulsion (AOAE) is generally defined herein as a water-based emulsion of oil and soluble albumin protein.

[0121] Native edestin protein isolate (NEPI) is generally defined herein as the product of the protein isolation process disclosed herein, and can refer to NEPI in liquid, slurry, and powder forms, as understood by those of skill in the art in the appropriate context for its use.

[0122] All of the products described in the flow charts can exist in a variety of physical forms, including liquid, gel, or solid, as will be understood by those of skill in the art in appropriate relation to their use.

[0123] The present disclosure may relate to compositions containing natural edestin protein isolates (NEPIs) containing edestin or edestin-like proteins, as well as methods of extracting and using NEPIs to produce meat and dairy analogs. The present disclosure solves the problems of the prior art in isolating hemp protein, preparing raw material inputs, and processing raw material inputs to produce superior plant-based meat and dairy analogs. The compositions and processes of the present disclosure include processes for isolating hemp kernel protein, pasteurizing, forming sols, forming gels, texturizing, and producing meat and dairy analogs. The processes of the present disclosure result in meat or dairy analogs with superior properties when compared to existing products or similar products produced using known technology.

[0124] In addition to isolating proteins, this document is based on methods and materials for producing plant-based products that more closely replicate meat products, including the texture, juiciness, fibrousness, and texture uniformity of animal meat. Described herein is a process for producing meat analogs, which may include protein defibration or denaturation, selecting proteins based on their properties and fat-retaining ability. Additionally, the processes described herein include a method of preparing an extrusion mixture prior to extrusion, incorporating water and fat with the selected protein in such a manner that the water and fat form a liquid matrix with the protein (also referred to herein as a liquid-fat hydrosol, hydrosol, extruder or extrusion input, and input material). Additionally, the processes described herein include a method of extruding or otherwise heating the liquid matrix. The process of extruding the liquid matrix includes feeding the liquid matrix to a pump at a first end of an extrusion chamber. The liquid matrix is ​​fed into the extrusion chamber of the extruder, and the extruder is set to parameters for the liquid matrix.

[0125] As disclosed herein, NEPI can be extracted from hemp grain or other grains, nuts, or seeds that contain edestin or edestin-like proteins; however, hemp grain is currently believed to be the only source of edestin. In one embodiment, hemp grain is wet milled and subjected to aqueous extraction, thereby producing an insoluble edestin-containing extract, referred to herein as NEPI, and an albumin oil-water emulsion.

[0126] The process according to the present disclosure can produce a pasteurized functional hemp grain protein concentrate, which can be a concentrated liquid obtained from the manufacturing line or from centrifugation and decanting, or a NEPI powder, which in some embodiments contains low or no trypsin inhibitors and has high nutritional value and functionality. The process may not use isoelectric extraction, alkaline or CO2 solubilization methods. It is believed that the texturizable protein NEPI concentrate or NEPI powder is produced by utilizing the natural pH and oil content of the hemp grain in combination with water, oil extraction and separation of albumin. The emulsion forming ability of soluble albumin allows it to form an emulsion that can be easily separated from the insoluble edestin by centrifugation. No freeze drying, pH re-adjustment, or separation by ultrafiltration is required. Additionally, fiber and chlorophyll may be removed during the NEPI process. Maintaining a low temperature, preferably between 33°F and 38°F, promotes the insolubility of globulins and also promotes the coagulation of albumin.

[0127] One aspect of the present disclosure relates to the isolation of edestin and edestin-like proteins from plant materials, including hemp kernels.Edestin is found in hemp plants; in particular in hemp kernels.Hemp kernels are believed to be the most common or only source of edestin, although other plants may contain edestin.

[0128] The edestin extract composition or natural edestin protein isolate (NEPI) prepared according to the method of the present disclosure can be used to make a protein-containing composition. The NEPI can preferably be composed of about 80% dry basis protein. In some embodiments, the NEPI can include at least 65% dry basis protein, and in some embodiments, at least 90% dry basis protein. Thus, a NEPI can be defined as an edestin-containing composition produced according to the method described in the present disclosure that results in a product having the functional characteristics described in the present disclosure. The aqueous oil albumin emulsion (AOAE) described in the present disclosure can be further processed to produce other plant-based products containing hemp oil or grain oil and albumin.

[0129] The present disclosure can be practiced using any suitable grain, seed or plant material that contains edestin or edestin-like proteins, which may be homologous or have similar structure and function.

[0130] The grains used in this disclosure may be substantially full fat plant grains, i.e. grains that have not been defatted or pressed prior to milling. In some embodiments, the grains may be partially defatted. Partially defatted grains include any plant material from which at least a portion of the fat has been removed.

[0131] As known to those skilled in the art, substantially full-fat hemp grain may have a fat (or oil) content of 10% or more by weight. In this disclosure, the terms fat and oil may be used interchangeably. Suitably, the fat content of substantially full-fat grain is at least about 10%, 15%, 20%, 30%, 40%, or even 50% by weight. The fat content of hemp grain is usually at least 30%. The fat content of partially defatted plant material may be greater than about 5%, 10%, or 15% by weight. The edible portion of hemp grain after removing the husk contains an average of 46.7% oil and 35.9% protein.

[0132] As shown in FIG. 1, hemp grain 102 can be selected for use in the structured protein food process 100. Whole hemp grain 101 and dehulled hemp grain 105 can be used. Pasteurized whole hemp grain 103 produced by a pasteurization process 107 can also be used. Hemp grain 102 used in accordance with the present disclosure can be prepared for processing by any suitable means, including but not limited to drying, conditioning to achieve an equilibrium moisture level, dehulling, cracking, and washing with countercurrent air suction, screening methods, pasteurization that does not damage viable seeds, or other methods known in the art. Hemp grain 102 can be selected from any type of hemp plant, but in the present disclosure, Cannabis Sativa, which contains 0.3% or less THC, is preferably used. The hemp grain 102 may be whole hemp grain or dehulled (dehulled) hemp grain 102, which may be dehulled prior to processing in the structured protein food process 100, thereby producing dehulled hemp grain 150, as shown in FIG. 4.

[0133] Referring now to FIG. 1, hemp grain 102 in a structured protein food process 100 undergoes a native edestin protein isolation process 200 (shown in FIG. 2) to extract native edestin protein isolate (NEPI) 250. The native edestin protein isolate (NEPI) slurry 252 or powder 254, or NEPI 250, can be used to produce a structured protein food 120, which can be a meat or dairy analog. Conventional methods of extracting hemp protein or edestin from hemp grain or producing hemp protein isolate may result in aggregation of edestin and albumin or protein denaturation, and may not produce a satisfactory structured protein food or meat analog. However, NEPI 250, when used as the only protein source in a meat analog without combining it with soy or other types of plant-based protein isolates, can produce a superior novel meat analog, as described by Zahari (Zahari et al., 2020).

[0134] As shown in FIG. 1, in some embodiments, NEPI 250 can be pasteurized 104 and mixed with water 106 to form a protein hydrosol 108. NEPI 250 can be combined with preheated water 106 to form a protein hydrosol 108 (as shown in FIG. 6). NEPI 250 should be present at least 20% w / w to water up to 80% w / w or more in water. Allow protein to fully hydrate. Hydration times vary. High shear mixing is preferred to facilitate hydration.

[0135] Oil can then be added 110 to the protein hydrosol, followed by high shear mixing 112. In some embodiments, after high shear mixing 112, the mixture may optionally be incubated without mixing 113. The addition of oil 110 and mixing 112 produces a protein-fat hydrosol 114.

[0136] The protein-fat hydrosol 114 is used as an input to a means for heating the protein-fat hydrosol to harden the product 116. Hardening may involve heating by means including microwave, steam tunnel, oven, retort, and extrusion (as shown in Figures 7 and 8). Heating to harden may also include other means for heating protein or starch based foods to form hardened products, as known to those skilled in the art. When the protein fat hydrosol 114 is hardened, a structured protein food product 120 is produced. The structured protein food product 120 may be an analogue of a meat or dairy product.

[0137] As shown in FIG. 2, to produce NEPI 250, hemp grain 102 can be added to cold water 202 to form hemp grain mixture 204. The extraction temperature during milling and throughout the native edestin protein isolation process 200 can be more preferably 35° F., or between 33° F. and 38° F., or less than about 120° F., and added to hemp grain 102 to form hemp grain mixture 204. Hemp grain may be extracted with an aqueous solution, preferably water. As used herein, the term "aqueous solution" includes water that is substantially free of solutes (e.g., tap water, distilled water, or deionized water) and water that contains solutes. According to the present disclosure, the aqueous solution may be free of additives such as salts, buffers, acids, bases, and demulsifiers. In some embodiments, the aqueous solution may have an ionic strength lower than that which would alter the structure of the protein. Some water may be used.

[0138] The process does not require adjustment of the pH to isolate the NEPIs. Preferably, the pH is maintained near neutral, between 6.5 and 7, throughout the structured protein food process 100. In one embodiment, the pH of the solution does not change to a substantial extent during milling of the grain.

[0139] The hemp grain mixture 204 may be wet-milled 206 substantially as described in U.S. Patent No. 7,678,403 to Mitchell. In one embodiment, the milling 206 of the hemp grain may be performed using a Silverson rotor-stator mill. The wet-milling 206 may be performed as part of an aqueous extraction process. Preferably, the aqueous wet-milling 206 may be performed for a suitable period of time, and more preferably, the wet-milling 206 is performed for a suitable period of time. As one skilled in the art would understand, longer extraction periods may be used. In some embodiments, enzymes may be used to aid in the process. For example, liquefaction may be accomplished using an alpha-amylase enzyme with dextrinizing activity to produce a liquefied slurry. Such enzymes may include amylases, or other carbohydrases known in the art of food processing. The present disclosure, in one aspect, utilizes a method of aqueous wet-milling to separate fats stored within the hemp grain 102 without disrupting the chloroplasts and releasing chlorophyll into the oil. Calcium chloride may be added to the NEPI 250 to improve flavor after centrifuge decant 222.

[0140] After aqueous wet milling of the hemp grain 206, the extract can be separated from at least a portion of the insoluble by-products or fibrous slurry 210 (e.g., insoluble fiber fraction) using a mesh. In some embodiments, the hemp grain slurry 208 can be sieved in two steps. Sieving can remove unwanted impurities that impart unpleasant color or taste to the edestin. Insoluble fiber can be removed by a first sieving step. Surprisingly, another undesirable product that can be removed by sieving without substantially affecting the protein yield is chlorophyll from the chloroplasts of the hemp grain and dehulled hemp, which can cause undesirable color, taste, and fat oxidation in the oil or protein fraction. In some embodiments, the chlorophyll-containing particles can be removed in a second sieving step 212. After sieving 212, chloroplast and fiber sludge may be present in the retentate along with raw hemp milk having a fat to protein ratio of approximately 1:3:1 on the DSB in the filtrate.

[0141] In a first sieving step, the hemp grain slurry can be sieved 212 to remove husk, in some embodiments, through 30 mesh. The by-product of the first sieving step can be a fibrous slurry 210. In a second sieving step 212, the hemp grain slurry can be sieved 212 to remove chloroplasts at about 170 mesh, or in some embodiments, between 160-200 mesh, or in some embodiments, between 200-220 mesh to remove chloroplasts, or chlorophyll-containing material and remaining fiber. Mesh size 150 openings may generally be too large, allowing undesirable material, such as fiber and chlorophyll-containing particles, to enter the filtrate. Surprisingly, chlorophyll-containing particles remain at a size larger than the pore openings of 170 mesh, while most of the protein-containing particles pass through this size mesh. Sieving through different sized meshes separates the chloroplasts, protoplasts, or other chlorophyll-containing particles from the hemp oil and protein-containing fractions, resulting in a light yellow final oil product.

[0142] Chloroplasts isolated by the edestin extraction process 100 can be used as a dietary supplement in some embodiments. According to the process of the present disclosure, chlorophyll-containing particles 214 are selectively removed from the hemp grain slurry 208 while protein-containing particles are allowed to pass through to the filtrate. The method is effective for both whole hemp grain, where the husk has not been removed prior to aqueous wet milling, and for dehulled hemp grain.

[0143] After sieving the hemp grain slurry through a 170 mesh to remove chlorophyll-containing particles 212, the resulting product is a mixture of aqueous oil albumin emulsion (AOAE) and edestin 220, which may also contain some of the other components of the hemp grain 102. The AOAE and edestin mixture 220 is decanted 222 by centrifugation, resulting in NEPI 250 and AOAE 230. After separation from NEPI 250, AOAE 230 can be further processed to produce albumin 550 and hemp oil 560, as shown in FIG.

[0144] NEPI 250, in some embodiments, can be comprised of about 76% protein, 2% oil, 4% fiber, 1% carbohydrate, and 17% ash. AOAE 220 can be comprised of about 14% protein, 76% oil, 3% fiber, 4% carbohydrate, and 3% ash. NEPI can be preferably comprised of about 80% dry basis protein. In some embodiments, NEPI can contain at least 65% dry basis protein, and in some embodiments, at least 90% dry basis protein. Thus, NEPI can be defined as an edestin-containing composition produced according to the methods described in this disclosure that results in a product having the functional characteristics described in this disclosure. In some embodiments, NEPI can contain at least about 65%, 75%, 85%, or 90% protein on a dry weight basis.

[0145] Table 2 shows the summary analysis data of the nutritional composition of NEPI 250 and commercial hemp protein products. Table 2 shows that NEPI 250 product, like VICTORY HEMP, has high protein content and protein-to-fat ratio. Other commercial products have much lower protein content and protein-to-fat ratio. This indicates that NEPI 250 and VICTORY HEMP may be much better than other products among the products tested.

[0146] Figures 9-11 show SDS PAGE gel data of the NEPI 250 product and the commercial product showing protein composition, structure, and integrity (non-reducing conditions are shown in Figure 9 and reducing conditions are shown in Figure 10). For Figure 9, 910 is edestin dimer and 920 is albumin. For Figure 10, 930 is edestin acidic subunit, 940 is edestin basic subunit, and 950 is albumin. Figure 11 shows prior art SDS PAGE data showing the known molecular weight of edestin and edestin products under similar conditions. Lanes are identified below and apply to Figures 9 and 10. M=molecular weight standard 1=DP-276 HempLife Powder SD HPI 2=DP-276 HempLife Powder SD HPI 3=DC-344 HempLife Liquid Concentrated HPI 4=GH-350 Good Powder Hemp HPI 5=A-560 Anthony's Powder HPC 6=LP-643 Dehulled HempLife SD Powder HPI 7=VH-794 Victory Hemp Powder V70 HPI 8=N-950 Nutiva Powder HPC 9=N-950 Nutiva Powder HPC

[0147] Figure 11A shows a prior art SDS PAGE from hemp proteins published by Mamone and Wang (Mamone et al., 2019; Wang and Xiong, 2019). Figure 11B shows a prior art SDS PAGE from hemp proteins published by Shen (Shen et al., 2020).

[0148] In summary, Figures 9-10 show that the NEPI 250 product has a different protein composition than other commercial products, generally more structurally intact, with VICTORY HEMP being the closest in terms of native edestin content and non-degraded protein products. Interestingly, as expected, the NEPI 250 product was substantially free of albumin. In this disclosure, it is hypothesized that albumin impedes the ability of hemp protein isolate to form a well-structured protein food 120 with superior textural properties. This theory is supported by the texture profile analysis data shown in Table 2, where the NEPI product has a much better hardness and chewiness compared to commercial hemp protein products. It is also possible that the superior native structural features of NEPI 250 edestin contribute to the formation of the unexpectedly superior textural properties of NEPI 250 shown in Table 2. The superior structural preservation of the native state of edestin is further shown in Table 3, and Figures 12 and 13 show differential scanning calorimetry data for the product.

[0149] Table 3 shows differential scanning calorimetry thermograms that provide structural information about the edestin contained in NEPI 250 and commercial products. DSC thermograms of two NEPI products (Figure 12) and two commercial hemp protein powders, VICTORY HEMP and NUTIVA (Figure 13). Based on the DSC results, the NEPI products are superior in terms of structure compared to the commercial products, indicating that the edestin in NEPI 250 is in a more native state than the commercial products.

[0150] Compared to hemp protein isolates produced by conventional means, the quality of edestin in NEPI 250 is superior, as described above in the background art. Furthermore, compared to the process of the present disclosure, the protein extraction methods of the prior art have significant drawbacks and limitations. For example, the salt extraction and dialysis in the HMI process do not remove residual phenols from the final product. Furthermore, HMI is not very commercially viable.

[0151] The disclosed process has many advantages over the prior art. The disclosed process may release phenols and tocopherols from NEPI 250 and AOAE 230. The disclosed process may make hemp oil 560 more oxidatively stable. The disclosed process may separate phenols from hemp oil 560 during aqueous wet milling, thereby providing stability.

[0152] The process of the present disclosure differs from conventional methods of extracting protein from hemp grain in that conventional methods generally involve crushing the grain to extract oil and produce a hemp grain cake, which can then be milled and sieved to produce a flour. The resulting cake or flour may contain aggregated edestin and albumin, along with oil, carbohydrates, phenolics, and minerals. In some cases, the seeds can be dry-milled to produce a flour directly.

[0153] Mechanical processes involving high temperatures or pressures, such as crushing the grain, can cause chemical bonds to form between edestin and albumin. Crushing whole or dehulled hemp kernels can cause edestin and albumin to aggregate.

[0154] High pressure can alter protein structure and cause protein aggregation. According to Yang, high pressure modification of proteins involves changes in the secondary, tertiary, and quaternary structures of proteins from the native state through intermediate states to a fully denatured state (Yang et al., 2016). High pressure alters protein structure mainly through changes in non-covalent electronic interactions, hydrophobic interactions, and hydrogen bonds. High pressure can also result in the formation of new disulfide bonds, which can stabilize denatured proteins or cause protein aggregation (Yang et al., 2016).

[0155] Heat is also known to change the structure of proteins. Heat generated by friction during grain milling can change the structure of proteins. Heat can lead to protein denaturation and the formation of protein aggregates. Aggregation between edestin and albumin can occur during dry milling, where temperatures can reach over 100°C.

[0156] In one embodiment, the NEPI can then be heated to a temperature of about 145°F for about 30 minutes to pasteurize the product. In some jurisdictions, 145°F may be the legal lower limit for pasteurization. In one embodiment, the temperature can be maintained at about 145°F, or between 145°F and 155°F to prevent granulation. In this disclosure, it has been observed that granulation occurs at a temperature of about 158°F. In NEPI, granulation may occur at temperatures well below the denaturation temperature of edestin, for example, about 158°F, which has been shown to be about 95°C. It is very important to pasteurize the NEPI at a temperature lower than those typically used by those skilled in the art for pasteurization of plant proteins for use in food products. Those skilled in the art conventionally pasteurize protein isolates at temperatures that cause significant granulation in this disclosure in order to process the product quickly. The pasteurized NEPI 270 is the result of washing and dilution with cold water 232.

[0157] As shown in FIG. 3, after pasteurization 104 is complete, the NEPI 250 can be spray dried via the NEPI spray drying process 300 or stored refrigerated as a concentrate for use in the production of structured protein food 120. The solids content of the NEPI concentrate immediately after centrifugal decanter separation ranges from about 35% to 45%, making it a thick paste that is difficult to pump. At this point, cold water is added to reduce the solids content of the NEPI 250 concentrate, preferably to about 30%, and make the slurry easier to pump quickly through heated pipes maintained at a temperature not exceeding 158° F. The dilution allows for more turbulence, improving heat distribution when heating to 145° F, allowing pasteurization without the formation of undesirable overheated protein aggregates and granules in the finished dried edestin product. Prior to spray drying, the NEPI concentrate can be held in a tank at about 145° F, or at the pasteurization temperature. Spray drying 306 can then be performed at a higher spray drying 306 temperature or at a temperature where existing products can reach about 158° F. or higher, which may cause protein aggregation and result in a functionally inferior NEPI 250. This protein aggregation may be visible at about 100 kDa on a non-reducing SDS-PAGE gel (shown in FIG. 9) where bands other than the expected edestin or hemp grain protein bands are visible. Bands present at higher molecular weights than the expected about 50 kDa band for edestin dimer under non-reducing conditions may represent aggregation caused by excess heat during spray drying 300. Thus, in some embodiments, one potential way to determine if the maximum spray drying 300 temperature is below the temperature at which significant protein aggregation occurs may be to identify unexpected higher molecular weight bands on a non-reducing SDS-PAGE gel. Microwave drying is another method that can be used in the present disclosure; during microwave drying, the NEPI 250 is kept at a low temperature, such as between 130 F and 140 F, while moisture is removed under vacuum pressure.

[0158] FIG. 4 illustrates a process for adding color to structured protein food 120. White and dark meat analog process 400 can produce either white meat NEPI 412, which can replicate chicken or fish, or dark meat NEPI 422, which can replicate beef or chicken thigh. To produce white meat NEPI 422, dehulled hemp grain 105 can be used. In one embodiment, dehulled hemp grain 105 can be subjected to native edestin protein isolation process 200, which results in white meat NEPI 412, which can be used in structured protein food process 100 to produce a white meat replica. To produce dark meat NEPI 412, whole hemp grain 101 can be used. In one embodiment, whole hemp grain 101 can be subjected to native edestin protein isolation process 200, which results in dark meat NEPI 412, which can be used in structured protein food process 100 to produce a dark meat replica. In one embodiment, the use of some whole hemp grain and some dehulled hemp grain, where the whole hemp grain is used at a concentration of about 20-30% by weight relative to the amount of dehulled hemp grain, may result in a blood meat NEPI 412 or a medium color NEPI 432. In one embodiment, the husk previously removed by dehulling the hemp grain may be reintroduced to the dehulled hemp grain 105 to add color, where in one embodiment, the husk may be added to the dehulled grain 105 in an amount of about 10-15% by weight relative to the dehulled hemp grain to achieve a blood meat color, producing a medium color NEPI 422.

[0159] FIG. 5 shows an oil and albumin extraction process 500. The product of the native edestin protein isolation process 200, AOAE 230, can be a process to produce albumin 550 and hemp oil 560. In the oil and albumin extraction process 500, the AOAE 230 can be evaporated to a concentrate 506. The product is homogenized 504 and pasteurized by heating 530. It can be useful to clarify the AOAE 502. Heating to 180F 520 may break the emulsion. Evaporate to preferably more oil than water 506. Cool to near or frozen 508. Centrifuge in a creamery separator 510 to obtain albumin 550 or hemp oil 560.

[0160] FIG. 6 illustrates a hydrosol formation process 600 in which NEPI 250 can be combined with preheated water to form a protein hydrosol 108 substantially as described in FIG. 2. In the hydrosol formation process 600, water preheated to about 135° F. can be added to the NEPI 250 and mixed 106 under high shear to form the protein hydrosol 108. The protein hydrosol can be pasteurized at 145° F. before, during, or after formation of the protein hydrosol. Pasteurized conditions must be maintained or created after the production of the NEPI 250. The pasteurized product 104 can be prepared by first hydrating the NEPI 250, or otherwise maintaining an appropriate degree of hydration of the NEPI 250 and maintaining pasteurized conditions to the greatest extent possible when spray drying 306 to form a NEPI powder 308. In one embodiment, the amount of preheated water added to the NEPI 250 can be about 3 parts water to 1 part NEPI by dry solids weight of the solution. In some embodiments, the NEPI can be frozen 310 in a refrigerator and lyophilized 312 to produce a NEPI powder 308. Heating 111 the hydrosol to 130 F can be useful. Heating the oil to 110-115 F can be useful.

[0161] In some embodiments, the preheated water may be tap water, and in some embodiments, tap water sourced from Lake Erie, and may be substantially free of solutes (e.g., tap water, distilled water, or deionized water). Salt should not be added to the solution during the hydration and protein preparation process, as it may destroy the structure of the protein hydrosol 108 or protein-fat hydrosol 114. Salt may be added after hardening, but not before hardening. In some embodiments, the protein hydration and unfolding may be carried out at 100°F to 135°F, or in some embodiments, between 100°F to 155°F (without being bound by theory, so that the protein structure is slightly altered or unfolded to allow for proper interaction with the oil during the formation of the protein-fat hydrosol 114); alternatively, in other embodiments, the protein hydrosol formation may be carried out at lower temperatures, but the temperature must be higher than the low temperature at which the protein cannot be hydrated and unfolded. Preferably, the temperature during the hydration and protein preparation steps should remain as close as possible to 145°F or the pasteurization 104 temperature without reaching temperatures that may cause protein aggregation and granulation. Once the protein hydrosol is formed, the preheat oil 109 may be heated to between 110°F and 115°F in some embodiments, between 100°F and 155°F in other embodiments, or in some cases may be held at a temperature above what would be considered too low to allow the addition of oil to destroy the protein hydrosol structure, but below the temperature at which protein-fat hydrosol granules 114 would result.

[0162] In some embodiments, the protein-fat hydrosol 114 can be produced by combining fat with a warm suspension of hydrated protein (e.g., protein isolate containing edestin) having a pH between 6.5 and 7.8 (e.g., pH 7.5). Rapid mixing, such as in a Waring blender or handheld homogenizer, or homogenization of this mixture forms an emulsion. The physical properties of the protein-fat hydrosol 114 can be controlled by varying the type of protein, protein concentration, pH level during homogenization, speed of homogenization, and fat to water ratio.

[0163] To form the protein-fat hydrosol 114, a polyunsaturated fatty acid (PUFA) oil or fat, which may preferably be coconut oil or fat, may be heated to just above the melting point of the fat and added to the protein hydrosol 108. Without being bound by theory, the fat may form a layer surrounding the hydrated native edestin, thereby forming a liquid matrix that essentially encapsulates the hydrated protein, i.e., the protein-fat hydrosol 114, forming a hydrated protein-in-oil emulsion that effectively creates a thick and stable gel. Effectively, the oil may seal and protect the hydrated protein structure. Hydrated proteins can hold significantly more fat in the gel state than dry proteins. In general, as discussed in this application, it has been found that native globular proteins that are first hydrated and then gently heated below their denaturation temperature can hold up to twice their weight in fat. The water content of the protein-fat hydrosol may range from about 30% to about 70% by weight in some embodiments. Moisture content refers to the amount of water in a material as measured by analytical methods, calculated as a percentage of mass change after the water evaporates from a sample.

[0164] In any of the methods or compositions described herein, the protein fat hydrosol 114 may include flavorings or other added ingredients. The following ingredients may be optionally added: fat soluble or other flavor systems, salts including sodium chloride, plant based albumin sources, plant based insoluble or soluble fiber, typically less than 2% by weight based on the finished protein fat hydrosol 114. Starch may be added alone or in combination with other soluble carbohydrates including complex carbohydrates or sugars, as appropriate, at levels up to about 10% by weight, more preferably less than 5% by weight. Supplementary ingredients may be added to the protein fat hydrosol 114 prior to hardening for the purpose of improving and modifying flavor or texture. Fiber may be added to reduce the "squeakiness" of the structured protein food product 120.

[0165] In one embodiment, the protein-fat hydrosol 114 may, in one aspect, comprise about 15% to about 25% by weight, more preferably about 18% to about 22% by weight of protein, where the protein may be a natural oil seed protein; in one embodiment, about 75% to about 85% by weight of the protein isolate comprises globular protein, preferably the protein isolate comprises less than 15% by weight of albumin, more preferably less than 5% by weight of albumin. More importantly, the globular protein may be in its natural state and may have a significant amount of the amino acid cysteine, preferably in amounts greater than casein or soy protein isolate. The balance of the protein composition may, in some embodiments, be primarily minerals such as calcium and phosphorus. Natural oil seed globular protein may preferably have a significant amount of cysteine.

[0166] The protein-fat hydrosol 114, in one embodiment, can contain from about 40% to about 70% water by weight, or more preferably from 40% to 60% water by weight.

[0167] The protein-fat hydrosol 114, in one embodiment, can contain from about 0% to about 35% fat by weight; the ratio of saturated to polyunsaturated fatty acids (PUFAs) is between 100% saturated fat and 100% PUFAs by weight. Combining fats between these two amounts results in a variety of unique textures not previously reported, depending on the amount of protein used in combination with the fat.

[0168] The protein-fat hydrosol 114 may optionally include about 0% to about 5% by weight starch in some embodiments. The amount of starch added may depend on the amount of water added beyond the amount of water added to the protein required for hydration of the protein.

[0169] The protein-fat hydrosol 114 can be formed by manually or mechanically mixing the ingredients to form the protein-fat hydrosol 114. Preferably, the hydrated protein is first warmed to just below the granulation temperature of the protein, the oil and / or melted fat is added, and the mixture is preferably gently homogenized.

[0170] In one embodiment, the protein-fat hydrosol 114 can be mixed at a temperature between 120°F and 150°F. The temperature range for subjecting the protein to a heated environment without destroying the gel or matrix formed has been found to be between 70°C and 100°C. These temperatures are significantly lower than the extrusion temperatures typically required for extrusion of traditional meat analogue proteins such as soy. The denaturation and fiberization temperatures of soy protein under conditions typically used in extruders range from about 130°C to 140°C. In accordance with the present disclosure, good texturing can be obtained by oven heating the protein-fat hydrosol 114 and / or by pressure cooking (retorting) the protein-fat hydrosol 114 to aggressively harden the protein.

[0171] The physical properties of the protein-fat hydrosol 114 are those of the hydrosol. The viscosity depends on the oil, fat, and water and protein content. More water substantially reduces the viscosity even at low protein to fat ratios. Similarly, very low protein to fat ratios and low water can result in very high viscosity. The quality and choice of fat and protein systems also have a significant effect on the viscosity.

[0172] Formation of the protein-fat hydrosol 114 can be performed below the denaturation point of the native protein. However, according to the present disclosure, storage at that temperature is undesirable since the protein-fat hydrosol 114 is not microbiologically stable. It is preferable to immediately heat treat it to harden the protein shape. The liquid matrix can also be cooled in a heat exchanger or other methods to below 6° C. for storage prior to further processing.

[0173] 7 illustrates a retort process for NEPI 770 resulting in a structured protein food product 120. The NEPI protein-fat hydrosol 114 is dispensed into formed TETRAPAK 200 mL containers 702, and in one embodiment, each container is filled with 180 g. The tops can be sealed 704 using a TETRA RECART machine. The filled NEPI protein-fat hydrosol can then be placed 706 into a retort machine. The NEPI protein-fat hydrosol can then be heated 708 under retort conditions to harden 710. In some embodiments, this process results in a structured protein food product 120.

[0174] With respect to retort according to the present disclosure, Figures 14-18 show photographs of the results of retort processing of various NEPI products and commercial hemp protein powders. Each figure includes a close-up of the retort processed products. Boiled chicken was used as a standard. Table 6 below shows the results of the texture profile analysis of the retort processed hemp products. Tables 7 and 8 show the colorimetric data of each product produced and tested by retort processing, using boiled chicken breast as a standard.

[0175] 14-18 show photographs of retorted NEPI dehulled powder 250, the solids having a protein to fat (NEPI 250 to coconut oil) ratio of about 2:1 and a solid to liquid (water) ratio of about 2:3. After preparation of the protein-fat hydrosol, the retorted product was produced as known to one of skill in the art.

[0176] Figure 14A is a photograph of a cross section of boiled chicken breast. Figure 14A is a magnified photograph of the cross section of the boiled chicken breast of Figure 14A; Figure 14C is a magnified photograph of the cross section of the boiled chicken breast of Figure 14B.

[0177] FIG. 15A is a photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain concentrate; FIG. 15B is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp concentrate of FIG. 15A; FIG. 15C is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain concentrate of FIG. 15B in accordance with the present disclosure.

[0178] FIG. 16A is a photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain powder; FIG. 16B is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain powder of FIG. 16A; FIG. 16C is a magnified photograph of a cross section of a retort meat analog using NEPI dehulled hemp grain powder of FIG. 15B in accordance with the present disclosure.

[0179] FIG. 17A is a photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder; FIG. 17B is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder of FIG. 17A; FIG. 17C is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder of FIG. 17B in accordance with the present disclosure.

[0180] FIG. 18A is a photograph of a cross section of a retort meat analog using HEMPLAND dehulled hemp grain powder; FIG. 18B is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp powder of FIG. 18A; FIG. 18C is a magnified photograph of a cross section of a retort meat analog using VICTORY HEMP dehulled hemp grain powder of FIG. 18B in accordance with the present disclosure.

[0181] Figure 8 illustrates a process for extruding NEPI 250 to produce a textured structured protein food 120. Figure 8 illustrates the steps of providing an extruder having a heated auger, preferably in one embodiment a hollow steam heated auger 800, or other type of heated auger extruder. In one embodiment, the extruder may be a POWERHEATER PH 100 provided by SOURCE TECHNOLOGY. The technology used in this machine that can be utilized in the present disclosure is described in U.S. Patent Nos. 10,893,688, 10,624,382, 10,149,484, 10,092,013, 10,028,516, 9,931,603, U.S. Patent Application Publication Nos. 2010 / 0062093, 2011 / 0091627, 2019 / 0299179, 2020 / 0113222, 2020 / 012095, and 2020 / 02680205, which are incorporated by reference in their entireties. The POWERHEATER PH 100, in this disclosure, has a hollow auger design that allows steam to be introduced into the auger to heat the auger and provide a more uniformly heated protein-fat hydrosol, allowing better control of the temperature of the auger and the inner wall of the extrusion pipe or chamber, which is very important for properly setting up this disclosure. Conventional extruders, such as those developed by CLEXTRAL or WENGER, have been tested according to this disclosure but did not provide a satisfactory end product. The conventional extruders caused the protein-fat hydrosol of this disclosure to adhere to the inner wall of the extruder pipe.

[0182] The POWERHEATER PH 100 is known to be used with fiberized input materials, but is generally used to set starch as opposed to protein as the input material. Since extrusion of protein hardeners is generally performed at temperatures well above 100°C, protein hardener input materials are not considered to be used with the POWERHEATER PH 100. However, the protein-fat hydrosols of the present disclosure are effectively textured and fiberized by the POWERHEATER PH 100 at 75°C, which is accomplished at relatively low temperatures between about 75°C-85°C, with the auger and extruder preheated to between 75°C-85°C, and extrusion occurring in the range of about 70°C-95°C. In one embodiment, the protein-fat hydrosol extruder uses an 8mm screw size rather than a 3mm screw size using the POWERHEATER PH 100 at 75°C. The protein-fat hydrosol can be dosed into the POWERHEATER PH 100 using a suction or stuffing pump, and the starting temperature can be about 85° C. 802. After pumping the protein-fat hydrosol into the extruder 804, extrusion can proceed at about 75° C.-85° C., and the protein-fat hydrosol does not adhere to the inner walls of the extrusion pipe 806. This process produces a textured structured protein food 120. The textured structured protein food 120 extruded according to the present disclosure has been demonstrated in testing to have a texture, fiber, and color similar to cooked chicken breast, and has superior and unexpected properties in view of the prior art and the knowledge of one of ordinary skill in the art.

[0183] 19 shows a photograph of extruded NEPI and boiled chicken breast pieces from dehulled flour according to the present disclosure, extruded on a POWERHEATER PH 100 as described above, showing similarity in texture and fiber. The boiled chicken breast 1910 is shown next to an extruded NEPI 250 chicken product 1920 made and processed from spray dried dehulled hemp grain NEPI according to the present disclosure. This result is unexpected, coming from using hemp grain with only three ingredients, NEPI 250, coconut oil, and water, in a ratio of 2:1:3, respectively.

[0184] It has been found that in most extrusions, including the extrusion of soy-based meat analogs, the protein to fat ratio is typically greater than 10:1. Thus, extruded, denatured, and fiberized soybeans are unable to retain much fat. However, a hydrated gel of a natural globular protein such as edestin, according to the present disclosure, can retain up to twice its weight in fat, even after the formation of a set or solid form of the gel produced by application of radiant, microwave, or other forms of heat, including direct heating or extrusion.

[0185] According to the process of the present disclosure, the protein-fat hydrosol 114 can be cured to a solid state at temperatures between about 70° C. and 100° C., depending on the concentration of protein in the system. The low curing temperatures are consistent with denaturation of the native protein of NEPI 250.

[0186] According to the present disclosure, the solid structure formed during extrusion is allowed to cool and is representative of a hardened product, but with incomplete denaturation, similar to uncooked protein or "raw" meat. Further heating of the "uncooked" protein further denatures the protein, enhancing its shape, elasticity, texture, etc., and eventually releasing some moisture as well. According to the process of the present disclosure, it is not desirable to heat the product to the extent that significant amounts of water are released from the hardened product in the extruder. Rather, it is desirable to simply solidify the gel and protein shape or texture. In one embodiment, the present disclosure describes a process for preparing a structured protein food 120 in an extruder that resembles a raw meat or dairy analog, or raw animal meat. Further cooking of the raw meat analog by traditional or commercial means toughens and toughens the meat.

[0187] The process according to the present disclosure contrasts with existing technologies that create a meat-like texture by using fully denatured proteins that are then co-blended with fats, starches, and other binding agents, including other proteins, to form the appearance of a hamburger-type material, where this type of set is achieved during cooking primarily through the gelation of added raw proteins such as starch or gluten.

[0188] The final texture of the structured protein food product 120 may depend on the properties of the liquid matrix, including the ratio of protein, fat, and water, as well as the extrusion conditions. As described herein, the extruded mixture of isolated plant proteins may be referred to as the structured protein food product 120, which may be a meat analog, and the fibrousness and tensile strength of the meat analog may be controlled by co-variation of extrusion parameters such as temperature, pressure, throughput, and die size. For example, a combination of lower extrusion temperature, medium / low throughput, and smaller die favors the production of a highly fibrous tissue with low tensile strength, while a combination of higher extrusion temperature, higher throughput, and larger die favors the production of a less fibrous tissue replica with very high tensile strength.

[0189] The fiber quality and tensile strength of the meat analogs can also be adjusted by modifying the composition of the extrusion mixture, for example, by increasing the ratio of isolated plant protein to fat and water or by reducing the moisture content in the extrusion mixture, meat analogs with finer fibers and greater tensile strength can be produced.

[0190] Extrusion of the liquid matrix involves feeding the liquid matrix into an extruder. In some embodiments, the extruder may be a SOURCE TECHNOLOGY POWERHEATER PH 100. CLEXTRAL and WENGER twin screw extruders have been tested, but without satisfactory results. According to the process of the present disclosure, cooling is important in extrusion to achieve a temperature below 21°C, which allows the saturated fats to harden easily in the structure and allows the product to be cooled more efficiently to refrigeration or freezing temperatures.

[0191] For each product, the wet ingredient blend is transferred to a feeder and the liquid matrix is ​​metered through the extruder feed port at a constant input rate. In traditional extrusion, the dry protein product is fed into the input of the machine. As the dry product moves through the machine, water and fat are introduced through separate inputs. In contrast, during the process according to the present disclosure, the hydrated protein and oil are mixed first, as described herein above, to tightly control the chemical reactions that occur during the formation of the protein-fat hydrosol 114. Thus, in some embodiments, additional water, starch, or fat may or may not be added to the extruder during extrusion. In some embodiments, fiber may also be added.

[0192] In conventional extrusion of plant-based meat analogs, adding water and fat before starting extrusion can result in undesirable steam release as water escapes from the product as the temperature increases. Therefore, in the present disclosure, the process of adding water and fat is tightly controlled during extrusion. In the process according to the present disclosure, the liquid matrix extrusion mixture is specifically designed to prevent water from being released from the product by forming a gel. During preparation of the liquid matrix according to the present disclosure, oil is added to the hydrated protein to form an emulsion gel that prevents water from being released from the product during extrusion, which would otherwise cause steam to be released from the machine. The formation of a gel also allows for the maintenance of high moisture in the liquid matrix during extrusion and in the final product, which is desirable for the excellent texture of the structured protein food product 120.

[0193] The temperature during extrusion is important for the resulting product. The temperature should be gradually increased and maintained between about 70°C and 100°C, or between 100°C and 110°C. In conventional extrusion, the temperature in the extruder is generally above 130°C. In the disclosed process, the low temperature prevents the destruction of the protein-fat hydrosol 114, thereby allowing the molecular structure of the compound to remain substantially or partially intact. The temperature of the protein-fat hydrosol 114 is preferably maintained at about 75°C to 85°C to harden the protein-fat hydrosol 114, and then cooled to reduce the temperature to less than 21°C during the extrusion process. In the disclosed process, it is important to maintain a lower temperature than that used during conventional extrusion. Here, the temperature is increased only to a temperature that allows the setting of disulfide bonds so that the fat is fully incorporated between all the peptide layers of the protein. The residence time within the extruder or heated environment must be sufficient to allow the input temperature of the liquid matrix to reach between 70°C and 110°C, preferably between 75°C and 85°C.

[0194] Preferably, the extruder rotates the protein-fat hydrosol 114 at a relatively low screw speed, measured in revolutions per minute (rpm), during extrusion to form a meat analog that maintains a gel structure and maintains a high degree of moisture in the product. The screw speed can be carefully monitored to prevent temperature buildup and disruption of the chemical structure of the liquid matrix.

[0195] To prevent destruction of the structure of the loose protein-fat hydrosol 114 formed by the encapsulation of hydrated proteins and fats, it may be essential to move the gel slowly through the thermal system to maintain initial gel hardening (partial protein denaturation) with some shape formation and some fiberization. Fermentation (as occurs in cheese making), or complete cooking and denaturation, eventually occurs during subsequent use of the product. In some embodiments, the finished extruded product, having a moisture content between 35% and 75% by weight, can then be fully cooked at high temperatures by conventional or commercial cooking processes, fermented for microbiological stability, refrigerated or frozen for a period of time to obtain the desired finished texture before consumption, if desired. Additional relevant extrusion parameters may include die diameter, die length, product temperature at the end of the die, and feed rate.

[0196] The final product after extrusion may have a structure more similar to animal meat than conventional or known structured protein foods such as meat and dairy analogs. Without being bound by theory, extrusion of the protein-fat hydrosol 114 according to the present disclosure may allow the proteins to form substantially aligned protein fibers, which may be defined as continuous filaments of discrete lengths composed of proteins held together by intermolecular forces, such as disulfide bonds, hydrogen bonds, electrostatic bonds, hydrophobic interactions, peptide chain entanglements, and Maillard reaction chemistry that forms covalent crosslinks between protein side chains. The strength of the cured product after the initial extruder is not complete or as strong as it could be. In fact, it may be desirable to remove the completed heat-set product and subject it to further heating by direct or indirect heat, common cooking methods, such as boiling, baking, frying, roasting, microwaving, fermenting, and pressing (as in cheese production involving salting and acid addition), to name a few, to complete the strength and shape of the initial cured product.

[0197] The preparation and extrusion conditions of the protein-fat hydrosol 114 by the process of the present disclosure may, in some embodiments, allow the substantially aligned protein fibers to retain up to about 50% fat by weight within the protein. Thus, the final product is not oily and has a mouthfeel and fat release during chewing that is closer to animal meat than existing meat analogs. Mouthfeel may refer to a combination of properties such as moistness, chewiness, bite force, disintegration, and fattiness that provide a satisfying sensory experience.

[0198] The expected final structure of the structured protein food 120 may vary based on the composition of the protein-fat hydrosol 114. In one embodiment of the present disclosure, the expected final composition of the structured protein food 120 is shown in Table 4 by weight of protein, weight of carbohydrate (if present), weight of lipid, and weight of water, along with other potential ingredients. Table 4 shows the physical properties of the structured protein food 120 shown in Table 4. Table 5 shows the physical properties of the structured protein food 120 shown in Table 4. After extrusion is complete, the product can be cooled, shaped or cut. Post-processing steps can be performed on the extruded product.

[0199] Meat analogs, also referred to herein as structured protein foods 120, can be produced from the protein-fat hydrosol 114 by methods other than extrusion. Additional methods of producing meat analogs from the protein-fat hydrosol 114 include the application of mechanical energy (e.g., shear, pressure, friction), radiation energy (e.g., microwave, electromagnetic), and thermal energy (e.g., heating, steam texturization).

[0200] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0201] Example 1 Preparation of native edestin protein isolate (NEPI) Hemp kernels were obtained from Hemp Oil Canada, Manitoba Canada and River Valley Specialty Farms, Manitoba Canada. Dehulled kernels were obtained from River Valley Specialty Farms and whole kernel kernels were obtained from Hemp Oil Canada.

[0202] HHG contained 5.5% by weight moisture, 46% by weight dry basis Kjeldahl protein, 35% by weight dry basis fat, and a protein to fat ratio by weight of 1.3 to 1. WHG contained 8.8% by weight moisture, 22% by weight dry basis Kjeldahl protein, 30% by weight dry basis fat, and a protein to fat ratio by weight of 0.7 to 1.

[0203] 1000 pounds of HHG was mixed with 5000 pounds of water at 34°F in an 800 gallon stirred tank. The HHG was wet milled while maintaining a temperature between 34°F and 38°F. The hemp slurry was milled in a Silverson rotor-stator tank at a rate of 56 gallons per minute for 30 minutes to wet mill the HHG. The diluted slurry was held for an average time of 30 minutes. The extract was separated from the insoluble by-products using a SWECO 60 inch screen size 120 mesh to remove the majority of the solids. The permeate of the 120 mesh screen was then passed over a 200 mesh screen on another SWECO vibratory screen to obtain a slurry which was then transferred to a 500 gallon jacketed tank to maintain the temperature of the slurry between 34°F and 38°F. The slurry was then fed to a DELAVAL centrifuge decanter at a rate of 13 gpm to obtain a separation of the edestin solids from the AOAE emulsion. The AOAE emulsion was then passed through a tubular heat exchanger system for pasteurization at a maximum temperature of 185F for 10 minutes. The AOAE was then held in a 900 gallon tank for processing. The edestin solids at 40% solids were diluted to 30% solids with cold water and pumped through a preheated tubular system set at less than 150F, exiting the system at 146F into a jacketed holding tank with a temperature of 145F in the jacket. After 30 minutes, the material was passed through a heat exchanger to cool to 35F and placed in a tote in a refrigerator for further processing and drying with a spray dryer.

[0204] 1000 pounds of HHG was mixed with 5000 pounds of water at 34°F in an 800 gallon agitated tank. The HHG was wet milled while maintaining a temperature between 34F-38F. The hemp slurry was milled in a Silverson rotor-stator tank at a rate of 48 gallons per minute for 30 minutes to wet mill the WHG. The diluted slurry was held for an average time of 30 minutes. A two-stage SWECO 60 inch screen, size 60 mesh, was used to separate the extract from the insoluble by-products and remove the husk. A 200 mesh screen was installed on the second stage of the SWECO and the slurry from the Silverson was passed first through the 60 mesh to remove the husk and immediately dropped to the top of the 200 mesh screen, which removed the chloroplasts and fines. The rate through the SWECO was approximately 6 gpm and the screened slurry was sent directly to a jacketed 500 gallon jacketed tank where the temperature of the slurry was maintained between 34F-38F. Once the tank was full, the slurry without husk, fiber, or chloroplasts was fed at 13 gpm to a DELAVAL centrifuge decanter to obtain separation of edestin solids from the AOAE emulsion. The AOAE emulsion was then pasteurized through a tubular heat exchanger system at a maximum temperature of 185F for 10 minutes. The AOAE was then held in a 900 gallon tank for processing. The light brown edestin solids at 40% solids exiting the decanter were diluted to 30% solids with cold water and pumped through a preheated tubular system set at less than 150F, which exited the system at 146F and into a jacketed holding tank with a temperature of 145F in the jacket. After 30 minutes, the material was passed through a heat exchanger to cool to 35F and placed in a tote in a refrigerator for further processing and drying with a spray dryer. Dry matter basis yield of NEPI based on starting WGH weight was 15% or 79% of theoretical. AOAE yield was 25.3% on DSB and husk, fiber and chloroplast fractions were 46.9% on DSB. Overall recovery was 92%. NEPI yield from HHG was 30% or 86% of theoretical. AOAE yield was 40.9% on DSB and husk, fiber and chloroplast fractions were 22.5% on DSB.The overall recovery was 98%. Analyses of the NEPI products obtained from WGH and HHG are shown in Tables 1 and 2 below. [Table 1]

[0205] NEPI concentrate prepared by the disclosed process still exhibits high microbial activity prior to pasteurization and spray drying to powder while maintaining process temperatures below 38F. (See Table 1). Incoming raw materials, whether hemp grain or dehulled hemp, typically range in total plate count (TPC) from 2,000 TPC to 250,000 TPC. In protein-rich aqueous media, it is important to maintain temperatures below 42F, preferably below 38F. Despite the lower temperatures, if not pasteurized soon after the start of the aqueous milling, the TPC will continue to increase, leading to protein damage. The short duration of the process and the ability to pasteurize both the AOAE and edestin slurry immediately after separation by the centrifuge decanter are essential elements in the process. The resulting edestin product is pasteurized at a low temperature of 145F and retains its gelling function as previously described. The AOAE can be heated to much higher temperatures, above 145F and more preferably above 195F, for short periods of time, which is advantageous for further processing by centrifugation to remove residual insoluble solids, followed by emulsion breaking to separate the aqueous albumin and oil phases. The success of pasteurization of the NEPI product in its final powder form is reflected in the product's TPC in Table 1. [Table 2]

[0206] The DSC thermograms are shown in Table 3. The structure of NEPI, as determined by DSC thermograms (partially shown in Figures 12A-12B and 13A-B), can be compared to the following commercially available products: [Table 3]

[0207] Further structural and compositional analysis of NEPI and commercial hemp protein products, as determined by SDS-PAGE gel electrophoresis, is shown in Figures 9 and 10.

[0208] Example 2 spray drying NEPI The NEPI cooled slurry from Example 1 was sent to a commercial spray dryer for drying. The powder was dried using an ALFA LAVAL type spray dryer with a nozzle capable of removing 1200 lbs of water per hour. The refrigerated product was pumped to a jacketed 250 gallon tank and the water temperature was set to keep the jacket at 155F. The tank had a slow agitator and it took several hours to heat up about 200 gallons of 30% concentrated edestin slurry. Once the product was up to temperature it was sent to another tank which fed into the dryer. It should be noted that the NEPI dried very easily without sticking to the walls of the dryer. The final outlet temperature of the dried product was 85F. The composition of the dried product is shown in Table 2 below for each NEPI (WG and HHG) product from Example 1.

[0209] Example 3 Preparation of protein-fat hydrosols from NEPI and commercial hemp powders The protein hydrosol is easily made by adding 14 lbs of water preheated to 140F in a 5 gallon plastic bucket. Slowly add 14 lbs of NEPI dry powder to the water while stirring using a 1 / 4 horsepower handheld industrial homogenizing bar. Maintain homogenization until all of the powder has been added. The temperature is now 130F and after holding for about 15 minutes, add 7 lbs of canola oil all at once and briefly mix the mixture with the homogenizing bar for about 1 minute or until the slurry appears well blended and the oil is incorporated as a uniform emulsion.

[0210] Example 4 Protein-fat hydrosol formulations and properties of various types of meat and dairy analogues Example 4 discloses a formulation containing a liquid matrix that can be used to produce different types of meat analogs. According to the disclosure, depending on the ratio of protein, fat and water, different types of meat analog articles can be obtained, including plant-based meat analog targets that replicate seafood, white meat, dark meat, egg and cheese. [Table 4]

[0211] With respect to Table 4, the moisture content target is between 35% and 75% by weight. A minimum of 70% by weight of spherical natural vegetable protein has an albumin content of less than 15% by weight, preferably less than 5% by weight. The temperature of the liquid matrix should be maintained at 140°F from mixing blending until processing. In Table 4, the ability of the natural seed oil protein to be natural edestin allows the amount of fat to be varied to obtain different types of meat analogs.

[0212] The structural characteristics of the resulting products are similar to those of the materials they replicate. For example, the texture of the seafood was white in color and had a very elastic structure similar to raw shrimp or scallops. The white meat was white in color and had a texture similar to that expected from a partially cooked chicken fillet. The dark meat was somewhat light brown in color and also had a texture similar to chicken thigh meat, being fatty and moister than the white meat. The eggs were similar to that expected for scrambled eggs and were white in color. The cheese was similar to cheese curds and indeed produced a squeak sound when biting into pieces similar to fresh cheese curds. [Table 5]

[0213] Example 5 Production of structured protein foods by retort Retort conditions were from a temperature of 77F to a peak of 270F over 15 minutes, decreasing to 95F in 15 minutes. Pressure was 0.20 bar at 1 minute, increased to 3.0 bar at 4 minutes, and decreased to 0.8 bar at 15 minutes. The machine used was a Sundry RETORT Type: AP-95, Serial Number: 705. [Table 6] TIFF2024505310000049.tif41162 [Table 7] [Table 8]

[0214] [Table 9]

[0215] Example 6 Producing structured protein foods by extrusion The protein-fat hydrosol from Example 3 was used in a Power 100 Source Technology extruder set at a flow rate of 6 lb / min and a 3 MM screw auger diameter at 185 F to create a structured gel with the appearance and texture of white chicken meat. See Figure 19 for a comparative photo of white chicken meat and the extruded hydrogel structured protein food product.

[0216] The present disclosure unexpectedly demonstrates that a surprisingly excellent hemp-based structured protein product can be produced using only three ingredients: hemp kernels, oil, and water.Hemp meat analogs produced according to the present disclosure are shown herein to reproduce chicken meat to a surprising degree in color, texture, and taste.Some commercially available protein products claim to produce excellent meat analogs, but when used for this purpose, they have not been compared to natural edestin protein isolate in taste, color, or texture.

[0217] No commercial products have been found that use only hemp protein to produce meat analogs. Furthermore, the prior art teaches that hemp protein alone is not a promising protein for producing structured protein foods such as meat and dairy analogs. This disclosure demonstrates that this is not the case.

[0218] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the claims.

Claims

1. A composition comprising a hydrogel structured protein product, said hydrogel structured protein product comprising edestin protein, fat, and water, said hydrogel structured protein product being a meat analog having textural characteristics similar to meat.

2. The composition of claim 1, wherein the edestin protein forms the major protein component in a protein-based hydrogel, and optionally, the composition has a hardness of at least 2500 and a chewiness of at least 2000 as measured by conventional texture profile analysis.

3. The composition of claim 1, wherein the ratio of edestin protein to fat in the hydrogel structured protein product is between 4:1 and 0.5:

1.

4. The composition of claim 1, wherein the hydrogel structured protein product is a fiberized product.

5. The composition of claim 1, wherein the hydrogel structured protein product contains less than 5% by weight albumin, and optionally is substantially free of albumin.

6. The composition of claim 1, wherein the hydrogel structured protein product comprises 35% to 75% wt / w water, the hydrogel structured protein product comprises 15% to 25% edestin protein, and the hydrogel structured protein product comprises 0% to 35% wt / w fat.

7. (a) selecting hemp grains; (b) aqueous wet-milling the hemp grains to produce a hemp grain slurry; (c) sieving to remove insoluble fiber from the hemp grain slurry; (d) centrifuging and decanting the hemp grain slurry; (e) separating a fraction containing the native edestin protein isolate from a liquid supernatant fraction containing soluble albumin and hemp oil; (f) producing an aqueous native edestin protein isolate mixture, said aqueous native edestin protein isolate mixture comprising native edestin protein isolate and water; (g) heating the aqueous native edestin protein isolate mixture to form a protein hydrosol; (h) combining the protein hydrosol with an oil to form a protein-fat hydrosol; and (i) extruding or heating the protein-fat hydrosol to produce a hydrogel structured protein food product.

2. A process for producing the composition of claim 1 by

8. 8. The process of claim 7, wherein the native edestin protein isolate contains less than 5% by weight albumin, and optionally is substantially free of albumin.

9. The process of claim 7, wherein the hydrogel structured protein product is produced by adding the protein-fat hydrosol to an extruder, the extruder being set at a temperature of between about 75°C and 95°C to produce the hydrogel structured protein product.

10. 8. The process of claim 7, further comprising adding water to the aqueous native edestin protein isolate mixture to produce a native edestin protein isolate aqueous slurry, and heating the native edestin protein isolate aqueous slurry to produce the protein hydrosol.

11. The process of claim 7, further comprising holding the protein hydrosol at a temperature between 63°C (145°F) and 68°C (155°F) for at least about 30 minutes to pasteurize the protein hydrosol.

12. The process described in claim 7, wherein the natural edestin protein isolate contains at least about 70% weight / weight edestin.

13. The process of claim 7, wherein the sieving includes sieving through a mesh size of 160 to 200 to selectively remove husk and fiber and substantially all chlorophyll-containing particles from the hemp grain slurry such that chlorophyll-containing particles are not visible without magnification in the native edestin protein isolate after sieving and decanting.

14. Use of edestin protein in increasing the hardness and chewiness of a composition comprising a hydrogel structured protein product, the hydrogel structured protein product comprising edestin protein, fat, and water, the hydrogel structured protein product being a meat analog having textural characteristics similar to meat.

15. The use described in claim 14, wherein the edestin protein is in a native or non-denatured state, and optionally, the hardness is at least 2500 and the chewiness is at least 2000, as measured by conventional texture profile analysis.

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