Natural decarboxylase proteins with excellent gel properties for preparation of food and cosmetics

Abstract

The present disclosure provides a structurally related family of pro-gelling decarboxylase homologues for use in commercial foods and cosmetics. The GPDH protein is expressed in cells in plants, animals and eukaryotic microorganisms in a trace form, and plays a catalytic role in a mevalonic acid pathway. Members of the GPDH family are structurally associated with one another through a series of strictly conserved amino acid sequence motifs. The ability to promote gelation when GPDH is used as a commercial product ingredient has been unclear yet before. The low gelation onset temperature (about 50 DEG C) and very low critical gelation concentration (4%) make the GPDH proteins of the present disclosure particularly suitable as functional protein substitutes in food and personal care products compared to most commonly used plant proteins and plant protein isolates.

Description

Related Applications

[0001] This application claims the benefit of priority of U.S. provisional application 63 / 411,112, filed September 29, 2023. The entire contents of this priority application are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present patent disclosure relates generally to the identification of natural sources of new product ingredients. A series of plant and microbially derived proteins are provided that have improved gelation properties suitable for use in commercial food, cosmetic, and other manufactured products. BACKGROUND

[0003] To improve the health of consumers and the planet, the market is rapidly growing in demand for food and cosmetics that are more ethically sourced, more sustainable, and more nutritious.

[0004] The food industry is currently undergoing a revolution as companies are working to create a new generation of plant-based products to meet consumer demand. The current focus is on developing plant-based foods that mimic traditional foods such as meat, fish, eggs, dairy products, and their derivatives. A key challenge is how to find ingredients from plant or microbial sources to mimic the appearance, texture, flavor, mouthfeel, and functionality required for traditional foods. The texture and mouthfeel of meat and dairy alternatives depend in part on gelation.

[0005] The cosmetics and beauty industry is undergoing a revolution to achieve more sustainable sources of ingredients and production. Goals include reducing the amount of synthetic ingredients, relying on natural ingredients instead of synthetics, and reconfiguring products to reduce water and energy consumption in the production process. In this context, the challenge is to find natural ingredients for cosmetics to achieve the target properties currently imparted by synthetics. SUMMARY

[0006] The present disclosure provides a structurally related family of gelation promoting decarboxylase homologs (GPDH) that can be used in commercial food and cosmetics. GPDH proteins are expressed at low levels intracellularly in plants, animals, and eukaryotic microbes and play a catalytic role in the mevalonate pathway.

[0007] Members of the GPDH family are structurally associated with a series of amino acid sequence motifs that are strictly conserved in GPDH proteins of diverse biological origin. A prototype of GPDH is the gelatinous protein SGP2A, which is a diphosphomevalonate decarboxylase MVD1 from Arabidopsis thaliana. Another GPDH prototype is the gelatinous protein SGP2B, which is a diphosphomevalonate decarboxylase MVD1 from Saccharomyces cerevisiae S288C.

[0008] The ability of GPDH to promote gelation as a food or cosmetic ingredient was previously unknown. The data provided in this disclosure demonstrate that recombinantly produced and purified GPDH has superior properties compared to commonly used plant proteins and isolates: specifically, reliable purity, low gelation onset temperature (about 50°C), low critical gelation concentration (4%), and little to no melt-back phenomenon. Natural GPDH that has already entered the food chain as part of an ingredient should face fewer regulatory hurdles as a food additive. For the above reasons, GPDH is well suited to replace gel and texture improving ingredients in meat, dairy, egg replacement products, and cosmetics. Food and its preparation

[0009] Food, ingredients, and additives containing a gel-promoting decarboxylase homolog (GPDH) at a concentration of 0.2% to 10% or 1% to 20% by weight of the dry ingredients in the food or ingredient are described in more detail below. This does not include whole organisms that naturally contain GPDH, such as yeast that might be added to a food for other reasons. The distinguishing method is to state that the GPDH is recombinantly produced, that the GPDH has a non-naturally occurring sequence, or that less than 1%, 2%, or 5% of the other proteins in the product or additive are from the organism in which the GPDH with essentially the same sequence is naturally expressed.

[0010] Also disclosed is a method of gelling or thickening a food during manufacture or upon heating, but including in the food a purified or recombinantly produced gel-promoting decarboxylase homolog (GPDH) at a concentration of 1% to 20% by weight of the dry ingredients in the food.

[0011] Also disclosed is a method of manufacturing a food that is free of animal products by recombinantly expressing GPDH, optionally purifying the recombinantly produced GPDH from other proteins produced by the host cell; and then preparing a food containing 1-20% by weight of the recombinantly expressed GPDH.

[0012] Also disclosed is a method of preparing or improving food products (e.g., increasing nutritional value and / or reducing environmental impact) in standard formulations by using GPDH in place of one or more previously used gelling or thickening agents: e.g., methylcellulose, carboxymethylcellulose (CMC), pectin, xanthan gum, guar gum, locust bean gum, carrageenan, starch, tapioca, and proteins obtained from chickpea, pea, lentil, egg, milk, wheat protein, and gelatin.

[0013] Any of these products and methods include the manufacture, distribution, and use of food products such as meat patties, meatballs, sausages, and chicken nuggets, plant ice cream, pies or tarts, cakes, soups, and frozen or refrigerated dough.

[0014] A meat substitute, replacement, or imitation is a food product in which one or more animal meats are replaced by plant or tissue-based ingredients selected to have similar texture and / or flavor. For example, a meat substitute can contain, e.g., at least 10% by weight protein content, where at least 75% of the protein content is a mixture of plant proteins and / or one or more tissue cultures; and optionally at least 5% by weight fat content, where at least 75% of the fat content is one or more oils isolated from a crop or culture. Optionally, the protein content and fat content form muscle and fat tissue imitations assembled in the product in a manner that approximates the physical organization of meat.

[0015] After cooking, the meat substitute or product made from the flavor additive preferably has a meat-related aroma and / or taste. For example, a meat-related flavor can be imparted by including 0.2% to 5% by weight of a heme-containing protein or a porphyrin-binding protein. The meat substitute typically contains a sugar such as glucose, ribose, fructose, lactose, xylose, arabinose, glucose-6-phosphate, maltose, and galactose, and mixtures of two or more thereof. To further enhance flavor, the product can also include a non-protein sulfur-containing compound such as cysteine, cystine, selenocysteine, thiamine, methionine, and mixtures of two or more thereof.

[0016] An ice cream containing a plant-based GPDH typically has at least 5% by weight protein content, where at least 75% of the protein content is a mixture of plant proteins and / or one or more tissue cultures; at least 5% by weight fat content, where at least 75% of the fat content is one or more plant-derived oils; and at least 5% by weight natural sweetener (and / or artificial sweetener to provide a desired sweetness). When frozen in an environment of -5 to -25°C, the composition remains mixed and has the mouthfeel of ice cream. Personal care products, pharmaceuticals and other uses

[0017] The following discloses in greater detail ingredients for cosmetic and personal care products that contain a gel-promoting decarboxylase homolog (GPDH) at a concentration of 1% to 20%, 1% to 5%, or at least 5% by weight of the product or ingredient, but less than 1%, 2%, or 5% of other proteins from the organism that naturally expresses GPDH. Depending on the context, the GPDH can improve or thicken the texture of the product or ingredient, or promote or stabilize the emulsification of its ingredients.

[0018] Also disclosed is a method of improving the texture, thickening, or emulsification of a cosmetic or personal care ingredient during production, which method comprises including in the product or ingredient a purified or recombinant GPDH at a concentration of 1% to 20% by weight of the product or ingredient. Also disclosed is a method of improving a cosmetic or personal care ingredient, which method comprises preparing the product using a formulation in which one or more previously used ingredients are replaced with a GPDH at a concentration of 1-20% by weight of the product or ingredient. For example, the previously used ingredient can be hyaluronic acid (HA), methyl or ethyl cellulose, hydroxypropyl methyl cellulose, gum, wax, or other currently used ingredients listed below.

[0019] The cosmetic or personal care product can be a moisturizer, eye or skin cosmetic preparation, lip balm, lip salve, emulsion, cleansing milk, skin cream, shaving cream, oral hygiene product, facial care product, skin care preparation, or tanning or sunscreen preparation. In addition to or instead of texture improvement, thickening, and emulsification, the GPDH can increase or improve viscosity, color or colorfastness, antibiotic activity, sun protection factor (SPF), water resistance, gloss, stability of activity, moisturizing activity, film formation, smoothness, lubricity, pearlescence, and physical structure.

[0020] Also disclosed below are pharmaceutical and nutraceutical products containing a pharmaceutically active agent or a nutritional ingredient in combination with a gel-promoting decarboxylase homolog (GPDH) as a pharmaceutical excipient, wherein the GPDH is at a concentration of 1% to 20% by weight of the dry ingredients in the composition. Also included are pharmaceutical and nutraceutical products containing a pharmaceutically active agent or a nutritional ingredient encapsulated in a capsule or granule, wherein the capsule or granule comprises a gel-promoting decarboxylase homolog (GPDH) at a concentration of 5-75% by weight.

[0021] More generally, the present disclosure provides an industrial product for commercial sale or public use that contains a gel-promoting decarboxylase homolog (GPDH) at a concentration of 0.2% to 10%, 1% to 20%, 1% to 5%, or at least 5% by weight of the dry ingredients in the product, but less than 1%, 2%, or 5% of other proteins from the organism that naturally expresses GPDH. In principle, any industrial product that contains a gelling, texture-improving, thickening, or emulsifying ingredient can benefit from the selection and optimization of GPDH as presented in the present disclosure. Characteristics of GPDH

[0022] In any of the products and methods mentioned above, each GPDH can have one, two, three or more than three of the following structural and functional features in any combination: comprises Figure 9B an amino acid sequence of any A and / or B and / or C and / or D and / or E and / or F and / or G motif defined in the above; an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95% or 99% identical to any of SGP2A (SEQ ID NO: 1), SGP2B (SEQ ID NO: 30) or any other sequence related to either (as determined by the BLAST algorithm (SF Altschul et al., 1990; J. Mol. Biol. 2015: 403-410); produced by recombinant expression; containing a sequence identical to a naturally occurring protein or a gelation-promoting, thickening or texture-improving portion thereof, preferably expressed in a non-vertebrate animal, non-animal or plant, fungus or cyanobacterium; may or may not have the enzymatic activity of diphosphomevalonate decarboxylase.

[0023] In any of these products and methods, the GPDH can have any of the desired gelation properties or functions set forth in this disclosure in any combination, including but not limited to: a critical gelation concentration of no more than 4%, 6% or 8%; a density of at least 1.1 g / cm 3 ; a gel T onset of 55 to 75 °C; 40 to 70 °C, or more than 40, 50 or 60% a final gel strength of at least 500 Pa or 2,000 Pa, or between 1,000 and 10,000 Pa, for a 12% (w / w) GPDH solution; a final gel elasticity of 1-15% or at least 4% critical strain for a 12% (w / w) GPDH solution.

[0024] In any of these products and methods, the GPDH can be an Arabidopsis or yeast protein and / or have the fold or three-dimensional structure of SGP2A or SPG2B or other members of the diphosphomevalonate decarboxylase family. Typically, the GPDH is recombinantly produced and isolated prior to being added as an ingredient to a food, cosmetic, or other product. The GPDH can have been fragmented, mutated, hydrolyzed, digested, denatured, cross-linked, conjugated to other substances, or otherwise industrially processed prior to or after being added to the substance or manufactured product. Alternatively or additionally, the product has less than 0.2%, 1%, 2%, or 5% of other proteins from the organism that naturally expresses the GPDH.

[0025] Other aspects, embodiments, features, and characteristics of the invention, its products, its manufacture, and its use are described in the following sections, figures, and appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1A and 1B A description of the structure and physiological function of SGP2A, a diphosphomevalonate decarboxylase MVD1 from Arabidopsis thaliana, EC 4.1.1.33, is provided. It is the prototype of the gel-promoting decarboxylase homologs (GPDH) of the present disclosure. Figure 1C Known and predicted associations of MVD1 with other proteins are shown. Figure 1D Homologs and isoforms of SGP2A in other plants are listed.

[0027] Figure 2A The appearance of a gelatinized batch of SGP2A is shown, as is the corresponding gelatinization heat map. Figure 2A The gelatinization tendencies of a 12% weight / weight solution of powder batch PL51B under food-related conditions of different protein concentrations, salt concentrations, and pH values are also shown. Figure 2B The purity of recombinantly produced SGP2A in crude lysate or purified protein samples is shown, as determined by SDS gel electrophoresis. Figure 2C The appearance of a gelatinized batch of SGP2B is shown.

[0028] Figure 3 is a flowchart showing a protocol for preparing other GPDHs and testing their relevant properties as food ingredients.

[0029] Figure 4A and Figure 4B are plots of the storage modulus (G') of SGP2A and SGP2B, respectively, as a function of temperature, compared to other compounds commonly used for gelatinization in foods: specifically, methylcellulose, Solanic 200 potato protein isolate, and ovalbumin.

[0030] Figure 4C The effect of pH and sodium chloride concentration on the solubility of SGP2B in 1% weight / volume protein dispersion is shown graphically. SGP2B is highly soluble at pH 6-8, independent of salt concentration, making it highly suitable for use in food formulations.

[0031] Figure 4D The gelation propensity of SGP2B formulations is summarized. SGP2 combines a low gelation onset temperature (about 50°C) and a low critical gelation concentration (4%) compared to most commonly used plant proteins and plant protein isolates.

[0032] Figure 4E and Figure 4F are images of meatball prototypes made using 2% (weight / weight) recombinant SGP2B before or after cooking.

[0033] Figure 4G The hardness of meatballs containing recombinant SGP2A or SGP2B was compared to meatballs containing methylcellulose. During cooking, the recombinant proteins caused the ingredients to gel, resulting in a good texture of the cooked product.

[0034] Figure 5 is a chart showing plant and animal proteins currently used in the food industry and some of their food applications (adapted from JT Martins et al., Front. Sus. Food Sys. 2018, 2:77).

[0035] Figures 6A to 6C The amino acid sequences of prototype GPDH proteins designated SGP2A are provided (SEQ ID NOs: 1-3). Figure 6D and 6E The second GPDH sequence designated SGP2B is provided (SEQ ID NOs: 30 and 31).

[0036] Figure 7 is a comparison of the amino acid sequences of SGP2A (SEQ ID NO: 1) and SGP2B (SEQ ID NO: 30). Although the amino acid sequences of SGP2A and SBP2B are only about 40% identical to each other, the highlighted regions are motifs that are closely shared by members of the GPDH family.

[0037] Figures 8A to 8K The amino acid sequences of several species and strain homologues of SGP2A are provided (SEQ ID NOs: 4-14).

[0038] Figure 9AThe domain organization of certain diphosphomevalonate decarboxylases in the GPDH class is shown. Figure 9B Amino acid motifs (SEQ ID NOs: 15-29) identified as common features of GDPH proteins during the course of this project are shown.

[0039] Figure 10 A sequence similarity network of selected GPDHs, indicating the degree of sequence identity between different diphosphomevalonate decarboxylases. DETAILED DESCRIPTION

[0040] The present disclosure provides for the first time a family of gel-promoting decarboxylase homologs (GPDHs) that can be used as food gelling agents. Some of these proteins are enzymes: specifically, diphosphomevalonate decarboxylases (EC 4.1.1.33). The gelation properties of the GPDH family were previously unknown. GPDHs can replace the gelling ingredients currently in use, with superior gelation properties and other beneficial properties. 1. Superior performance of GPDH of the disclosure compared to currently used gelling agents

[0041] The food development and cosmetic industries are seeking clean-label gelling agents and texture improvers of non-animal origin with better properties than the gelling agents currently in use. Methylcellulose is the most commonly used gelling agent in plant-based meats, and other alternatives (such as polysaccharide gums) have poor consumer attitudes and questionable nutritional value. Gelling agents such as methylcellulose exhibit undesirable properties such as “melting back”, i.e. a significant decrease in gel strength (or complete loss of gel structure) upon cooling of a heat-reversible gel.

[0042] In contrast, the GPDHs of the present disclosure are naturally occurring proteins, a source of digestible protein that is clean label. Some GPDHs are naturally plant products, meaning that they are already a small part of existing food systems. As a gelation-inducing food ingredient, GPDHs are more nutritionally valuable than gum and cellulose gel ingredients. GPDHs have a low critical gel concentration (<6%), a low gelation onset temperature (T onset <70°C), and no melting back. GPDHs can provide binding, viscosifying, texture-improving, and moisturizing functions to foods. GPDHs have potential value for application in a variety of food categories, such as plant-based and cell-grown meats, baked goods, dairy products and their derivatives, beverages, soups, and sauces.

[0043] Previously known methylcellulose alternatives are citrus fiber, potato protein isolate, and rapeseed protein isolate. Production of these alternatives can be adversely affected by supply fluctuations due to single cultivation or disease-related, or have inconsistent organoleptic properties (bitter or plant-like taste). Production of GPDH can employ a rigorously controlled process flow, including optimization of energy usage, waste control and management, thereby providing more reliable product consistency. Recombinantly produced GPDH has extremely high standards in uniformity, batch-to-batch consistency, food safety, sanitation, and final ingredient quality. 2. Discovery of GPDH and its beneficial properties as a food ingredient

[0044] Gel properties of the GPDH family were discovered by characterizing a prototype SGP2A. This prototype was identified as part of a project to identify gel proteins with desirable heat-onset hydrogel properties.

[0045] As a first step in discovering new gelation-inducing agents, a protein sequence selection starting database was created. Protein sequences were obtained from public databases and screened by searching for a series of desirable characteristics. Filters were then applied to exclude proteins with undesirable characteristics. Three-dimensional structures were obtained from the Protein Data Bank (PDB), which is compiled and provided by the World wide Protein Data Bank Foundation, located in Piscataway, NJ. To reduce the number of structure alignments of near-variant variants, redundant protein sequences or fragments of other sequences in the database were removed, as were homologues with greater than 90% amino acid sequence identity. Proteins that are known to interact with other proteins, thereby complicating their expression, testing, or use as a food ingredient were also removed. Otherwise, no restrictions were placed on the natural biological function of each protein in the database. The curated dataset included approximately 45,000 protein sequences.

[0046] To search the database for promising gelation candidates, 29 reference proteins were used that are known or suspected by the inventors to have beneficial gelation properties. Proteins in the database were algorithmically aligned in pairs with each of the 29 reference proteins. Each reference protein produced a list of proteins with structural similarity, with a p-value selection threshold of <0.05. SGP2A was identified as having some degree of structural similarity to one of the 29 reference proteins. Through cluster analysis, SGP2B was determined to have superior gel-forming properties and was structurally related to SGP2A.

[0047] Following identification, SGP2A and SGP2B were further evaluated to assess whether they could be tested empirically in the laboratory and confirmed that they did not have known toxicity or allergenicity. It was determined that SGP2A does not require any chaperone molecules or post-translational modifications to fold properly. On this basis, SGP2A was selected as a candidate for recombinant expression and testing. 3. Role of SGP2A and SGP2B in cell biology

[0048] SGP2A and SGP2B are annotated in the UniProt and GenBank databases as isozymes of diphosphomevalonate decarboxylase MVD1 in Arabidopsis thaliana (Mouse-ear cress) and Saccharomyces cerevisiae S288C (brewer's yeast), respectively. Diphosphomevalonate decarboxylase (EC 4.1.1.33), also known as mevalonate diphosphate decarboxylase, is an enzyme that catalyzes the chemical reaction ATP + mevalonate diphosphate → diphosphate + pyrophosphate + isopentenyl diphosphate.

[0049] Figure 1A and 1B Several descriptions of the structure and physiological function of SGP2A, a diphosphomevalonate decarboxylase MVD1 from Arabidopsis thaliana, EC 4.1.1.33, are provided. It is the prototype of the gel-promoting decarboxylase homologs (GPDH) of the present disclosure. Figure 1C Known and predicted associations of MVD1 with other proteins are shown. Data from the STRING database of the ELIXIR Core Data Resource: R. Drysdale et al., F1000Research 2018, 7(ELIXIR): 1711. Figure 1D Homologues and isoforms of SGP2A in other plants are listed.

[0050] Mevalonate diphosphate decarboxylase catalyzes the last step of the mevalonate pathway. The mevalonate pathway is responsible for the biosynthesis of isoprenes from acetate. This pathway plays a key role in a variety of cellular processes by synthesizing isoprenoid compounds such as sterols like cholesterol and non-steroidal isoprenoid compounds like polyols, hemoglobin A, tRNA isopentenyltransferases, and ubiquinone. The enzyme belongs to the lyase family, specifically carboxyl lyases that cleave carbon-carbon bonds.

[0051] Many different organisms utilize the mevalonate pathway and mevalonate diphosphate decarboxylase, but for different purposes. ML Barta et al., Biochemistry. 51(28):5611-5621. In Gram-positive bacteria, isopentenyl diphosphate is the final product of mevalonate diphosphate decarboxylase and is an important intermediate in the biosynthesis of peptidoglycan and polyisoprene.

[0052] Higher eukaryotes and plants also use the mevalonate pathway. Mevalonate diphosphate decarboxylase is mainly found in the liver of mammals, where most mevalonate is converted to cholesterol. Some cholesterol is converted into steroid hormones, bile acids, and vitamin D. Mevalonate is also converted into reaction intermediates such as polyhydroxy compounds, ubiquinone, tRNA isopentenyltransferase, and farnesylated and geranylated proteins. (DDHinson et al., J. Lipid Res. 38(11): 2216-23.)

[0053] The ability of GPDH to promote gelation when added to food, cosmetics or other products is unpredictable from its previously known enzymatic and physiological functions. 4. Scientific methods for forming gels with food ingredients and evaluating gel properties

[0054] The formation of food gels is a transformation from a solution to a gel state, during which viscoelasticity undergoes abrupt changes, and solid-state properties also change. During gel formation, continuous and discontinuous phases interconvert. Tables 1A and 1B show the experimental methods for determining gel properties (adapted from S. Banerjee et al., Crit. Rev. Food Sci. Nutr. 2012; 52:334-346).

[0055] These tests are independent of the sample geometry and the instrument used. Rheological properties measured under large deformation include fracture, failure, and rupture characteristics, such as stress / strain, typically determined by uniaxial compression and tension. The rheological or textural properties of starch gels are mostly studied in model systems to understand the fundamental factors and mechanisms involved in gelation and to characterize the gel properties.

[0056] Rheological properties depend on the presence of a molecular network. The relationship between stress (force per unit area) and strain (deformation caused by the applied force) in a gel during compression can be measured. Young's modulus, or elastic modulus, is the ratio of stress to strain when a material is tested within its linear elastic limit. The maximum stress a gel can withstand is its breaking strength (RS). When forces are applied from all directions (isotropically), the bulk modulus (K) can be obtained, yielding the volume change per unit initial volume.

[0057] Different types of gels can be formed depending on the molecular characteristics and properties. For example, hydrogels are networks of polymer chains that are insoluble in water, sometimes in the form of colloidal gels, where water is the dispersion medium. Organogels are non-crystalline, non-glassy, thermoreversible solid materials composed of a liquid organic phase entrapped in a structural network. Aerogels are solids formed by drying of a gel, with the characteristics of unobstructed shrinkage, high porosity, and high surface area.

[0058] Aerogels are colloidal gels with a gas as the dispersion medium. Some polysaccharides, usually derived from food, show properties intermediate between a polysaccharide solution and a true gel, and form weak gels. Weak gels behave as elastic gels under low deformation conditions. When the deformation is large enough or the shear rate is high, they break irreversibly and flow. Fluid gels are formed when a hot hydrocolloid dispersion is cooled and solidifies in a quiescent state. The dispersion separates into small particles rich in polymer and regions poor in polymer, which form the interstitial space between the particles. 5. General methods for protein expression and purification

[0059] Recombinant production of proteins, such as GDPH, is usually achieved by genetically modifying a suitable expression host, which is generally genetically modified to incorporate DNA or to carry a plasmid designed to express the protein of interest constitutively or by induction. Table 2 lists suitable organisms for recombinant expression of candidate proteins. The choice of host organism takes into account the ability of the host to express large amounts of soluble protein, as well as post-translational modifications that can affect the function of the protein, such as the addition of carbohydrates and / or interchain cross-linking.

[0060] The advantage of eukaryotic expression systems is that post-translational processing of candidate proteins can be performed similar to that used in nature or in industrial production, such as glycosylation and interchain cross-linking. The advantage of prokaryotic expression systems is ease of implementation and high yield. During development, a variety of systems can be used: for example, expression in E. coli for screening assays, and expression in eukaryotes for later development and testing. Some expression systems, such as yeast, are suitable for use in both stages.

[0061] Common purification methods include centrifugation, filtration, affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity capture, isoelectric precipitation, liquid-liquid phase separation (LLPS), lyophilization, and dialysis. One method can be used as a single step or in combination with other methods as needed to achieve the desired level of purity. Once purity is achieved, the protein will be processed into a final state compatible with characterization methods by standard methods. For example, certain detection methods can require a powdered protein, while other characterization methods can require the protein in aqueous solution. Protein Purification, 2nd Edition, P. Bonner, 2018; and High-Throughput Protein Production and Purification, R. Vincentelli, ed., 2019.

[0062] To facilitate protein purification for initial testing of GPDH candidates, the recombinant protein can be expressed with an affinity-bound proprietary tag. The tag can be any feature added to the protein during expression that can be used as a handle for affinity purification using a conjugated binding partner. Examples include amino acid sequences added internally or to both ends of the native protein sequence as well as carbohydrates. After the tagged protein is immobilized on an affinity surface, fermentation byproducts can be washed away. Then, depending on the tag used, the purified target protein is eluted from the resin using competitive binding or a change in conditions such as pH. 6. Production and purification of GPDH

[0063] GPDH can be produced using standard E. coli expression and purification methods for initial testing. For example, SGP2A is overexpressed using the pET28a(+) vector and expression is induced using the lactose induction promoter system. Construction of the plasmid includes a C-terminal 6X-HIS tag for detection and purification.

[0064] To obtain protein for physicochemical characterization, strains containing the SGP2A coding sequence are grown at 30°C in the range of 2 mL to 10 L using either 24 deep well plates or shake flasks (depending on volume). Standard Luria Broth (LB) media with IPTG induction or commercially available auto-induction media (such as MagicMedia TM ) results in overexpression of SGP2A. After approximately 24 hours of induction, cells are harvested by centrifugation. Cells are then suspended in a 50 mM Phosphate Buffer, 500 mM Sodium Chloride, pH 7.5 solution for cell lysis. Depending on scale, standard protocols for sonication or pressure homogenization are used to lyse the cells. Cell debris is then removed by centrifugation.

[0065] ​The solubilized protein was collected and clarified using 0.45 μΜ cut-off filtration. The protein was purified using immobilized metal affinity chromatography and fractions were eluted using an elution gradient of 0 to 250 mM imidazole over 8 column volumes to separate the protein from impurities. Fractions containing SGP2A were identified by SDS-PAGE analysis and pooled together. The protein was concentrated approximately 10-fold using a tangential flow filtration cassette with a 10 kDa molecular weight cut-off. The exchange buffer was then exchanged four times in succession with 18.2 mega ohm resistivity water at a ratio of 1 :40 using methods such as dialysis to remove salts from SGP2A. The protein was dried by lyophilization for subsequent characterization and use.

[0066] Figure 2A and 2C FIGS. 1 and 2 show the appearance of gel preparations of purified SGP2A and SGP2B, respectively.

[0067] Figure 2B FIG. 3 shows the purity of His-tagged isolated protein SGP2A as determined by SDS polyacrylamide gel electrophoresis. A major single band was observed corresponding to a mass between 50 and 75 kDa.

[0068] Figure 3 FIG. 4 is a flow chart showing a protocol that can be used to prepare other GPDH preparations and test them for relevant properties as a food ingredient.

[0069] Production scales of protein SGP2A and other GPDHs can be scaled up for commercial production as a food or cosmetic ingredient using other synthesis and purification protocols. Production methods can include replacing the recombinant expression host with any Generally Recognized as Safe (GRAS) host such as Pichia or Aspergillus. The protein can be produced by fermentation in a bioreactor that can range in size from 1 liter to 10,000 liters. When used as a food ingredient, the artificial affinity tag must be removed and the protein purified by other methods. If the protein is soluble and intracellular, the cells must be lysed and the cell debris removed, which can be accomplished by centrifugation and filtration.

[0070] The protein can then be recovered by standard separation techniques, such as fractionation, filtration or simultaneous use of several separation techniques. If the protein is secreted from the host cell, cell lysis can be omitted. The biochemical properties of the protein can be used to guide the selection of such steps. For example, a protein with a molecular weight of 60 kDa can be used to select a filtration method with a molecular weight cutoff less than the protein, so that SGP2A remains in the retentate, while a filtrate with a molecular weight cutoff greater than the protein will allow the protein to pass into the permeate. The isoelectric point of the protein (calculated value for SGP2A protein is 6) can be used to guide the pH at which the protein can become unstable and precipitate from solution. This precipitation step can be used to separate GPDH from cell debris and non-target proteins. 7. General principles of protein characterization

[0071] Purified proteins that are candidates for GPDH can be tested for various physicochemical properties as listed in Table 3.

[0072] Purified candidate gel proteins can be tested for various functional properties as listed in Table 4. 8. Evaluation of gel properties of GPDH

[0073] Various SGP2A preparations were subjected to specific assays to test gel properties by several standard tests. Thermal Shift Assay (TSA)

[0074] Using the fluorophore SYPRO-Orange TM Thermal shift analysis was performed on the His-tag purified preparation of expressed SGP2A in solution using differential scanning fluorimetry using the fluorophore SYPRO-Orange (ThermoFisher) and an Applied Biosystems 7500 RT-PCR thermocycler with a fluorescence reader using excitation and emission wavelengths of 472 nm and 570 nm, respectively. Purified gelatinized candidate proteins can be tested for physicochemical properties. According to the BioRad protein thermal shift protocol, the protein sample was diluted to a concentration of 1 mg / mL in pure water and then mixed with the appropriate buffer and sodium chloride solution to obtain the pH and sodium chloride concentrations shown in the table below. The buffer concentration was 50 mM for all samples.

[0075] Analysis of the protein melting curve was done using the internal software, T m The maximum of the derivative from the raw melting curve data was assigned. Results are shown in Table 5. Chemical and physical properties (powder)

[0076] After preparation-scale purification and lyophilization, the recovered SGP2A protein was ground into a powder using a mortar and pestle to create the final protein / ingredient batch. The final batch was analyzed for color and ingredients, and the results are summarized in Table 6.

[0077] Powder color was measured by reflectance spectrophotometry (Konica Minolta CM-5) using ~0.2 g of powder loaded into a mini-leaf cup for measurement. CIE L*a*b* color values are the average of nine replicate measurements. Powder color can change over time (e.g., oxidation) or be related to certain ingredient parameters (e.g., fat content), and powder color can vary between batches. Solution properties

[0078] Solubility of SGP2A and other GPDHs can be determined as follows. The protein was dissolved in ultrapure water to make a concentrated (15% w / w) SGP2A stock solution. The solution was homogenized by test tube rotation and vortexing, then stored overnight. The pH and conductivity of the stock solution were measured to better quantify the extent of removal of buffer salts after upstream diafiltration / dialysis and to track batch-to-batch variation in this property.

[0079] To monitor batch-to-batch variation and to provide quantitative data on solution turbidity, the stock solution was diluted (1 : 10) with water, and the NanoDrop® 2000 / 2000c UV-Vis Spectrophotometer was used to collect UV-Vis spectra (200-800 nm). Insoluble material was separated by a benchtop centrifuge, and the UV-Vis spectrum was (re)measured. TM Spectrophotometer. Insoluble material was separated by a benchtop centrifuge, and the UV-Vis spectrum was (re)measured.

[0080] The primary structure, higher-order structure, and structural homogeneity of SGP2A in the final powder batch can affect functional properties and performance. The homogeneity of denatured SGP2A can be determined by measuring its apparent molecular weight on SDS-PAGE, or using size-exclusion chromatography. Protein expression assay (1 : 100 dilution of SGP2A in 8 M urea). Gel properties

[0081] Thermal gelation of SGP2A protein solutions was initially tested using a qualitative gelation assay. Small volumes of protein solution were heated slowly to 92 °C in a temperature controlled water bath and then cooled to room temperature. Thermal gelation of protein solutions can also be assessed by inverting, resistance to puncture, and ability to support a spherical mass. By assessing the gel formation tendency of small volumes of 12% w / w protein solution buffered to pH values of 4 to 7.5 with 20 mM citric acid / phosphoric acid plus NaCl concentrations of 0 to 300 mM, a solvent dependent gel thermal map was determined. The critical gelation concentration was determined by measuring the gelation tendency of 0% to 12% SGP2A (w / w) solutions dissolved in water or 20 mM phosphate pH 7.5, 300 mM NaCl. Results are shown in Table 7A. Similar SGP2B assay results are shown in Table 7B.

[0082] Figure 2A Gel appearance of SGP2A of batch PL51B1 is shown, as well as the gel thermal map at a concentration of 12% (w / w) in solution. The appearance of SGP2A gels was similar to the appearance of the protein in all test conditions. While no significant effect of pH on solubility or gelation was observed, the isoelectric point of SGP2A (6.33) is within the pH range (pH 3-8) associated with most food systems. The stability of SGP2A protein near its isoelectric point can be reduced. Therefore, some changes in solubility and / or SGP2A heat-set gel structure can occur in this pH range. For a discussion of micellar versus branched gels, the reader is referred to Y. Cao and R. Mezzenga, Nature Food 2020, 106:118.

[0083] Small-amplitude oscillatory shear (SAOS) rheology was used to determine the dynamic rheological properties of SGP2A solutions / gels as a function of temperature. M. H. Tunick, J. Agric. Food Chem. 2011, 59: 1481-1486, doi.org / 10.1021. Data were collected using an Anton Paar strain-controlled rheometer, model MC302, equipped with a PP25 (25 mm) parallel plate measuring system, operating under small deformation conditions (0.1% strain, constant angular frequency of 10 rad / sec). Rheological properties as a function of temperature were measured in 3 stages: (1) temperature ramp from 30 °C to 95 °C at a rate of 5 °C / min; (2) hold at 95 °C for 5 minutes; (3) temperature ramp from 95 °C to 50 °C at a rate of 5 °C / min. Following the 3rd stage (cooling), a strain-amplitude sweep (0.01-100% strain) was performed at constant frequency (10 rad / sec) to probe the linear viscoelastic region of the gel and the shear-induced breakdown of the gel's upper structure.

[0084] Figure 4A The plot of storage modulus (G') versus temperature for SGP2A, methylcellulose, Solanic 200 potato protein isolate, and egg white protein is shown by small-amplitude oscillatory shear rheology. Due to the solution-gel transition experienced by SGP2A during the first stage temperature ramp, its storage modulus (G') increased substantially. The gelation onset temperature T onset = 50 °C was estimated from the point at which G' began to rapidly increase above its baseline value at lower temperatures.

[0085] Similarly, Figure 4B The plot of storage modulus (G') versus temperature for SGP2B is shown. The gelation onset temperature for this protein was lower, at 55 °C, which is the point at which G' rapidly increased above its baseline value at lower temperatures. The gelation onset temperature of 55 °C was slightly higher than for SGP2A (55 °C), but lower than for Solanic 200 potato protein or egg white protein. The storage modulus of SGP2B increased relatively rapidly during the solution-gel process as the solution transitioned to a gel. The storage modulus of the SGP2B gel was 2241.7 Pa as the gel formed and G' approached its maximum value during the temperature ramp. This was close to the range of G' values (10 3 to 10 4 Pa) exhibited by the model gelling systems of Solanic 200, egg white protein, and methylcellulose.

[0086] The relatively low T onsetThe values are consistent with the low Tm values observed in the thermal migration test. After the solution-gel transition, a maximum gel strength of 601 Pa was reached during the temperature ramp, which increased to a final gel strength of 2127 Pa after the 2nd (95 °C hold) and 3rd (95 °C to 50 °C) stages of the temperature cycle. The increase in gel strength during cooling is typical of many thermal gel proteins and is in stark contrast to the “melting back” phenomenon observed in thermally reversible methylcellulose gels, where G’ decreases as the gel cools.

[0087] After the temperature ramp, strain sweep measurements were performed to probe the integrity of the SGP2A gel superstructure. The strain at which the storage modulus deviated from the linear viscoelastic region by more than 5% was defined as the critical strain or “elasticity” of the gel - the point at which the gel structure began to break down. The final strength of the gel was defined as the G’ value of the linear viscoelastic region (Tables 7A and 7B).

[0088] The critical gel concentration of standard commercially available plant-based gelling proteins ranges from 5% (canola cruciferin) to 18% (pumpkin seed globulins), with the critical gel concentration generally higher than 10% (L. Grosssman et al., Annu. Rev. Food Sci Technol. 2021; 12: 93-117). In addition to the generally higher critical gel concentration, the food functionality of plant-based proteins is often limited by their tendency to exhibit higher denaturation temperatures (80 °C to 120 °C) (D. J. McClements et al., Compr. Rev. Food Sci. Food Safety 2021 May 30) and corresponding higher initial gel temperatures.

[0089] Figure 4C The effect of pH and sodium chloride concentration on the solubility of SGP2B in a 1% weight / volume protein dispersion was summarized. The protein concentration detected in the supernatant is expressed as a percentage of the original protein concentration in the buffered 1% weight / volume starting solution for each data point.

[0090] Many plant proteins isolated from seeds and legumes have a solubility of less than 50% when evaluated in a similar manner (K. Ma et al., Foods 2022, 11(4): 594). Figure 4CThe data in Figure 6 show that the solubility of dispersed SGP2B powder is greater than 60% at all pH values measured in low salt solutions (0 to 50 mM NaCl). The solubility is greatest (greater than 70%) observed under acidic and basic conditions (pH 3 to 3.5 and pH 6 to 8). This indicates that SGP2B protein powder has a high solubility, especially in buffers with a salt content of no more than 50 mM. At higher salt concentrations (500 mM NaCl), the solubility is still high at pH 6 to 8, but drops sharply at pH 4 to 5, which is consistent with the formation of aggregates.

[0091] Due to the high solubility of SGP2B at pH 6 to 8, independent of salt concentration, it is very suitable for use in food formulations.

[0092] Figure 4D The gelation propensity of SGP2B formulations was summarized under a set of extended food-related conditions, investigating a range of protein concentrations, salt concentrations, and pH values. The notation indicates whether the solution formed a gel (+) or remained a solution (o) after thermal cycling. All measurements were performed in duplicate.

[0093] The effect of salt and pH on the gelation results is most pronounced near the critical gelation concentration of the protein. At a SGP2 concentration of 2% w / v, no gel was formed under any of the tested conditions. At a concentration of 4% w / v, a gel was formed under 11 out of 12 tested conditions. At a concentration of 6% w / v, a gel was formed under all conditions. Thus, the critical gelation concentration is slightly below 4%. SGP2 shows robust gelation that is insensitive to pH or salt when the concentration is 4% w / v or higher.

[0094] The combination of low gel onset temperature (about 50°C) and low critical gel concentration (4%) means that SGP2A and other GPDHs are suitable as functional animal protein replacements in meat, dairy, and egg replacement products, compared to most commonly used plant proteins and plant protein isolates. 9. Molecular mechanisms of gel formation in foods and other products

[0095] The gelation process mechanism can differ for different gelling agents, resulting in specific attributes. S. Banerjee et al., Crit. Rev. Food Sci. Nutr. 2012; 52:334-346. Some or all of the attributes described in this section can be reproduced with GPDH, depending on the specific case, with appropriate adjustments to the gelation conditions.

[0096] Gelatin melts on heating and solidifies on cooling again. It forms a semi-solid gel with water. Gelation is controlled by partial reorganization of triple helices in collagen during cooling. In the first step, polypeptide chains orient to induce reactive sites. Subsequently, the other two chains in the vicinity of the reactive sites coagulate to form triple helices. Heat-induced whey protein gelation is a typical globular protein gelation and proceeds through a series of transitions: (i) denaturation (unfolding) of the native protein, (ii) aggregation of the unfolded molecules, (iii) chain formation from the aggregates, (iv) network formation from the chains.

[0097] Heating of soy flour or milk and then adding salt (Ca ++ or Mg ++ ) causes gelation of soy proteins, forming a gel or curd. Casein molecules have a strong hydrophobicity. Sub-micelles are held together by hydrophobic bonds and salt bridges. Enzymatic hydrolysis of k-casein by rennet releases CMP (casein peptides) and causes micelles to aggregate, resulting in rennet gelation. Egg white and egg yolk of liquid egg can both form gels after heating. Gel formation has two steps: denaturation, followed by aggregation of denatured proteins.

[0098] Gelation of brown algae occurs upon addition of multivalent cations at low pH (<4). Gular acid residues adopt a bent conformation, providing effective binding sites for cations. Unlike most other gelling polysaccharides, brown algae gels have a cold-set character. The gelling properties of pectin depend largely on the degree of esterification. High methoxyl pectin gels in the presence of sugar or other kosmotropic agents (such as sugars, polyols or monohydric alcohols).

[0099] Gelation of agar is a heat-reversible process that occurs due to the formation of hydrogen bonds. Agar needs to be heated to enter a dispersed state. Upon cooling, the hot dispersion coagulates into a gel. The molecules undergo a coiled helix transition, followed by helix aggregation (Stanley, 2006). Carrageenan is an ionic polymer and forms a helical gel upon cooling in the presence of salts (electrolytes), especially K + ions. The cations K + , Rb + , Cs + and NH4 + promote helix formation and gelation. Gelation of carrageenan generally involves the formation of ordered regions by the polymer chains through the formation of intermolecular double helices. These regions subsequently aggregate, leading to gelation, mediated by specific binding of gel-promoting cations.

[0100] The gelation mechanism of gellan gum is based on a coiled model. In aqueous solution at high temperature, gellan polymer is in a disordered, single-coil state. Cooling the gellan gum solution promotes the formation of a triple left-handed double helix stabilized by internal hydrogen bonds. The coil-helix conformational transition occurs in the temperature range of 30 to 50°C, depending on the ionic strength of the dispersion. After this transition, the gellan gum double helix can associate to form junction zones in the presence of cations, which can polymerize and form a three-dimensional gel network interconnected by the solution, which is converted to a gel in the process.

[0101] Xanthan gum gels in the presence of electrolytes over a wide range of pH values and at high temperatures. The gel forms upon cooling. Xanthan gum and the polymeric mannose chains associate together after the xanthan coil-helix transition. Locust bean gum forms a gel upon cooling. The polymeric mannose chains associate during the coil-helix transition, involving galactose-deficient regions. 10. Screening for other functional and physicochemical properties

[0102] In investigating and testing other members of the GPDH family, users will want to assess their gelation properties as described above. They can also want to determine whether the candidate has other desirable functions or properties, thereby increasing the desirability of the candidate - and whether it has one or more undesirable functions or properties, thereby decreasing the desirability of the candidate or eliminating it from contention. By way of illustration, desirable properties can include one or more of the following: ease of expression, ease of purification, stability on storage, mixability, and one or more desirable flavor or sensory properties. Undesirable properties can include one or more of the following: allergenicity or immunogenicity, incompatibility with other food ingredients, undesirable physiological effects, and undesirable flavor.

[0103] Computer algorithms can be used to predict some of these properties. For example, allergenicity can be predicted by the following methods: L. Zhang et al., Bioinformatics 2012, 28:2178-2179; L. Wang et al., Foods 2021, 10:809, doi.org / 10.3390; and S. Saha et al., Nucl. Acids Res. 2006, 34, doi:10.1093. Immunogenicity can be predicted according to the algorithm of N. Doneva et al., Symmetry 2021: 13, 388, based on MHC binding motifs and T and B cell epitopes. Toxicity can be predicted according to S. S. Negi et al., Sci. Reports 2017: 7, 13957-1 and Y. Jin et al., Food Chem. Toxicol. 2017; 109: 81-89. Flavor aspects can be predicted according to P. Keska et al., J. Sensory Studies 2017: e12301; F. Fritz et al., Nucleic Acids Res. 2021 Jul 2; 49(W1): W679-W684, and S. Ployon et al., Food Chem. 2018 Jul 1; 253: 79-87. Other properties are assessed by laboratory or in situ testing. 11. Commonly used gelling agents in foods that can be replaced with GPDH

[0104] GPDHs according to the present disclosure can be used to replace gelatin and thickening agents commonly used in food products. Typical gel components that can be replaced include methylcellulose, carboxymethylcellulose (CMC), pectin, gums (agar, xanthan gum, guar gum, locust bean, k-carrageenan), starches (corn, potato, tapioca flour), and other proteins (chickpea, pea, fava bean, egg white proteins, milk proteins, wheat gluten, and gelatin).

[0105] Figure 5 Some plant and animal proteins currently used in the food industry are shown (adapted from JT Martins et al., Front. Sus. Food Sys. 2018, 2:77). Animal-derived proteins used in food products include gelatin, collagen, silk, elastin, albumin, and milk proteins such as casein, a-lactalbumin, b-lactoglobulin, and lactoferrin. Plant-derived proteins used in food products include corn protein, soy protein, lectin, fibrin, pea protein, rice protein, and wheat protein. As food ingredients, these proteins can facilitate gelation or increase the nutritional content of food products. They can also be used for packaging (in the form of biodegradable or edible films), formation of nanoparticles, and encapsulated delivery of food or pharmaceuticals.

[0106] Hydrocolloids can be added to food products for thickening. D. Saha et al., J Food Sci Technol. 2010 Dec;47(6):587-597. The thickening process involves the non-specific entanglement of conformationally disordered polymer chains. Thickening occurs above a critical concentration (overlap concentration, C*). Below this concentration, the polymer dispersion exhibits Newtonian behavior, but above this concentration non-Newtonian behavior is exhibited. Hydrophilic colloids used as thickening agents in various food systems include starch, modified starch, xanthan gum, galactomannan (such as guar gum and locust bean gum (LBG)), gum arabic or locust bean gum, karaya gum, sea buckthorn gum, and carboxymethylcellulose (CMC). The thickening effect depends on the type of hydrophilic colloid used, the concentration, the food system used, and the pH and temperature of the food system. 12. Texture profile analysis of a model food

[0107] Gelation properties of GDPH in food products can be determined by preparing test food products and mechanically measuring the performance characteristics.

[0108] Test meatballs were prepared using the following ingredients:

[0109] The steps to make the patties were as follows: Soak the TVP in the first portion of water for 30 minutes. Dissolve the test protein in the second portion of water, add the citrus fiber and salt, and then emulsify with the melted coconut oil. Add the potato starch and soy protein to the soaked TVP and mix, then add the emulsion and mix until fully incorporated. Store the mixture at a temperature of 40°F for 1 hour. Shape into 10 g meatballs. Bake at 375F for 8 minutes, confirming internal temperature of 165-170F. Store in insulated container and perform TPA at 150-155°F.

[0110] Figure 4E and 4F are images of the meatballs made from the above recipe before cooking and after cooking. The addition of SGP2 in the recipe allows the dough to form the desired shape and can be produced on a large scale. The prototype meatballs reacted well during cooking, maintaining their shape and becoming firm.

[0111] The AMETEK TMThe performance characteristics of these prototype patties were objectively measured using a Brookfield CTX Texture Analyzer. Texture Profile Analysis (TPA) was accomplished by a double compression test using a 5 kg load cell at a rate of 0.5 mm per second to achieve 40% deformation. The reading was the peak force in Newton (N) at the first compression. In addition, total work (mJ), chewiness (N), gumminess (N), springiness, cohesiveness, and adhesiveness (mJ) were measured. Hardness was often the most differentiating measurement.

[0112] Figure 4G The hardness of meatballs containing 4% of the recombinant proteins SGP2A or SGP2B was compared to meatballs containing a common binder (2% methylcellulose). The gelling proteins performed well, gelling the ingredients during cooking, resulting in a cooked product with the desired final texture. 13. Foods with GPDH added as a gelling agent

[0113] The GPDH proteins are suitable for use as gelling agents in a variety of food manufactured products. The user can add GPDH to the food product in the appropriate mass ratio depending on the degree of gelling desired. This will depend on the other ingredients in the product, whether the product will be heated or otherwise processed by the consumer, and the particular GPDH chosen as the gelling ingredient.

[0114] In general, any concentration between 0.1% and 50% weight / weight of the dry food ingredients can be used. The range of 0.5% or 1% to 20% is more typical. When using the proteins SGP2A or SGP2B, the user can test formulations of 2% or 4% to 12% to produce the desired network formation, texture, and water / oil retention effects in different food systems. Given the better gel-forming propensity of SGP2A in citrate / phosphate buffers at pH 5.5 to 7.5 with 50 to 300 mM NaCI added, a range of 2% to 10% or 4% to 6% can be suitable for food formulations that are near neutral in pH and have higher salt concentrations. This range is comparable to the recommended range of potato protein isolate (2-4%) in the currently popular alternative meat products.

[0115] Here are some specific examples of ranges in food products: Plant-based ground meat: 10 to 20% weight / weight of the dry ingredients Plant-based kibbeh or kibbeh nayyeh: 20 to 30% weight / weight of the dry ingredients Vegan cake: 1 to 10% weight / weight of the dry ingredients Soup: 20 to 30% weight / weight of the dry ingredients Frozen / chilled dough: 10 to 20% weight / weight of the dry ingredients Plant-based meat patties and meatballs

[0116] As shown in the following recipes, SGP2A, SGP2B, and other GPDHs can be used as alternatives to gelling agents and binders in plant-based meats.

[0117] Meat substitutes and seasonings can be made from plant-based or other ingredients by combining approximately 60% (by weight) of muscle imitations (e.g., 62% (by weight) of dark muscle imitations and 38% (by weight) of white muscle imitations), approximately 30% (by weight) of adipose tissue imitations, and approximately 5% (by weight) of connective tissue imitations. (US Patent 10,863,761). Muscle tissue imitations can be prepared by combining a heme-binding protein (such as myoglobin or leg hemoglobin) (12 mg / mL) with approximately an equal volume of plant protein (150 mg / mL) in the presence of a cross-linking agent (such as transglutaminase) (approximately 1 wt% / volume). Adipose tissue imitations can be prepared by combining 8S globulin stored in mango seeds or pea globulin with oil (such as soybean oil or rice bran oil) under the action of transglutaminase, heating at approximately 95°C for 5 minutes, and then cooling. Adipose tissue imitations typically form an opaque, off-white gel with a smooth, homogeneous texture and no visible unintegrated liquid. Connective tissue imitations can be prepared as a combination of plant proteins or structural equivalents that mimic collagen or fascia-like fibers, or a combination of both.

[0118] Meat substitutes or seasonings may also contain sugars and / or sulfur-containing compounds that are not proteins. Sugars may be selected from glucose, ribose, fructose, lactose, xylose, arabinose, glucose-6-phosphate, maltose, and galactose, and mixtures of two or more of these. Sulfur-containing compounds may be selected from cysteine, cystine, selenocysteine, thiamine, methionine, and mixtures of two or more of these.

[0119] Meat-like flavor or aroma can be expressed during cooking, which releases at least two volatile compounds with meat-related aromas, such as 2-methyl-furan, bis(2-methyl-3-furanyl)disulfide, 2-pentyl-furan, 3,3′-dithiobis-2-methyl-furan, 2,5-dimethylpyrazine, 2-methyl-3-furanthiol, dihydro-3-(2H)-thiophenone, 5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2-thiophenecarboxaldehyde, 2-methylthiazole, dimethyl sulfide, decanal, 5-ethyldihydro-2(3H)-furanone, dihydro-5-pentyl-2(3H)-furanone, 2-octanone, 3,5-octadien-2-one, p-cresol, and hexanoic acid.

[0120] Methods and characteristics of the preparation of meat substitutes and condiments are outlined in U.S. Patent Nos. 3,815,823, 9,700,067, 10,863,761, 10,798,958 and 11,013,250, and EP3952661A1.

[0121] For example, minced meat can be prepared as follows; 1. Weigh all dry ingredients. 2. Weigh the liquid components separately. 3. Mix the liquid ingredients with the dry ingredients to form a dough. 4. Let the dough hydrate at room temperature for 20 minutes. 5. Making coconut oil beads: First, freeze the coconut oil, then use a fork, cheese grater, or blender to... Scrape 6. Add half of the coconut oil beads and stir well. 7. Add the remaining half of the coconut oil beads, and gently fold to create a marble pattern. 8. Cooking steps for plant-based meat patties: 9. Weigh the dough and divide it into 50-gram balls. 10. Press it into a patty shape using a patty press (2.5 inches in diameter, weigh again after measuring the diameter), or make it into meatballs (50g each). 11. Cook in a non-stick pan (150°C, 5 minutes per side, flip 3 times, until the internal temperature reaches 75°C).

[0122] The method for making plant-based meatballs is as follows: 1. Preheat oven to 400℉ 2. Shape the dough into balls (25g each) and place them on a baking sheet (using parchment paper). 3. Bake for 20-25 minutes. Flip the cake halfway through baking. Plant-based sausages

[0123] When making sausages, mix the following ingredients and stuff them into vegetarian casings:

[0124] The preparations are as follows: 1. Mix methylcellulose, SGP2A, or SGP2B with water until completely dissolved to make sausage glue. mixture 2. Weigh and mix the dry and liquid components separately. 3. Combine the liquid ingredients with the dry ingredients and binder, and mix in a stand mixer at medium speed to form a dough. 4. Let the dough hydrate for 20 minutes at room temperature 5. Using the sausage filling attachment on a stand mixer, fill the dough into a vegetarian casing and either chill in the fridge or cook on a flat pan. Plant-based chicken nuggets

[0125] The addition of baking powder and calcium chloride is to increase the water binding capacity of the protein and create air pockets in the dough.

[0126] Method of making is as follows: 1. Place all ingredients into a food processor and blend on low for 5 minutes to make the dough. 2. Mould and shape the plant-based dough nuggets 3. Steam the shaped dough at a temperature of 100°C for 14 minutes 4. Make the batter: mix flour and water in a 1:2 ratio as required 5. Coat in batter and fry for 1.5 minutes 6. Freeze at -20°C 7. Customer preparation: bake at 220°C for 15 minutes Plant-based ice cream

[0127] The interaction of protein and fat will affect the melt rate, stability (creaming), texture, overrun and viscosity properties.

[0128] Method of making is as follows: 1. In a pan mix the soya milk or pea protein powder with water and heat to 45-50°C while continuously stirring; 2. Add the remaining ingredients to the warm mixture while continuously stirring 3. Homogenise at 15000 rpm for 3 minutes 4. Place the mixture at a temperature of 5°C for 24 hours 5. Freeze the mixture while aerating (using an ice cream machine) for 20 minutes Plant-based fruit pie or tart

[0129] Method of making is as follows. For a fruit pie: 1. Preheat the oven to 350°F (175°C) 2. For the caramel: Place 1 cup of sugar in a saucepan and heat over medium-low heat until the sugar liquefies and turns a light brown colour. Pour the syrup into a round baking pan while it is still hot, making sure to spread it evenly 3. Blend all the ingredients with a blender until they are completely mixed and even 4. Place mixture in a baking dish with caramel, cover with foil. Place dish in a larger auxiliary container with water (water bath) 5. Bake for 60 minutes 6. Chill until cool 7. After chilling, carefully invert onto a plate.

[0130] For caramels: 1. Place all ingredients in a blender and blend until smooth 2. Cook over medium heat on a stove, stirring constantly, until caramels are thick 3. Pour into a dish and chill until cool Vegan vanilla cake

[0131] Preparation is as follows 1. Preheat oven to 350°F (175°C). 2. Hydrate SGP2A or SGP2B solution in water (concentration range 1.5-8%). 3. Mix vinegar and plant milk, let sit for 5 minutes 4. Mix flour, sugar, baking powder, baking soda, and salt 5. Add oil, vanilla, and protein solution to plant milk mixture 6. Add liquid ingredients to dry ingredients and mix until well combined 7. Pour batter into an 8-inch cake pan lined with oil paper 8. Bake for 36-38 minutes, until center is golden and set 9. Cool at room temperature Plant-based cookies

[0132] Preparation is as follows: 1. Preheat oven to 350°F (175°C) 2. Hydrate SGP2A in water 3. Beat butter and sugar with medium speed until light in color, about 4 minutes 4. Add SGP2A solution and vanilla extract and mix until fully incorporated 5. Add flour, salt, and baking soda and mix until well combined 6. Add chocolate chips and mix lightly 7. Divide into 100g portions and shape into balls, placing on a cookie sheet 8. Bake for 18-21 minutes Creamy mushroom soup

[0133] Preparation: 1. In a pan, sauté the mushrooms in oil for about 5 to 7 minutes. 2. Add the vegetable broth and seasonings and simmer on low heat. 3. In the meantime, dissolve SGP2A in water (5-15% w / w) according to the desired thickness. 4. Add the coconut milk and protein solution and stir until the soup thickens. 5. Season with salt and pepper. Freeze or refrigerate the dough

[0134] SGP2A, SGP2B and other GPDHs can be used to replace gums (xanthan gum, gum arabic, methylcellulose, guar gum) in frozen and refrigerated doughs.

[0135] The amount of water should be adjusted according to the protein concentration of the flour and the SGP2A or SGP2B. The preparation method is as follows. 1. Mix the ingredients on low speed until smooth, about 5 minutes 2. Let rest for 10 minutes 3. Divide the dough into 150g portions 4. Knead each portion until structure is formed, about 8 minutes 5. Proof at 28°C and 85% humidity until the dough volume doubles 6. Freeze quickly 7. Customer preparation: bake at 350°F for 20-25 minutes 14. Regulatory approval of GPDH as a food ingredient

[0136] After determining that a GPDH is further developed as a food ingredient, the user will ensure that all regulatory requirements are met before starting commercial sales. For example, new food additives and their products sold in the United States must obtain pre-market approval from the Food and Drug Administration (FDA). New additives are "generally recognized as safe" (GRAS) if there is generally available and recognized scientific data, information or methods that indicate that the new additive is safe and there is a possibility of confirmation of unpublished scientific data. The approval notice sent to the FDA Office of Food Additive Safety includes a brief description of the substance (chemical, toxicological and microbiological characteristics), the applicable conditions of use and the basis for GRAS recognition. Then, the FDA assesses whether the submitted notice provides sufficient basis for the GRAS determination. 15. The art and science of cosmetics, their preparation and use

[0137] There are thousands of cosmetic products on the market, each with a different combination of ingredients. In the United States alone, there are approximately 12,500 unique chemical ingredients approved for use in the production of personal care products. A typical product will contain 15-50 ingredients. Given that some consumers use 9 to 15 personal care products every day, consumers can leave up to 500 different chemicals on their skin every day through the use of cosmetics. O. Jones and B. Selinger, Aust. Acad. Sci. 2022.

[0138] Most cosmetics contain at least some combination of the following core ingredients: water, emulsifiers, preservatives, thickeners, emollients, pigments, fragrances, and pH stabilizers.

[0139] Distilled or ultra-pure water is a base ingredient in almost all types of cosmetics, including creams, lotions, makeup, deodorants, shampoos, and conditioners. It can dissolve other ingredients as a solvent and form an emulsion to maintain consistency.

[0140] Emulsifiers are used to prevent the separation of hydrophilic and hydrophobic ingredients in a formulation. Many cosmetics are based on emulsions - small droplets of oil dispersed in water or small droplets of water dispersed in oil. Emulsifiers are added to change the surface tension between water and oil, resulting in a product that is uniform in texture and well mixed. Emulsifiers used in cosmetics include polysorbates, laureth-4, and potassium cetyl sulfate.

[0141] Preservatives are added to cosmetics to extend shelf life and prevent the growth of microorganisms such as bacteria and fungi, which can spoil the product and potentially harm the user. Preservatives used in cosmetics are water-soluble and non-toxic. Preservatives can be natural or artificially synthesized, and their performance varies depending on the product formulation. Some preservatives are present in low amounts, around 0.01%, while others are present in high amounts, up to 5%. Commonly used preservatives include parabens, benzyl alcohol, salicylic acid, formaldehyde, and tetrasodium ethylenediaminetetraacetate.

[0142] Thickeners are used to give the product an attractive consistency and ease of use. Lipid thickeners serve to impart their natural thickness to the formulation. Examples include cetyl alcohol, stearic acid, and palm wax. Natural thickeners are polymers that can absorb water, swell, and increase the viscosity of the product. Examples include hydroxyethyl cellulose, guar gum, xanthan gum, and gelatin. Mineral thickeners can absorb water and oil to increase viscosity, but they have a different effect on the final emulsion compared to gums. Commonly used mineral thickeners include magnesium aluminum silicate, silica, and bentonite. Synthetic thickeners are commonly used in lotions and creams. The most common synthetic thickener is carbomer, which is a water-soluble acrylic acid polymer and can be used to form transparent gels. Other examples include cetyl palmitate and acryloyldimethyltaurate ammonium.

[0143] Emollients soften the skin of the user by preventing water loss. Emollients are widely used in lipsticks, creams, and cosmetics. Many different natural and synthetic chemicals can be used as emollients, including beeswax, olive oil, coconut oil, and lanolin, as well as petrolatum (Vaseline), mineral oil, glycerin, zinc oxide, butyl stearate, and diethylene glycol laurate.

[0144] Colorants and pigments are used in many cosmetics to highlight or change a person's natural skin tone. Mineral ingredients include iron oxides, mica flakes, manganese, chromium oxide, and coal tar. Natural pigments can come from plants, such as beetroot powder, or from animals, such as carmine, which is often used in red lipsticks. The two most common organic pigments are lakes and toners. Lakes are pigments that combine a dye color with an insoluble substance such as hydrated alumina. This makes the dye insoluble in water, making it suitable for cosmetics that require water resistance or waterproof properties. Toner pigments are organic pigments that are not combined with any other substance. Inorganic metal oxide pigments are generally duller than organic pigments, but are more heat and light resistant, and have more color permanence.

[0145] Shimmer effects can be produced by a range of materials. The most common of these are mica and bismuth oxychloride. The size of the particles used to make pearlescent and shiny effects will affect the degree of shine of the product. The smaller the particle (15-60 microns, 1 micron equals one millionth of a meter), the less shiny and more opaque the powder will be. The larger the particle (up to 500 microns), the more shiny and transparent the powder will be.

[0146] Liquid and cream cosmetics often have added fragrances to improve their appeal. 16. Target properties of cosmetics and use of GPDH as a texturizing or thickening agent

[0147] In general, proteins can be developed for use in cosmetics and other personal care ingredients to impart desired properties to the cosmetic or ingredient, or to enhance the ability of other ingredients to impart such properties. The target properties can include one or more of the following: emulsification activity, thickness, texture, viscosity, color or color retention, antibiotic activity, sun protection factor (SPF), water resistance, gloss, stabilization activity, moisturization activity, film formation, smoothness, lubricity, pearlescence, and physical structure.

[0148] The GPDHs of the present disclosure can be used in cosmetics as thickening or texturing agents. For example, they can be used to enhance the consistency and feel of the finished product. In currently marketed cosmetics, the texturing ingredients are usually polymers (polyacrylates, polysaccharides, or gums) or lipid derivatives (oils, esters, or wax derivatives). Texturing agents can be used to create or improve the texture of a product in any desired processing intermediate or final product. For example, texturing agents can give a cosmetic a creamy feel, transparency, thickness, and / or viscosity.

[0149] GPDH of the present disclosure can also be used in cosmetics as a thickening agent or thickener to increase the viscosity of a liquid. In some cases, the GPDH imparts this property while not substantially changing other properties. In other cases, it imparts other desirable properties to the cosmetic. Thickening agents are commonly used in cosmetics and other industrial products such as paints, inks, and explosives.

[0150] Thickeners can also improve the suspension of other ingredients or emulsions, thereby improving the stability of the product. For the above reasons and others, the thickening agents presented in the present disclosure can be used in cosmetics and personal hygiene products. Some thickening agents are gelling agents (gelatinizers) that form gels as colloidal mixtures (forming a weakly cohesive internal structure) dissolved in a liquid phase. Other thickening agents are mechanical thixotropic additives whose discrete particles adhere to or interlock with each other, preventing dispersion or flow when not needed.

[0151] GPDH of the present disclosure can also be used in cosmetics as an emulsifier. Cosmetic ingredients with emulsifying properties can be used in creams and lotions to mix water and oil. There are two types of emulsifiers. Oil-in-water (O / W) emulsifiers can encapsulate oil droplets in water, while water-in-oil (W / O) emulsifiers can encapsulate water droplets in oil. W / O emulsifiers can impart a greasy feel (e.g., night creams and sunscreens). O / W emulsifiers are more commonly used in moisturizing products (e.g., body lotions, day creams). O / W is the most common type of emulsion in cosmetic formulations. The emulsifying power of a water-soluble emulsifier is defined as the maximum amount of oil that can be dispersed in an aqueous solution containing a certain amount of emulsifier without the emulsion breaking or inverting into a water-in-oil emulsion. Emulsifying power can also be characterized by measuring the minimum amount of emulsifier required to form an emulsion, i.e., measuring the surface load (D), which is equivalent to the mass of emulsifier required to cover a unit area of the surface of the droplets.

[0152] GPDH of the present disclosure can also be used to impart other target properties to cosmetics, such as those listed above, to replace or complement the role of a texturizing agent, thickener, and / or emulsifier. 17. Commonly used ingredients in cosmetics and personal care products that can be replaced with GPDH

[0153] The most commonly used natural gum in cosmetics sold in the market today is xanthan gum, which acts as an emulsion stabilizer, film former, and adhesive. It is obtained by fermenting carbohydrates such as glucose with Xanthomonas campestris. Other commonly used ingredients include hydroxyethyl cellulose, locust bean gum, konjac gum, sclerotium gum, and hyaluronic acid. Alternatively or additionally, GPDH can also be used as an auxiliary ingredient commonly used or to impart additional desirable properties to the product.

[0154] Individual proteins can be used in cosmetics to form biopolymers and hydrogels. S. Mitural et al., J. Mat. Sci. (2020) 31 :50. Hydrogels are crosslinked networks of polymeric compounds characterized by high water absorption. Common biopolymer formers include collagen, chitosan, hyaluronic acid, and other polysaccharides.

[0155] In topical moisturizing formulations, individual proteins with high water binding and water retention capacity can be used as a replacement for hyaluronic acid (HA). Individual proteins can replace methyl or ethyl cellulose and hydroxypropyl methylcellulose for emulsion stabilizers, viscosity enhancers, or rheology modifiers. Strongly transparent protein gels can be used for moisturization and physical structure in topical mask applications. Individual proteins can be used to replace or enhance gums / waxes and provide high levels of thickening effect without creating stickiness.

[0156] Individual proteins can be used as a barrier agent / film former in cosmetics. Ronacare, Merck KGaA, Damstadt, Germany.

[0157] Individual proteins can also be used to replace silicones / siloxanes and provide a smooth and lubricious feel without the greasy feel. One of the industry workhorses, cyclomethicone, has recently been removed from the personal care market. Octamethylcyclotetrasiloxane (also known as D4) is a relatively inexpensive ingredient that gives skin creams a silky, non-greasy feel and hair a luxurious bounce and sheen. However, later studies showed that D4 has potential toxicity, can be washed off the hair and skin, and accumulate in marine environments. Therefore, a related compound, decamethylcyclopentasiloxane (D5), which has many of the beneficial properties of D4, replaced it. D4 levels in cosmetics ranged from a few tenths of a percent by weight to as high as 85% in some hair creams, but now regulators are beginning to worry that D5 can also be bioaccumulative and should be replaced with something else. M. S. Reisch, Chem. Eng. News, 2011.

[0158] GPDH of the present disclosure can be screened and developed to optimize its performance in any of the above situations. 18. Regulatory approval of GPDH as an ingredient in cosmetics and personal care products

[0159] In the context of the present disclosure, the term "personal care product" generally refers to any article intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to any surface or part of the human body to clean, beautify, correct bodily odors, or change the appearance, as well as any article used as an ingredient thereof. Personal care products include cleansing pads, colognes, cotton swabs, cosmetic pads, deodorants, eye liners, facial tissues, hairpins, lip glosses, lipsticks, lip balms, lotions, make-up, hand sanitizers, facial cleansers, body washes, nail files, pomades, perfumes, razors, shaving creams, skin creams, baby powders, toilet paper, toothpastes, facial care products, wet wipes, towels, and shampoos. In the present disclosure, the GPDH will be one ingredient in a product or ingredient that is a compounded liquid, cream, gel, lotion, colloid, powder, or dissolvable solid, which can optionally be used in conjunction with a dispensing device or personal care appliance.

[0160] Certain personal care products and ingredients are regulated as cosmetics by the U.S. Food and Drug Administration. The Federal Food, Drug & Cosmetic Act (FD&C Act) defines a cosmetic as "an article intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body or any part thereof, to clean, beautify, correct bodily odors, or change the appearance." Products included in this definition are skin moisturizers, perfumes, lipsticks, nail polishes, eye and facial makeup, shampoos, hair dyes, toothpastes, and deodorants, as well as any material intended for use as a component of these articles. The U.S. Food and Drug Administration classifies cosmetics as one of the following categories: 01. Baby products 02. Bath products 03. Eye makeup products 04. Perfume preparations 05. Hair preparations (not coloring) 06. Hair coloring preparations 07. Cosmetic preparations (non-eye) 08. Nail care preparations 09. Oral hygiene products (toothpaste and mouthwash) 10. Personal cleansing 11. Shaving products 12. Skin care preparations (creams, lotions, powders, and sprays); and 13. Sunscreen preparations

[0161] Certain personal care products and ingredients are regulated as drugs. The FD&C Act defines a drug as "an article intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease and articles intended for use in affecting the structure or any function of the body of man or other animals (except food)”. Over-the-counter drugs (OTC) are drugs that can be purchased without a doctor's prescription. Certain advertising claims can cause a product to be considered a drug, even if the product is marketed as a cosmetic. Such claims can qualify a product as a drug because of its intended use to treat or prevent disease, or to otherwise affect the structure or function of the body. Some examples claim that a product can restore hair growth, reduce cellulite, treat varicose veins, or activate cells. Other examples include skin protectants (such as lip balms and diaper ointments), mouthwashes that claim therapeutic effects, antiperspirants, and dandruff or acne treatments.

[0162] Some personal care products and ingredients meet both the FDA's definition of a cosmetic and a drug. This occurs when a product has two intended uses. For example, a shampoo is a cosmetic because its intended use is to clean the hair. A dandruff treatment is a drug because its intended use is to treat dandruff. Therefore, a dandruff shampoo is both a cosmetic and a drug because it cleans the hair and treats dandruff. Toothpastes containing fluoride, deodorants that are also antiperspirants, and moisturizers and cosmetics that claim to have sunscreen capabilities are all combinations of cosmetics / drugs. Such products must meet the requirements for both cosmetics and drugs.

[0163] In general, products classified as drugs must have premarket approval from the FDA, or meet the requirements of an exemption from the premarket approval requirements, i.e., these products are generally recognized as safe and effective, and not misbranded. Cosmetics and ingredients, with the exception of color additives, are not subject to the premarket approval requirements of the FDA. It is the responsibility of the cosmetic firm to ensure that its products and ingredients are safe before marketing.

[0164] Some personal care products can fall into other regulatory categories, including medical devices (such as certain hair removal and dermabrasion devices), dietary supplements (such as vitamin or mineral tablets or capsules), or other consumer products (such as manicure kits).

[0165] Cosmetic firms can register in the United States through the FDA's Voluntary Cosmetic Registration Program (VCRP). The VCRP assists the FDA in carrying out its responsibility for regulating cosmetics. The FDA uses this information to evaluate cosmetics on the market. Because product filing and establishment registration are not mandatory, the voluntarily submitted information provides the FDA with the best information available about cosmetic products and ingredients, their frequency of use, and the firms that manufacture and distribute them (Federal Register 73:76360, and 69:9339). 19. Use of GPDH in the production of pharmaceuticals

[0166] GPDH can be used as part of a drug or nutraceutical, for example, in combination with an effective dose of one or more pharmaceutically active agents or nutritional ingredients, optional ingredients such as pharmaceutically acceptable preservatives, and an aqueous solvent or excipient. The concentration of GPDH is 0.5% to 50% or 2% to 20% by weight of the final product. Users can wish to adjust the salinity and pH of the solution to facilitate gel formation at lower temperatures and / or to facilitate dissolution after administration. Because SGP2A and SGP2B are derived from plant species, regulatory agencies can be more amenable to using such proteins as part of a drug formulation rather than a synthetic gel. 20. Regulatory approval of pharmaceuticals

[0167] A drug or pharmaceutical product is a composition that contains at least one active agent that requires regulatory approval and provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body of man or animals. A nutraceutical refers to any substance or ingredient that is advertised as beneficial to health but is not regulated by the U.S. Food and Drug Administration.

[0168] FDA approval of a drug requires that the drug’s effects have been tested for safety and effectiveness in clinical trials or similar experiments and reviewed by the FDA’s Center for Drug Evaluation and Research (CDER). The drug is determined to provide a benefit to the target population that outweighs its known and potential risks. The drug approval process is conducted within a structured framework that includes: 1. Analysis of the target condition and existing treatments—FDA reviewers analyze the condition or disease the drug is intended to treat and evaluate current treatments to weigh the risks and benefits of the drug. For example, a drug intended to treat a patient with a life-threatening disease can be considered to have a benefit that outweighs the risks even if those risks would not be acceptable for a disease that does not threaten life, if there are no other treatments available. 2. Evaluation of benefits and risks from clinical data—FDA reviewers evaluate the clinical benefit and risk information submitted by the drug manufacturer, taking into account any uncertainties that can result from incomplete or imperfect data. Generally, the FDA expects drug manufacturers to submit results from two well-designed clinical trials to ensure that the results of the first trial are not the result of chance or bias. In some cases, particularly when the disease is rare and multiple trials are not possible, convincing evidence from one clinical trial can be sufficient. The evidence of benefit to the target population must outweigh any risks and uncertainties. 3. Strategies for managing risk - Risk management strategies include FDA-approved drug labels that clearly describe the benefits and risks of a drug, and how to detect and manage risks. In some cases, pharmaceutical manufacturers can be required to implement risk management and mitigation strategies (REMS). 21. Other uses of GPDH in industrial products

[0169] The ability of SGP2A, SGP2B, and other GPDHs to form robust gels with beneficial properties can be used in other contexts. GPDHs can be used as storage and / or transport media, insulators, or packaging materials for any industrial process that relies on or is facilitated by gels.

[0170] Proteins can be used as delivery vehicles of macro-, micro-, or nano- dimensions, releasing active compounds into the environment in a controlled manner when desired. The design of the delivery structure depends in part on the surface and bulk properties of the protein. For example, a drug delivery device can take advantage of swelling or shrinking capabilities when temperature or pH is changed, triggering the release of active compounds. This increases efficiency, cost-effectiveness, and the range of delivery functions, enabling users to adjust storage and release conditions to the desired outcome.

[0171] Thickeners are an important component of the paint and printing industries. These products require the use of rheology modifiers to prevent pigments from settling to the bottom of the can, resulting in inconsistent results. Water-based formulations are almost impossible except for a few pigments in India ink and a few others that are water-soluble, but these pigments have very low coverage, at best, to tint wood slightly. All modern paints and inks are shipped from the factory with some pigments added to increase opacity and control the mirror effect of the finish, from matte to high gloss, depending on the thickener used, but more on the particle size added as an opacity adjuster. Particle sizes of 1 pm and below would be the limit for high gloss, possibly limited to luxury car paints, and particles of about 100 pm.

[0172] In the petrochemical mining industry, gelling agents (curing agents) are used to react with spilled oil to form a rubbery solid. The gelled oil can then be removed from the surface of the water by skimming, suction devices, or fishing nets. In the production of explosives, gelling agents are used to convert liquid explosives into gels. Nitrocellulose and other nitro esters are often used. Many fuels used in combustion devices also need to be thickened to improve performance. Gelling agents currently in use include aluminum salts, polystyrene, and hydroxybis(2-ethylhexanoate aluminum).

[0173] The GPDHs of the present disclosure can be screened and developed to optimize their performance in any of the above contexts.

[0174] In a general sense, this disclosure provides an industrial product for commercial sale or public use containing a pro-gelling decarboxylase homologue (GPDH) at an effective concentration that results in the desired product suitability for the user. In principle, any industrial product containing, or that would benefit from, gelling, texture-improving, thickening, or emulsifying ingredients can benefit from the GPDH presented in this disclosure by selecting and optimizing it. 22. Amino acid sequences of SGP2A, SGP2B and other GPDH

[0175] Figures 6A to 6C The amino acid sequence of the GPDH prototype named SGP2A is shown (SEQ ID NO:1-3). Figure 6A The sequence was initially identified in the protein database PDB by comparing its sequence with other proteins that are considered to have gelling properties. Figure 6B This is an SGP2A sequence obtained from the UniProt database for presentation purposes. Figure 6C It is the actual protein expressed for testing, including subtle differences and the addition of a polyhistidine tag at the COOH end for easy purification.

[0176] Figure 6D and 6E The amino acid sequence of the second GPDH prototype is shown, named SGP2B (SEQ ID NO:30-31). Figure 6D The sequence was initially determined by comparing it with other proteins that are believed to have gelling properties. Figure 6E It is the actual protein expressed for testing, including subtle differences and the addition of a polyhistidine tag at the COOH end to facilitate purification.

[0177] Figure 7 This is a comparison of the amino acid sequences of SGP2A (SEQ ID NO:1) and SGP2B (SEQ ID NO:30). The highlighted regions correspond to the groups defined below, which are strictly conserved among naturally occurring proteins in the GPDH family.

[0178] GPDH, which is closely related to SGP2A and SGP2B in sequence identity, is also suitable for testing as a gelling agent in food. Table 9 lists strains or species homologs whose amino acid sequences are at least approximately 70% identical to the SGP2A sequence. (Sequences are listed in...) Figures 8A to 8K (SEQ ID NO:4-14). 23. Consensus amino acid sequence motifs in GPDH from different species

[0179] Figure 9AThe domains of diphosphomevalonate decarboxylase that are shared by homologs of SGP2A and SGP2B listed in Table 9 are shown. Starting at the N-terminus, there is a GHMP kinase N-terminal domain (“GHK_N”), followed by a 5-diphosphomevalonate decarboxylase C-terminal domain (“MDD_C”), and a short disordered region. The GHK_N domain is structurally conserved among eukaryotes and prokaryotes, and proteins containing this domain are kinases involved in multiple key metabolic pathways.

[0180] Figure 9B Amino acid motifs (SEQ ID NOS: 15-29) that were determined during the course of this project to be characteristic of the sequences shown in these alignments are shown.

[0181] Motifs Al, A2, and A3 are from the GHMP kinase N’ domain, which helped to define the GHMP kinase N’ domain. Motifs Bl, B2, B3, and motifs Cl, C2, and C3 are from the 5- diphosphomevalonate decarboxylase C-terminal domain, which helped to define that domain. The amino acids highlighted in light gray represent residues involved in the catalytic cycle of members of this enzyme family, and the lysine in black shading represents the catalytic residue that is essential for the native function of diphosphodecarboxylases.

[0182] Motifs Al, Bl, and Cl were determined by sequence alignment, which was generated during the course of the project to characterize certain features of candidate GPDHs. The results of this alignment are shown in the U.S. provisional application from which this disclosure claims priority. The amino acid sequences of thirty-five proteins were extracted from the Pfam protein database provided by the European Molecular Biology Laboratory, version 35.0 (November 2021, 19632 entries). J. Mistry et al., Nucleic Acids Research (2020) doi:10.1093 / nar / gkaa913. The extracted sequences were identified in Pfam as having sequence patterns (hidden Markov models) corresponding to both the GHMP kinase N’-terminal domain (PF00288) and the 5-diphosphomevalonate decarboxylase C-terminal domain (PF18376). The sequences were aligned using the fast Fourier transform algorithm MAFFT. K Katoh et al., Nucl Acids Res. 2002; 30:3059-3066. All sequences were determined to have significant similarity to either the SGP2A or SGP2B sequences by the BLAST algorithm (S F Altschul et al., 1990; J. Mol Biol. 2015: 403-410).

[0183] Motifs A2, B2, and C2 were identified through sequence alignment of the amino acid sequences of 42 additional candidate GPDH proteins. These proteins were extracted from the UniProtKB database (UniProt Consortium, 2021; Nucl. Acids Res. 215:403-410) and possess PFAM domains containing the N'-terminal domain of GHMP kinase (PF00288) and the C-terminal domain of 5-bisphosphate mevalonate decarboxylase (PF18376). Using the BLAST algorithm (SF Altschul et al., 1990; J. MolBiol. 2015:403-410), all sequences were determined to have significant similarity to either SGP2A or SGP2B sequences.

[0184] Motifs A3, B3, and C3 were identified through sequence alignment of amino acid sequences with 60 other candidate GPDH proteins. These proteins were extracted from the UniProtKB database (UniProt Consortium, 2021; Nucl. Acids Res. 215:403-410) and possess PFAM domains containing the N'-terminal domain of GHMP kinase (PF00288) and the C-terminal domain of 5-bisphosphate mevalonate decarboxylase (PF18376). All sequences were determined to have significant similarity to SGP2A or SGP2B sequences using the BLAST algorithm (SF Altschul et al., 1990; J. MolBiol. 2015:403-410).

[0185] By comparison Figure 7 The amino acid sequences of SGP2A and SGP2B shown in the figure identify motifs D, E1, E2, F, G1, and G2.

[0186] Figure 10 This is a sequence similarity network for the selected GPDH. It's a diagram illustrating the degree of pairwise sequence identity between different diomyol-acetone decarboxylases, where each circle represents a specific sequence. The length of the line between sequences reflects the degree of sequence similarity. Sequences used for the network were collected by aggregating all UniProtKB sequences that match the sequence patterns of the N-terminus of GHMP kinase and the C-terminus of 5-bisphosphate mevalonate decarboxylase, according to the definition of PFAM. Larger nodes represent proteins annotated as mevalonate decarboxylases in SwissProt. Edges represent pairwise sequence similarity determined by BLAST, with a bit score greater than or equal to 100 (equivalent to approximately 30% sequence identity). Nodes are density-coded by the percentage of identity between the protein sequence they represent and protein X (as shown in the example). 24. Unique attributes of GPDH with one or more of the above motifs

[0187] In a range of species, proteins with similar functions often have homologues and heterologues with closely related amino acid sequences. The GPDH family is unusual in that sequence identity of species and strain homologues can be as low as 40% ( Figure 7 ). Even so, naturally occurring proteins generally have substantially the same domains and share amino acid motifs that are strictly conserved throughout the plant kingdom. The GPDH definition given and claimed in this disclosure is therefore quite conservative, encompassing a very limited number of related proteins.

[0188] This can be illustrated by querying a large protein database using the structural definition presented in the previous section.

[0189] Shiru (the owner of the present invention) has built its own database, which at the time of writing this document contains over 400 million protein sequences from many public and private databases. A subset of this database (the Plant+ subset) is proteins derived from plants, fungi and cyanobacteria.

[0190] These data show that GPDH is a very rare selective protein: in the Plant+ database, less than 0.01% of all proteins have any of the motifs A1, A2, A3, B1, B2, D, E1, E2, F and G2. Fewer organisms have one of these motifs and some others. SGP2A and SGP2B are representative in this genus due to having common motifs.

[0191] The numbers in Table 10 are not high compared to the amino acid homology definitions that often appear in patent publications. For example, if a protein is identified as having 90% sequence identity to a sequence of 100 contiguous amino acids, there are (100! / 10! / 90!) combinations of positions that can differ, with 20 amino acids differing at each position. Within the 10% limit, the total number of possible sequence streams is approximately 3.5 x 10 14 . 25. Optional changes to the amino acid sequence to further optimize the performance of the GDPH protein

[0192] In some cases, the natural proteins upon which GDPH is based may possess enzymatic activities or binding affinities that are detrimental to human consumption. In such cases, users can choose to alter the amino acid sequence of the natural protein or a portion thereof to remove this activity or add or delete glycosylation sites. The altered form can be designed empirically, for example, by randomly mutagenizing the natural sequence or a portion thereof and testing the functional properties of the altered protein. Alternatively, the altered form can be rationally designed with reference to the known three-dimensional structure of the protein and its suspected functional domains, altering one, two, three, five, ten, or more amino acids, or those between 1 and 5, 11 and 10, or 1 and 25, by substitution, addition, or deletion, thereby removing unwanted binding sites or residues essential to catalytic sites, or causing the protein to refold so that enzyme substrates or enzyme cofactors do not bind. 26. Definition and scope of the GPDH family

[0193] As used in this disclosure, the term "progel-forming decarboxylase homolog" ("GPDH") refers to a family of proteins that share structural similarity with the prototype gelling enzyme SGP2A (SEQ ID NO:1) or SGP2B (SEQ ID NO:30). For the purposes of this disclosure, a protein falls within the definition of GPDH if it possesses one or more of the structural features mentioned below and has a measurable gel-forming ability or the ability to gel or thicken mixtures of food ingredients containing it. In addition to promoting gel formation, individual GPDHs of this disclosure may or may not possess enzymatic activity.

[0194] Unless otherwise expressly stated, the acronym GDPH used in this disclosure is for convenience only and does not imply the function, characteristics, or physiological role of members of the GPDH family. Some GPDHs are naturally occurring, meaning they are produced by organisms whose GPDH-encoding genes have not been genetically modified. GPDHs can be full-length gene products, including splice variants, or fragments of gene products produced during normal expression and manipulation. They can have... Figure 9A This refers to any one or two of the domains shown. Additionally, users can create artificial fragments, fusion proteins, and amino acid variants that are structurally and functionally identical to the definition of GPDH.

[0195] Unless otherwise stated, the GPDH used in accordance with this disclosure has one or more of the following structural features: like Figure 9B As shown, they have at least one motif selected from A1, A2, and A3 and / or at least one motif selected from B1, B2, and B3 and / or at least one motif selected from C1, C2, and C3 and / or at least one motif D. The amino acid sequences of E1, E2, F, G1 and G1; According to the BLAST algorithm (SF Altschul et al., 1990; J. Mol Biol. 2015: 403-410), their amino acid sequence has at least 60%, typically at least 70% (possibly at least 80%, 90%, 95%, or 98.5%) identity to any one or more of the sequences of SEQ ID NOs: 1-14 and 30-31.

[0196] In addition to sharing motifs and / or other structural features with SGP2A and / or SGP2B, the GPDH of the present disclosure also has the property of facilitating the gelation or thickening of a food ingredient mixture comprising the GPDH, either at the time of manufacture or when the food is cooked or otherwise processed by a consumer.

[0197] If, during formulation, the GPDH is capable of increasing the gelation properties of a product relative to a product having the same ingredients except for the GPDH, then the GPDH has the property of facilitating the gelation of a product composed of multiple ingredients. Alternatively or additionally, the GPDH can also facilitate the gelation of a product after heating to a temperature exceeding T onset Values and cooling relative to the same product before heating. This characterization can be performed at any concentration of dry weight concentration of all ingredients in the product between 0.1% and 20% (or 1% and 12%, or 5% and 12%). Quantitatively, the storage modulus of the product will increase by at least 200, 500, 1,000, 5,000, or 10,000 Pascals (Pa).

[0198] If, during formulation, the GPDH is capable of increasing the gelation properties of a product relative to a product having the same ingredients except for the GPDH, then the GPDH has the property of facilitating the gelation of a product composed of multiple ingredients. Alternatively or additionally, the GPDH can also facilitate the gelation of a product after heating to a temperature exceeding T 27. Incorporation of references

[0199] Each of the publications and patent documents cited in this disclosure is incorporated herein by reference to the same extent as if each such publication or document was specifically and individually indicated to be incorporated by reference. 28. Explanations and implementation

[0200] While the foregoing technique has been described in some detail for the purposes of clarity and the understanding of the inventors' contribution to the art, it will be appreciated that certain changes and modifications can be practiced within the scope of the desired claims, except as they have been particularly noted otherwise. The theory underlying the production, action and evaluation of the various products and components presented in this disclosure document is provided for the reader's interest and possible enlightenment only, and is not intended to limit the practice of the claimed invention.

[0201] While the GPDH described in this disclosure was developed by the inventors and the owners of this technology as a gelation agent with superior properties, SGP2A, SGP2B and other GPDHs mentioned in the following claims can be used for food manufacturing for any reason, including but not limited to gelation. Information about the physiological role of SGP2A, SGP2B and other GPDHs is historical information, and unless otherwise explicitly stated, does not limit the use of SGP2A, SGP2B and their homologues or any other product falling within the definition of GPDH as a food ingredient. The reader can use the technology presented in this disclosure for any appropriate purpose.

[0202] While various aspects of the present application have been described with reference to specific examples and drawings, it will be appreciated that changes and modifications can be made within the scope of the claimed invention, as can occur to those ordinarily skilled in the art, without departing from the spirit or scope of the claimed invention in its application to satisfy particular environmental or usage requirements.

Claims

1. A food product or additive comprising a gel-promoting decarboxylase homolog (GPDH) at a concentration of 1-20% by weight of the dry ingredients in the food product or additive, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20), and Motif C3 (SEQ ID NO: 23), wherein the food product or additive comprises less than 2% by weight of other proteins naturally expressed by the organism.

2. The food product or additive of claim 1, wherein the GPDH causes the food product or additive to gel or thicken during manufacture or upon heating.

3. A method of causing a food product to gel or thicken during manufacture or upon heating, the method comprising including in the food product a purified or recombinant gel-promoting decarboxylase homolog (GPDH) at a concentration of 1-20% by weight of the dry ingredients in the food product, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20), and Motif C3 (SEQ ID NO: 23).

4. A method of improving a food product, comprising preparing the food product using a recipe in which one or more previously used gelling or thickening agents are replaced with a gel-promoting decarboxylase homolog (GPDH) at a concentration of 1-20% by weight, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20), and Motif C3 (SEQ ID NO: 23).

5. The method of claim 4, wherein the gelling or thickening agent replaced by the GPDH is one or more of the following: methylcellulose, carboxymethylcellulose (CMC), pectin, xanthan gum, guar gum, locust bean gum, carrageenan, starch, tapioca, butterfly pea flower, fava bean flower, egg, milk, wheat protein, and gelatin.

6. The product or method of any preceding claim, wherein the food product or additive is a meat patty, meatball, sausage, or chicken nugget form of a meat substitute, a plant-based ice cream, a pie or a cake, a cake, a soup, or a frozen or refrigerated dough.

7. The product or method of any preceding claim, wherein the food product or additive is a meat substitute or a condiment, which further comprises: a) a protein content of at least 10% by weight, wherein at least 75% of the protein content is a mixture of plant protein and / or one or more tissue cultures; and b) a fat content of at least 5% by weight, wherein at least 75% of the fat content is one or more plant-derived oils; and optionally c) a heme protein or a porphyrin-binding protein content of 0.2-5% by weight. wherein, upon cooking, the food or product made with the flavor additive has a meat-related aroma and / or taste; and / or wherein the protein content and the fat content form a muscle mimic and a fat tissue mimic combined in the product in a manner approximating the physical organization of meat.

8. A product or method according to any preceding claim, wherein the food is a plant-based ice cream, which further comprises: a) a protein content of at least 5% by weight, wherein at least 75% of the protein content is plant protein and / or a mixture of one or more tissue cultures; and b) a fat content of at least 5% by weight, wherein at least 75% of the fat content is one or more plant-derived oils; and c) a sweetener of at least 5% by weight; maintains the mix and has the mouthfeel of ice cream when frozen at -5 to -25°C.

9. A cosmetic or personal care product ingredient comprising a gel-promoting decarboxylase homolog (GPDH) at a concentration of 1-20% by weight, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20), and Motif C3 (SEQ ID NO: 23), wherein the cosmetic or personal care product ingredient comprises less than 2% by weight of other proteins naturally expressed by the organism.

10. A product or ingredient according to claim 9, wherein the GPDH improves or thickens the texture of the product or ingredient, or facilitates or stabilizes the emulsification of its ingredients.

11. A method of improving, thickening, or emulsifying the texture of a cosmetic or personal care ingredient during production, the method comprising including in the cosmetic or ingredient a purified or recombinant gel-promoting decarboxylase homolog (GPDH) at a concentration of 1-20% by weight of the cosmetic or ingredient, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20), and Motif C3 (SEQ ID NO: 23).

12. A method of improving a cosmetic or personal care ingredient, comprising preparing a product using a formulation in which one or more previously used ingredients is replaced with a gel-promoting decarboxylase homolog (GPDH) at a concentration of 1-20% by weight of the cosmetic or ingredient, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20), and Motif C3 (SEQ ID NO: 23).

13. A method according to claim 12, wherein the previously used ingredient is hyaluronic acid (HA), methyl or ethyl cellulose, hydroxypropyl methyl cellulose, gum, or wax.

14. The product, ingredient or method of any one of claims 9 to 13, which is a moisturizer, eye or skin cosmetic preparation, lip stick, lip balm, emulsion, cleansing milk, skin cream, shaving cream, oral hygiene product, facial treatment, skin care preparation or tanning or sun protection preparation.

15. The product, ingredient or method of any one of claims 9 to 14, wherein the GPDH has the effect of increasing or improving one or more of the following properties of the product or ingredient: emulsifying activity, thickening, texture, viscosity, color or color fixation, antibiotic activity, sun protection factor (SPF), water resistance, gloss, stabilizing activity, moisturizing activity, film forming, smoothness, lubricity, pearlescence and physical structuring.

16. The product, ingredient or method of any one of claims 9 to 15, wherein the GPDH comprises the same sequence as a naturally occurring protein or a gelation-promoting, thickening or texture-improving portion thereof.

17. A method of formulating a pharmaceutical active agent or a nutritional ingredient into a unit dose of a pharmaceutical or nutraceutical product, the method comprising combining an effective amount of the agent or ingredient with a compatible excipient comprising a GPDH, wherein the concentration of the GPDH in the composition is 1 to 10% by weight of the dry ingredient, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20) and Motif C3 (SEQ ID NO: 23).

18. A method of formulating a pharmaceutical active agent or a nutritional ingredient into a unit dose of a pharmaceutical or nutraceutical product, the method comprising encapsulating an effective amount of the agent or ingredient in a capsule or granule comprising a gelation-promoting decarboxylase homolog (GPDH) at a concentration of 5 to 75% by weight of the capsule or granule, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20) and Motif C3 (SEQ ID NO: 23).

19. A method of causing a commercial product or an ingredient thereof to gel or thicken during manufacture or in use, the method comprising incorporating into or causing the product to comprise a GPDH at a concentration of 1 to 20% by weight of the dry ingredient, wherein the GPDH is a protein or protein fragment naturally expressed by a non-animal organism and whose amino acid sequence comprises Motif A3 (SEQ ID NO: 17), Motif B3 (SEQ ID NO: 20) and Motif C3 (SEQ ID NO: 23).

20. The method of claim 19, wherein the product is selected from: - food products, food ingredients and food flavorings; - cosmetic and personal care product ingredients; and - pharmaceutical and nutraceutical products.

21. The product or method of any one of claims 1 to 20, wherein the GPDH comprises Motif Al (SEQ ID NO: 15), Motif Bl (SEQ ID NO: 18), and Motif CI (SEQ ID NO: 21).

22. The product or method of any one of claims 1 to 20, wherein the GPDH further comprises any one or more of Motif D (SEQ ID NO: 24), Motif E2 (SEQ ID NO: 26), Motif F (SEQ ID NO: 27), and Motif G (SEQ ID NO: 29).

23. The product or method of any one of claims 1 to 20, wherein the GPDH comprises Motif A3 (SEQ ID NO: 17), Motif C3 (SEQ ID NO: 20), Motif E (SEQ ID NO: 26), and Motif G2 (SEQ ID NO: 29).

24. The product or method of any one of claims 1 to 20, wherein the GPDH has at least 70% identity to SGP2A (SEQ ID NO: 1).

25. The product or method of any one of claims 1 to 20, wherein the GPDH has at least 70% identity to SGP2B (SEQ ID NO: 30).

26. The product or method of any preceding claim, wherein the GPDH is produced by recombinant expression.

27. The product or method of any preceding claim, wherein the GPDH has been fragmented, mutated, hydrolyzed, digested, denatured, cross-linked, or conjugated to another substance prior to being added to the food, additive, cosmetic, ingredient, pharmaceutical, or commercial product.

28. The product or method of any preceding claim, wherein the GPDH has the enzymatic activity of a diphosphomevalonate decarboxylase.

29. The product or method of any preceding claim, wherein the amino acid sequence of the GPDH is modified by one to ten amino acid substitutions, additions, and / or deletions.

30. The product or method of any preceding claim, wherein the GPDH is naturally expressed in a plant, fungus, or cyanobacterium.

31. The food or additive of claim 1 or the cosmetic or ingredient of claim 9, wherein the GPDH has a critical gel concentration of no more than 6%.

32. The food or additive of claim 1 or the cosmetic or ingredient of claim 9, wherein the T of the GPDH is between 40 and 70 °C. onset 40 to 70 °C.

33. The food or additive of claim 1 or the cosmetic or ingredient of claim 9, wherein a 12% w / w solution of the GPDH has a final gel strength of at least 2,000 Pa.

Images