Method for producing plant-based food substitute

Abstract

The present invention relates to methods of treating plant proteins with an enzyme to produce plant-based food products, including plant-based meat substitutes. More particularly, the methods involve the treatment of plant-based proteins using transglutaminase, sugar oxidase and catalase.

Description

Technical Field

[0001] The present invention relates to a method for producing plant-based food substitutes. More particularly, the present invention relates to the use of enzymes to treat plant proteins to produce plant-based food matrices. Background Art

[0002] With widespread concern about the potential risks of high meat consumption to the environment, animal welfare, and human health, consumers are increasingly seeking alternative products that can serve as meat substitutes. Furthermore, projected population growth poses additional challenges to the sustainability of the food system. These developments have prompted the scientific community and the food industry to develop meat substitutes produced from cereals and legumes to meet the growing demand for non-meat diets. While these plant-based meat substitutes are becoming increasingly accessible, significant gaps remain in mimicking the texture and sensory properties of meat.

[0003] To address these gaps, several food technologies have been introduced. One such technology uses transglutaminase to create a meat-like structure by promoting plant-protein crosslinking. Transglutaminase catalyzes crosslinking through an acyl transfer reaction between the γ-carboxamide group of glutamine residues and the ε-amino group of lysine residues, resulting in the formation of covalent inter- and intramolecular isopeptide bonds. This crosslinking between proteins enables the modification of protein functional properties, including gelling properties, solubility, and emulsification ability.

[0004] The meat-like gel structure produced by treating plant proteins with transglutaminase is only one aspect of creating a true plant-based meat substitute that is acceptable to consumers. Plant-based meat substitutes must also have a suitable texture, including mouthfeel. In addition, plant-based meat products must have an acceptable shelf life and color. Glucose oxidase is used in food and dairy applications to remove excess oxygen, thereby preventing or inhibiting microbial growth. Removing excess oxygen also prevents undesirable browning of foods. In the baking industry, glucose oxidase is used to modify wheat flour protein to improve crumb texture and dough strength. It has been suggested that glucose oxidase can be used to improve the texture of plant-based meats. However, it has been observed that glucose oxidase may have a negative impact on transglutaminase and the meat-like gel it produces.

[0005] There is an ongoing need for methods to improve the texture and shelf life of plant-based meats without negatively impacting gel strength. Summary of the Invention

[0006] In one aspect of the present invention, a method for producing a food containing plant protein is provided, the method comprising the steps of adding transglutaminase, saccharide oxidoreductase and catalase to a food raw material containing plant protein.

[0007] Optionally, the vegetable protein is soy, pea, soybean, fava bean, gluten or oat. Optionally, the vegetable protein is soy protein.

[0008] Optionally, the oxidoreductase is a hexose oxidase. Optionally, the hexose oxidase is derived from Hansenula polymorpha. Optionally, the hexose oxidase is an enzyme having at least 80% sequence identity with SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 85% sequence identity with SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase comprises an enzyme having at least 90% sequence identity with SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 95% sequence identity with SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 98% sequence identity with SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 99% sequence identity with SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase comprises an enzyme according to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

[0009] Optionally, the oxidoreductase is a glucose oxidase. Optionally, the glucose oxidase is derived from Aspergillus niger. Optionally, the glucose oxidase is an enzyme having at least 80% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Optionally, the glucose oxidase is an enzyme having at least 85% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Optionally, the glucose oxidase is an enzyme having at least 90% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Optionally, the glucose oxidase is an enzyme having at least 95% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Optionally, the glucose oxidase is an enzyme having at least 98% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Optionally, the glucose oxidase is an enzyme having at least 99% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Optionally, the glucose oxidase is an enzyme according to SEQ ID NO: 2 or a glucose oxidase-active fragment thereof.

[0010] Optionally, the transglutaminase is derived from Streptomyces mobaraensis. Optionally, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

[0011] Optionally, the catalase is derived from Aspergillus niger. Optionally, the catalase is an enzyme having at least 80% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Optionally, the catalase is an enzyme having at least 85% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Optionally, the catalase is an enzyme having at least 90% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Optionally, the catalase is an enzyme having at least 95% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Optionally, the catalase is an enzyme having at least 98% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Optionally, the catalase is an enzyme having at least 99% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Optionally, the catalase is an enzyme according to SEQ ID NO: 4, or a catalase-active fragment thereof.

[0012] Optionally, the food product is a meat substitute. Optionally, the meat substitute is a hot dog or pepperoni slices.

[0013] In another aspect of the present invention, an improved method for preparing a plant-based meat substitute is provided, the method having the following steps: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors and transglutaminase; b.) keeping the batter warm for less than 60 minutes; c.) molding the batter; and d.) baking the batter to provide the plant-based meat.

[0014] Optionally, the batter further comprises a texturized or structured protein derived from a plant. Optionally, the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat. Optionally, the plant protein is soy protein or pea protein.

[0015] Optionally, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

[0016] Optionally, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

[0017] Optionally, the incubation step lasts less than 30, 15, 10, 5, 2, or 1 minutes or is 0 minutes.

[0018] Optionally, the meat substitute is a hot dog, frankfurters, sausage, deli meat (loaf), bread, salami, meat loaf (meatloaf), pepperoni or barbecue. Optionally, the meat substitute is pepperoni.

[0019] Optionally, the transglutaminase is present in step a) in an amount of from about 5 to 500 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 10 to 400 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 20 to 300 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 25 to 250 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 30 to 200 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 50 to 150 mg / kg of vegetable protein.

[0020] Optionally, the plant-based meat has from 1% to 35% plant protein. Optionally, the plant-based meat has from 5% to 30% plant protein. Optionally, the plant-based meat has from 10% to 25% plant protein. Optionally, the plant-based meat has from 12% to 22% plant protein.

[0021] Optionally, baking step d) is performed at an initial temperature of 25°C to 70°C for a period of 10 to 80 minutes, followed by increasing the temperature until the meat substitute has an internal temperature of at least 90°C. Optionally, it is performed at this initial temperature for 20 to 60 minutes. Optionally, it is performed at this initial temperature for 30 to 45 minutes.

[0022] Optionally, the initial temperature is 35°C to 60°C. Optionally, the initial temperature is 45°C to 50°C.

[0023] In another aspect of the present invention, an improved method for preparing a plant-based meat substitute comprises the following steps: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloid, one or more meat flavors, and transglutaminase; b.) molding the batter; and c.) baking the batter to provide the plant-based meat. The improved method of the present invention does not have a holding step, thereby reducing complexity and lowering costs for manufacturers.

[0024] Optionally, the batter further has texturized or structured proteins derived from plants.

[0025] Optionally, the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat.

[0026] Optionally, the vegetable protein is soy protein or pea protein.

[0027] Optionally, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

[0028] Optionally, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

[0029] Optionally, the meat substitute is a hot dog, frankfurters, sausage, deli meat, bread, salami, meat loaf, pepperoni or barbecue. Optionally, the meat substitute is pepperoni.

[0030] Optionally, the transglutaminase is present in step a) in an amount of from about 5 to 500 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 10 to 400 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 20 to 300 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 25 to 250 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 30 to 200 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 50 to 150 mg / kg of vegetable protein.

[0031] Optionally, the plant-based meat has from 1% to 35% plant protein. Optionally, the plant-based meat has from 5% to 30% plant protein. Optionally, the plant-based meat has from 10% to 25% plant protein. Optionally, the plant-based meat has from 12% to 22% plant protein.

[0032] Optionally, baking step d) is performed at an initial temperature of 25°C to 70°C for a period of 10 to 80 minutes, followed by increasing the temperature until the meat substitute has an internal temperature of at least 90°C. Optionally, it is performed at this initial temperature for 20 to 60 minutes. Optionally, it is performed at this initial temperature for 30 to 45 minutes.

[0033] Optionally, the initial temperature is 35°C to 60°C. Optionally, the initial temperature is 45°C to 50°C.

[0034] In another aspect of the present invention, an improved method for preparing a plant-based meat substitute is provided, wherein there is no holding step, the steps comprising the steps of: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors and transglutaminase; b.) molding the batter; and c.) baking the batter to provide the plant-based meat.

[0035] Optionally, the batter further comprises a texturized or structured protein derived from a plant. Optionally, the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat. Optionally, the plant protein is soy protein or pea protein.

[0036] Optionally, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

[0037] Optionally, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Optionally, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

[0038] Optionally, the meat substitute is a hot dog, frankfurters, sausage, deli meat, bread, salami, meat loaf, pepperoni or barbecue. Optionally, the meat substitute is pepperoni.

[0039] Optionally, the transglutaminase is present in step a) in an amount of from about 5 to 500 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 10 to 400 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 20 to 300 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 25 to 250 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 30 to 200 mg / kg of vegetable protein. Optionally, the transglutaminase is present in an amount of from about 50 to 150 mg / kg of vegetable protein.

[0040] Optionally, the plant-based meat has from 1% to 35% plant protein. Optionally, the plant-based meat has from 5% to 30% plant protein. Optionally, the plant-based meat has from 10% to 25% plant protein. Optionally, the plant-based meat has from 12% to 22% plant protein.

[0041] Optionally, baking step d) is performed at an initial temperature of 25°C to 70°C for a period of 10 to 80 minutes, followed by increasing the temperature until the meat substitute has an internal temperature of at least 90°C. Optionally, it is performed at this initial temperature for 20 to 60 minutes. Optionally, it is performed at this initial temperature for 30 to 45 minutes.

[0042] Optionally, the initial temperature is 35°C to 60°C. Optionally, the initial temperature is 45°C to 50°C.

[0043] Biological sequence description

[0044] SEQ ID NO: 1 is the amino acid sequence of the mature protein of transglutaminase.

[0045] SEQ ID NO: 2 is the amino acid sequence of the mature protein of glucose oxidase (GOX).

[0046] SEQ ID NO: 3 is the amino acid sequence of the mature protein of hexose oxidase (HOX).

[0047] SEQ ID NO: 4 is the amino acid sequence of the mature protein of catalase.

[0048] SEQ ID NO: 5 is the amino acid sequence of CRC24210 SciTG2 precursor protein.

[0049] SEQ ID NO: 6 is the amino acid sequence of the mature protein predicted by CRC24210 SciTG2.

[0050] SEQ ID NO: 7 is the nucleic acid sequence of the CRC24210 SciTG2 synthesized / optimized full-length DNA.

[0051] SEQ ID NO: 8 is the protein sequence of CRC26523 SnoTG2 precursor protein.

[0052] SEQ ID NO: 9 is the protein sequence of the predicted mature protein of CRC26523 SnoTG2.

[0053] SEQ ID NO: 10 is the nucleic acid sequence of CRC26523 SnoTG2 synthetic / optimized full-length DNA.

[0054] SEQ ID NO: 11 is the protein sequence of CRC26802 SroTG1 precursor protein.

[0055] SEQ ID NO: 12 is the protein sequence of the predicted mature protein of CRC26802 SroTG1.

[0056] SEQ ID NO: 13 is the nucleic acid sequence of the synthetic / optimized full-length DNA of CRC26802 SroTG1.

[0057] SEQ ID NO: 14 is the protein sequence of CRC26807 SsyTG1 precursor protein.

[0058] SEQ ID NO: 15 is the protein sequence of the predicted mature protein of CRC26807 SsyTG1.

[0059] SEQ ID NO: 16 is the nucleic acid sequence of the CRC26807 SsyTG1 synthetic / optimized full-length DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Shown is the transglutaminase activity (y-axis) as reflected by the generation of ammonia measured using OPA in protein solutions containing different levels of GOX and catalase (x-axis).

[0061] Figure 2 Compression test results are shown to determine the effect of TG in combination with different levels of GOX and catalase on soy-based protein gels.

[0062] Figure 3 Compression test results are shown to determine the effect of TG in combination with GOX and catalase on soy-based protein gels at 8 or 24 h incubation periods.

[0063] Figure 4 Soy-based hot dog samples containing TG and varying levels of GOX and catalase are shown: T1 (0.4% TG only), T2 (0.4% TG, 0.025% GOX, 0.01% catalase), T3 (0.3% TG, 0.025% GOX), T4 (0.4% TG, 0.01% catalase).

[0064] Figure 5 Shown are texture analyses of plant-based hot dog samples containing TG and varying levels of GOX and catalase: T1 (0.4% TG only), T2 (0.4% TG, 0.025% GOX, 0.01% CAT), T3 (0.4% TG, 0.025% GOX), T4 (0.4% TG, 0.01% CAT).

[0065] Figure 6 Shown are texture analyses of plant-based deli meat samples using TG at different holding time points prior to cooking.

[0066] Figure 7 Shown is a comparison of texture analysis of plant-based deli meat samples using TG (different holding times).

[0067] Figure 8 Shown is a comparison of texture evaluations of plant-based pepperoni samples prepared using hydrocolloids and prepared using TG.

[0068] Figures 9A and 9B Shown is a comparison of texture analysis of plant-based hot dog samples prepared using hydrocolloids and prepared using TG: (A) cold sample and (B) re-cooked sample.

[0069] Figures 10A and 10B Shown are texture analyses of plant-based hot dog samples prepared using TG with different protein levels: (A) cold sample and (B) refrozen sample. DETAILED DESCRIPTION

[0070] definition

[0071] The term "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes." Amino acid sequences are represented using the standard amino-terminal to carboxyl-terminal orientation (i.e., N→C) using the conventional single-letter or three-letter code for amino acid residues.

[0072] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules that can encode polypeptides. Nucleic acids can be single-stranded or double-stranded. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0073] The term "plant protein" means a substrate protein from a plant, such as one or more soy protein isolates, on which transglutaminase can act, and does not include texturized or structured proteins derived from plants that are denatured and do not serve as substrates for transglutaminase.

[0074] Hardness is measured in grams (g). Tackiness is the product of hardness and cohesiveness. Chewability is the product of hardness, cohesiveness, and springiness. Cohesiveness is the work area during the second compression divided by the work area during the first compression. Springiness is the ratio of the product's original height.

[0075] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention also encompasses variants, homologues, derivatives and fragments thereof.The term "variant" is used to refer to a nucleotide sequence or amino acid sequence that differs from the wild-type sequence.

[0076] For example, variants can include substitutions, insertions, deletions, truncations, transversions and / or inversions relative to one or more positions of the wild-type sequence. Variants can be prepared using methods known in the art (e.g., site scanning mutagenesis, insertion mutagenesis, random mutagenesis, site-directed mutagenesis, and orthogenesis) and recombination methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences can be easily synthesized using methods known in the art.

[0077] In some aspects, a variant is a naturally occurring nucleotide sequence or amino acid sequence that is different from the wild-type sequence. For example, a variant can be a natural genetic variant.

[0078] In some aspects, the variant is an engineered variant. For example, the variant can be engineered by recombinant methods.

[0079] The target protein sequence of the present invention may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent substances. As long as the secondary binding activity of the substance is retained, deliberate amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0080] For example, conservative substitutions can be made according to the following table: Amino acids in the same box in the second column, and preferably amino acids in the same row in the third column, can be substituted for each other as shown in Table 1.

[0081] Table 1.

[0082]

[0083] The present invention also encompasses homologous substitutions (substitution and replacement are both used herein to mean the exchange of an existing amino acid residue with an alternative residue), i.e., equivalent substitutions, such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions may also occur, i.e., from one type of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine.

[0084] Substitutions can also be made with synthetic amino acids (e.g., unnatural amino acids), including: α* and α-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids (such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*), L-allyl-glycine*, β-alanine*, La-aminobutyric acid*, Lg-aminobutyric acid*, La-aminoisobutyric acid*, Le-aminocaproic acid # , 7-aminoheptanoic acid*, L-methionine sulfone #* , L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline# , L-thioproline*, methyl derivatives of phenylalanine (Phe) (such as 4-methyl-Phe*, pentamethyl-Phe*), L-Phe (4-amino) # 、L-Tyr(methyl)*、L-Phe(4-isopropyl)*、L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*、L-diaminopropionic acid # and L-Phe(4-benzyl)*.

[0085] For the purposes of the above discussion (relating to homologous or nonhomologous substitution), the symbol * is used to refer to the hydrophobic nature of the derivative, while # is used to refer to the hydrophilic nature of the derivative, with #* referring to amphiphilic character.

[0086] The variant amino acid sequence may comprise a suitable spacer group that can be inserted between any two amino acid residues of the sequence, including, in addition to amino acid spacers such as glycine or β-alanine residues, alkyl groups such as methyl, ethyl or propyl groups. Additional variant forms (involving the presence of one or more amino acid residues in the form of a peptoid) will be well known to those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues in which the α-carbon substituent group is on the nitrogen-atom of the residue, rather than on the α-carbon. Methods for preparing peptides in the form of a peptoid are known in the art, such as Simon RJ et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America] (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. [Biological Technology Trends] (1995) 13 (4), 132-134.

[0087] Nucleotide sequences used in the present invention can include synthetic or modified nucleotides therein. Various different types of modifications for oligonucleotides are known in the art. They include methylphosphonate and phosphorothioate backbones and / or add acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For purposes of the present invention, it will be appreciated that nucleotide sequences as herein described can be modified by any method available in the art. Such modifications can be performed to enhance the in vivo activity or lifespan of nucleotide sequences of the present invention.

[0088] Unless otherwise indicated, the present invention employs conventional biochemistry, molecular biology, microbiology and recombinant DNA techniques, which are within the capabilities of a person of ordinary skill in the art and are explained in the literature. See, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M.J. Gait (ed.), 1984, Oligonucleotide Synthesis: A Practical Guide. Approach [Oligonucleotide Synthesis: A Practical Method], IrI Press [IrI Press]; and D.M.J. Lilley and J.E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology [Enzymology Methods: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology], Academic Press [Academic Press]. Each of these general texts is incorporated herein by reference.

[0089] As used herein, "percentage (%) of sequence identity" means that a particular sequence has at least a certain percentage of amino acid residues that are identical to the amino acid residues in a specified reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

[0090]

[0091] Deletions are considered non-identical residues compared to the reference sequence. This includes deletions occurring at either terminus. For example, a variant having five amino acid deletions from the C-terminus of a mature 617-residue polypeptide would have a 99% sequence identity relative to the mature polypeptide (612 / 617 identical residues x 100, rounded to the nearest integer). Such a variant would be encompassed by variants having "at least 99% sequence identity" to the mature polypeptide.

[0092] All references cited in this specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references specifically referenced herein are hereby incorporated by reference.

[0093] Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention.

[0094] Enzyme production

[0095] Enzyme of the present invention can be produced in host cells, for example, by secretion or intracellular expression. After the enzyme is secreted into the cell culture medium, a cultured cell material (for example, a whole cell culture fluid) with the enzyme can be obtained. Optionally, the enzyme can be separated from the host cells, or even separated from the cell culture fluid, depending on the desired purity of the final enzyme. Suitable host cells include bacteria, fungi (including yeast and filamentous fungi) and plant cells (including algae). Particularly useful host cells include aspergillus niger, aspergillus oryzae (Aspergillus oryzae) or Trichoderma reesei (Trichoderma reesei). Other host cells include bacterial cells, for example Bacillus subtilis (Bacillus subtilis) or Bacillus licheniformis (B.licheniformis), and Streptomyces (Streptomyces), Escherichia coli (E.Coli).

[0096] carrier

[0097] Can construct the DNA construct of the nucleic acid comprising the enzyme encoding to express in host cell.Due to the degeneracy known in the genetic code, variant polynucleotides encoding the same amino acid sequence can be designed and prepared with conventional techniques.It is also well known in the art to optimize the codons for specific host cells.The nucleic acid encoding the enzyme can be incorporated into a vector.The vector can be transferred into a host cell using known transformation techniques (such as those disclosed below).

[0098] The vector can be any vector that can be transformed into a host cell and replicated in the host cell. For example, a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagation and amplification of the vector. The vector can also be transformed into an expression host so that the encoding nucleic acid can be expressed as a functional enzyme. The host cell used as an expression host can include, for example, filamentous fungi. The strain catalog of the Fungal Genetics Stock Center (FGSC) in the United States lists vectors suitable for expression in fungal host cells. Referring to FGSC, strain catalog, University of Missouri, website www.fgsc.net (latest modification time is January 17, 2007). A representative vector is pJG153, which is a promoterless Cre expression vector that can be replicated in a bacterial host. Referring to Harrison et al., (June 2011) Applied Environ.Microbiol [Application and Environmental Microbiology] 77:3916-22. pJG153 can be modified with conventional techniques to include and express nucleic acid encoding an enzyme.

[0099] The nucleic acid of codase can be operably connected to a suitable promotor, which allows transcription in the host cell.Promoter can be any DNA sequence dna showing transcriptional activity in the host cell selected, and can be derived from the gene of coding and host cell homologous or heterologous protein. Exemplary promoters for instructing the transcription (particularly in bacterial hosts) of the DNA sequence dna of codase are the promoter of the lactose operon of Escherichia coli, Streptomyces coelicolor (Streptomyces coelicolor) agarase gene dagA or celA promoter, Bacillus licheniformis α-amylase gene (amyL), the promoter of Bacillus stearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene (amyM), the promoter of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) α-amylase gene (amyQ), the promoter of Bacillus subtilis xylA and xylB genes etc. For transcription in fungal hosts, examples of useful promoters are promoters derived from genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae trisaccharide phosphate isomerase, or Aspergillus nidulans acetamidase. When the gene encoding the enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected from, for example, phage promoters including the T7 promoter and the bacteriophage lambda promoter. Examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal1 and Gal10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. cbh1 is an endogenous inducible promoter from Trichoderma reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2):158-65.

[0100] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence can be a DNA sequence naturally associated with the gene for the enzyme to be expressed or from a different genus or species. The signal sequence and promoter sequence comprising the DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbh1 signal sequence operably linked to the cbh1 promoter.

[0101] The expression vector may also comprise a suitable transcription terminator and, in eukaryotes, a polyadenylation sequence operably linked to the DNA sequence encoding the variant enzyme. The terminator and polyadenylation sequence may be suitably derived from the same source as the promoter.

[0102] The vector may further comprise a DNA sequence that enables the vector to replicate in the host cell. Examples of such sequences are the replication origins of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ702.

[0103] The vector may also comprise a selective marker, for example a gene whose product compensates for a defect in the isolated host cell, such as the dal gene from Bacillus subtilis or Bacillus licheniformis, or a gene that confers antibiotic resistance (e.g., such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance). In addition, the vector may comprise an Aspergillus selection marker, such as amdS, argB, niaD, and xxsC, a marker that confers hygromycin resistance, or selection may be achieved by co-transformation (such as known in the art). See, for example, International PCT Application WO 91 / 17243.

[0104] Intracellular expression may be advantageous in some respects, for example, when using certain bacteria or fungi as host cells to produce large amounts of enzyme for subsequent enrichment or purification. Extracellular secretion of enzymes into the culture medium can also be used to prepare cultured cell material containing isolated enzymes.

[0105] The expression vector typically comprises the composition of cloning vector, for example as the element that allows the carrier to replicate autonomously in selected host organisms and the detectable marker of one or more phenotypes for selecting purpose.Expression vector usually comprises control nucleotide sequence, such as promotor, operator, ribosome bind site, translation initiation signal and optionally suppressor gene or one or more activator genes.In addition, expression vector can comprise the sequence of encoding amino acid sequence, and this amino acid sequence can be by enzyme targeting host cell organelle (such as peroxisome) or target specific host cell compartment.This targeting sequence includes but is not limited to sequence SKL.For expressing under the guidance of control sequence, the nucleotide sequence of enzyme is operably connected to control sequence with about the appropriate manner of expressing.

[0106] The procedures for ligating the enzyme-encoding DNA construct, promoter, terminator, and other elements, respectively, and inserting them into a suitable vector containing the necessary information for replication are well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, and 3rd ed., 2001).

[0107] Host cell transformation and culture

[0108] The cell that comprises the separation of DNA construct or expression vector is advantageously used as the host cell of recombinant production according to enzyme of the present invention.By DNA construct (with one or more copies) being integrated in the host chromosome, can be easily transformed cell with the DNA construct of encoding enzyme.It is generally believed that this integration is favourable, because dna sequence dna is more likely to be stably maintained in the cell.Can, according to ordinary method, for example, by homology or heterologous recombination, DNA construct is integrated in the host chromosome.Alternately, can be with the expression vector transformed cell relevant to different types of host cells as above.

[0109] Examples of suitable bacterial host organisms are Gram-positive bacterial species such as the Bacillaceae family, including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species, such as Streptomyces murinus; lactic acid bacterial species, including Lactococcus species, such as Lactococcus lactis. lactis; Lactobacillus species, including Lactobacillus reuteri; Leuconostoc species; Pediococcus species; and Streptococcus sp. Alternatively, strains of Gram-negative bacterial species belonging to the Enterobacteriaceae family (including E. coli) or to the Pseudomonadaceae family can be selected as host organisms.

[0110] Suitable yeast host organisms can be selected from biotechnology-related yeast species, such as, but not limited to, yeast species, such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species, or species of Saccharomyces, including Saccharomyces cerevisiae, or species belonging to the genus Schizosaccharomyces, such as, for example, S. pombe species. The methylotrophic yeast species strain Pichia pastoris can be used as the host organism. Alternatively, the host organism can be a Hansenula species.

[0111] In filamentous fungi, suitable host organisms include species of Aspergillus, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori or Aspergillus nidulans. Alternatively, bacterial strains of Fusarium species (for example, Fusarium oxysporum) or Rhizomucor species (such as Rhizomucor miehei) can be used as host organisms. Other suitable bacterial strains include Thermomyces and Mucor species. In addition, Trichoderma species can be used as hosts. Suitable programs for transforming Aspergillus host cells include the program described in for example EP 238023. The enzyme expressed by the fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern may be the same as or different from that present in the wild-type enzyme. The type and / or extent of glycosylation may confer altered enzymatic and / or biochemical properties.

[0112] Deleting genes from an expression host can be advantageous, where the gene defect can be cured by a transformed expression vector. Known methods can be used to obtain fungal host cells with one or more inactivated genes. Gene inactivation can be accomplished by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene ineffective for its intended purpose such that the gene is prevented from expressing a functional protein. Any cloned gene from a Trichoderma species or other filamentous fungal host can be deleted, for example, the cbh1, cbh2, egl1, and egl2 genes. Gene deletion can be accomplished by inserting a form of the desired gene to be inactivated into a plasmid using methods known in the art.

[0113] The introduction of DNA constructs or vectors into host cells includes the following techniques, such as transformation; electroporation; nuclear microinjection; transduction; transfection, such as lipofection-mediated and DEAE-dextrin-mediated transfection; incubation with calcium phosphate DNA precipitation; high-speed bombardment with DNA-coated microparticles; and protoplast fusion. General transformation techniques are known in the art. See, for example, Sambrook et al. (2001), supra. The expression of heterologous proteins in Trichoderma is described in, for example, U.S. Patent No. 6,022,725. For the transformation of Aspergillus strains, reference is also made to Cao et al. (2000) Science [Science] 9:991-1001. Genetically stable transformants can be constructed using vector systems whereby the nucleic acid encoding the enzyme is stably integrated into the host cell chromosome. The transformants are then selected and purified by known techniques.

[0114] Preparation of Trichoderma species for transformation can, for example, involve preparing protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. [Contemporary Genetics] 16: 53-56. Mycelia can be obtained from germinated vegetative spores. Mycelia are treated with enzymes that digest the cell wall to produce protoplasts. Protoplasts are protected by the presence of osmotic stabilizers in the suspension medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, etc. Typically, the concentration of these stabilizers varies between 0.8M and 1.2M, for example, a 1.2M solution of sorbitol can be used in the suspension medium.

[0115] In some embodiments, the DNA is taken up into the host Trichoderma species strain. Typically, CaCl2 between about 10-50mM is used in the uptake solution. Additional suitable compounds include buffer systems such as TE buffer (10mMTris, pH 7.4; 1mM EDTA) or 10mM MOPS (pH 6.0) and polyethylene glycol. It is believed that polyethylene glycol allows cell membrane fusion, thereby allowing the content of the culture medium to be delivered to the cytoplasm of the Trichoderma species strain. This fusion often leaves multiple copies of the plasmid DNA that are integrated into the host chromosome.

[0116] Typically, Trichoderma species are transformed using protoplasts or cells that have been permeabilized, typically at 10 5 to 10 7 / mL, especially 2×10 6 The protoplasts can be grown in a suitable solution (e.g., 1.2 M sorbitol and 50 mM CaCl ) at a density of 100 μL or more. These protoplasts or cells can be mixed with the desired DNA in a suitable solution (e.g., 1.2 M sorbitol and 50 mM CaCl ). Typically, a high concentration of PEG is added to the uptake solution. 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, adding approximately 0.25 volume to the protoplast suspension is useful. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. can also be added to the uptake solution to promote transformation. Similar procedures can be used for other fungal host cells. See, for example, U.S. Patent No. 6,022,725 .

[0117] As used herein, protein identification ("JGI-PID") numbers for native Trichoderma genes refer to version 2 of the Trichoderma reesei QM6a genome sequence assembly generated by the Department of Energy Joint Genome Institute (JGI). (The Genome Portal of the Department of Energy Joint Genome Institute, Grigoriev et al., Nucleic Acids Res. 2012 Jan;40(Database Special):D26-32. doi:10.1093 / nar / gkr947). The JGI-assembled scaffold sequences and annotated genes have also been deposited in GeneBank (The National Center for Biotechnology) under nucleotide accession numbers GL985056.1 to GL985132.1.

[0118] Express

[0119] Methods for producing an enzyme of the present invention may comprise culturing the host cells as described above under conditions conducive for production of the enzyme, and recovering the enzyme from the cells and / or culture medium.

[0120] The substratum for culturing cells can be any conventional substratum suitable for the host cell growth under consideration and for obtaining enzyme expression. Suitable substratum and substratum components are available from commercial suppliers or can be prepared according to the formula announced (for example, as described in the catalogue of the American Type Culture Collection).

[0121] The enzyme secreted from the host cell can be used for full culture fluid preparation.In the method for the present invention, any culture method known in the art can be used to realize the preparation of the spent full fermentation liquid of recombinant microorganism, thus cause the expression of enzyme.Therefore, fermentation can be understood as being included in the shake flask culture, small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, fed-batch fermentation or solid-state fermentation) carried out under the condition that suitable substratum and enzyme can be expressed or separated in laboratory or industrial fermentor tank.Term " spent full fermentation liquid " is defined as the unfractionated contents of the fermentation material comprising substratum, extracellular protein (for example, enzyme) and cell biomass in this article.Should be understood that term " spent full fermentation liquid " also contains the cell biomass having used method cracking well known in the art or permeabilization.

[0122] Enzymes secreted from the host cells can be conveniently recovered from the culture medium by well-known procedures involving separation of the cells from the culture medium by centrifugation or filtration and precipitation of the proteinaceous components of the culture medium with the aid of a salt (such as ammonium sulfate), followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, and the like.

[0123] The polynucleotide encoding the enzyme in the vector can be operably linked to a control sequence that is capable of providing expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The control sequence can be modified, for example, by adding other transcriptional regulatory elements to make the transcription level directed by the control sequence more responsive to transcriptional regulatory factors. The control sequence can particularly include a promoter.

[0124] Host cells can be cultured under suitable conditions that allow expression of enzymes. The expression of these enzymes can be constitutive, so that they can be produced continuously, or inducible, requiring stimulation to start expression. In the case of inducible expression, protein production can be started when needed by, for example, adding an inducing substance, such as dexamethasone or IPTG or sophorose to the culture medium. Polypeptides can also be expressed in vitro in cell-free systems (such as TNT TM Produced recombinantly in the rabbit reticulocyte system (Promega).

[0125] The expression host can also be cultivated in a culture medium suitable for the host under aerobic conditions. A combination of shaking or stirring and ventilation can be provided, wherein production occurs at a temperature suitable for this host (e.g., from about 25°C to about 75°C (e.g., 30°C to 45°C), depending on the needs of the host and the production of the desired enzyme). Cultivation can occur from about 12 to about 100 hours or longer (and any hour value therebetween, e.g., from 24 to 72 hours). Typically, the pH of the culture fluid is about 4.0 to about 8.0, depending equally on the host's required culture conditions for the production of the enzyme.

[0126] Methods for enriching and purifying enzymes

[0127] Fermentation, separation and concentration techniques are well known in the art, and conventional methods can be used to prepare solutions containing enzyme polypeptides.

[0128] After fermentation, the fermentation broth is obtained and the microbial cells and various suspended solids (including the remaining crude fermentation material) are removed by conventional separation techniques to obtain an enzyme solution. Common methods include filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction, or chromatography.

[0129] It is desirable to concentrate the enzyme polypeptide-containing solution to optimize recovery. Using an unconcentrated solution requires increasing the incubation time in order to collect the enriched or purified enzyme precipitate.

[0130] Use conventional concentration technology to concentrate the enzyme solution until the desired enzyme level is obtained. The concentration of the enzyme solution can be achieved by any of the technology discussed in this article. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and / or ultrafiltration.

[0131] The enzyme solution is concentrated to form a concentrated enzyme solution until the enzyme activity of the concentrated enzyme polypeptide-containing solution reaches a desired level.

[0132] Concentration can be performed using, for example, a precipitant such as a metal halide precipitant. Metal halide precipitants include, but are not limited to, alkali metal chlorides, alkali metal bromides, and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide, and blends of two or more of these metal halides. The metal halide precipitant sodium chloride can also be used as a preservative.

[0133] The metal halide precipitation agent is used in an amount effective to precipitate the enzyme. After routine testing, it will be apparent to one of ordinary skill in the art to select at least an effective amount and an optimal amount of metal halide to effectively cause precipitation of the enzyme, as well as precipitation conditions, including incubation time, pH, temperature, and enzyme concentration, for maximizing recovery.

[0134] Typically, at least about 5% w / v (weight / volume) to about 25% w / v of the metal halide is added to the concentrated enzyme solution, and typically at least 8% w / v. Typically, no more than about 25% w / v of the metal halide is added to the concentrated enzyme solution, and typically no more than about 20% w / v. The optimal concentration of the metal halide precipitation agent will depend, among other things, on the properties of the particular enzyme polypeptide and its concentration in the concentrated enzyme solution.

[0135] Another alternative way to precipitate the enzyme is to use an organic compound. Exemplary organic compound precipitation agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitation agent can be performed before, simultaneously with, or after the addition of the metal halide precipitation agent, and the addition of the two precipitation agents, the organic compound, and the metal halide can be performed sequentially or simultaneously.

[0136] Typically, the organic precipitation agent is selected from the group consisting of: an alkali metal salt of 4-hydroxybenzoic acid (such as a sodium salt or potassium salt), and a straight or branched alkyl ester of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 12 carbon atoms, and a blend of two or more of these organic compounds. The organic compound precipitation agent can be, for example, a straight or branched alkyl ester of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 10 carbon atoms, and a blend of two or more of these organic compounds. An exemplary organic compound is a straight alkyl ester of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 6 carbon atoms, and a blend of two or more of these organic compounds. Methyl ester of 4-hydroxybenzoic acid, propyl ester of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid, and a blend of two or more of these organic compounds can also be used. Additional organic compounds include, but are not limited to, methyl 4-hydroxybenzoate (also known as methylparaben) and propyl 4-hydroxybenzoate (also known as propylparaben), which are also preservatives. For further description, see, for example, U.S. Patent No. 5,281,526.

[0137] The addition of an organic compound precipitation agent offers the advantage of a high degree of flexibility in precipitation conditions with respect to pH, temperature, enzyme concentration, precipitation agent concentration and incubation time.

[0138] The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by the metal halide precipitation agent. After routine testing, the selection of at least an effective amount and an optimal amount of the organic compound precipitation agent, along with the conditions of the precipitation to maximize recovery, including incubation time, pH, temperature, and enzyme concentration, will be apparent to one of ordinary skill in the art in light of this disclosure.

[0139] Typically, at least about 0.01% w / v of the organic compound precipitation agent is added to the concentrated enzyme solution, and often at least about 0.02% w / v. Typically, no more than about 0.3% w / v of the organic compound precipitation agent is added to the concentrated enzyme solution, and often no more than about 0.2% w / v.

[0140] The concentrated polypeptide solution containing the metal halide precipitation agent and the organic compound precipitation agent can be adjusted to a certain pH, which will necessarily depend on the enzyme to be enriched or purified. Typically, the pH is adjusted to a level close to the isoelectric point of the enzyme. The pH can be adjusted within a pH range of about 2.5 pH units below the isoelectric point (pi) to about 2.5 pH units above the isoelectric point.

[0141] The incubation time required to obtain an enriched or purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of the enzyme, and the specific precipitation agent or agents and their concentrations. Generally, the time effective for enzyme precipitation is between about 1 and about 30 hours; usually no more than about 25 hours. In the presence of an organic compound precipitation agent, the incubation time can still be reduced to less than about 10 hours, and in most cases even to about 6 hours.

[0142] Typically, the temperature during incubation is between about 4° C. and about 50° C. Typically, the method is performed at a temperature between about 10° C. and about 45° C. (e.g., between about 20° C. and about 40° C.). The optimal temperature for inducing precipitation varies depending on the solution conditions and the enzyme or precipitant(s) used.

[0143] The overall recovery of the enzyme precipitate of enrichment or purification and the efficiency of the process thereof are improved by stirring the solution comprising the enzyme, the added metal halide and the added organic compound. Stirring steps are performed during the addition of the metal halide and the organic compound and during the subsequent incubation period. Suitable stirring methods include mechanical stirring or shaking, vigorous ventilation or any similar techniques.

[0144] In some embodiments, the enzyme of the present invention is purified by filtration or purification of the enzyme.The enzyme of the present invention is purified by filtration or purification of the enzyme.After the incubation period, the enzyme is separated with the dissociated pigment and other impurities, and collected by conventional separation techniques such as filtration, centrifugal, microfiltration, rotary vacuum filtration, ultrafiltration, filter press, cross-membrane microfiltration, cross-flow membrane microfiltration, etc. The enzyme is further enriched or purified by washing the precipitate with water. For example, the enzyme is washed with water containing a metal halide precipitation agent or with a metal halide and an organic compound precipitation agent.

[0145] During fermentation, enzyme polypeptides accumulate in the culture broth. In order to separate, enrich or purify the desired enzyme, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or purification. In one embodiment, the cell-free culture broth is salted out using ammonium sulfate at about 70% saturation; the precipitated fraction at 70% saturation is then dissolved in a buffer and applied to a column (such as a Sephadex G-100 column), and eluted to recover the enzyme activity fraction. For further enrichment or purification, conventional procedures such as ion exchange chromatography can be used.

[0146] The enriched or purified enzyme can be prepared as a liquid (solution, slurry) or solid (granules, powder) final product.

[0147] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0148] In one aspect of the present invention, a method for producing a food containing plant protein is provided, the method comprising the steps of adding transglutaminase, saccharide oxidoreductase and catalase to a food raw material containing plant protein.

[0149] Preferably, the vegetable protein is soy, pea, soybean, fava bean, gluten or oat. More preferably, the vegetable protein is soy protein.

[0150] Preferably, the oxidoreductase is a hexose oxidase. More preferably, the hexose oxidase is derived from Hansenula polymorpha. In other preferred embodiments, the hexose oxidase is an enzyme having at least 80% sequence identity to SEQ ID NO: 3, or a hexose oxidase-active fragment thereof. More preferably, the hexose oxidase is an enzyme having at least 85% sequence identity to SEQ ID NO: 3, or a hexose oxidase-active fragment thereof. Even more preferably, the hexose oxidase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 3, or a hexose oxidase-active fragment thereof. More preferably, the hexose oxidase is an enzyme having at least 95% sequence identity to SEQ ID NO: 3, or a hexose oxidase-active fragment thereof. In yet more preferred embodiments, the hexose oxidase is an enzyme having at least 98% sequence identity to SEQ ID NO: 3, or a hexose oxidase-active fragment thereof. In yet more preferred embodiments, the hexose oxidase is an enzyme having at least 99% sequence identity to SEQ ID NO: 3, or a hexose oxidase-active fragment thereof. In a most preferred embodiment, the hexose oxidase comprises an enzyme according to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

[0151] In other preferred embodiments, the oxidoreductase is glucose oxidase. Preferably, the glucose oxidase is derived from Aspergillus niger. In other preferred embodiments, the glucose oxidase is an enzyme having at least 80% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. More preferably, the glucose oxidase is an enzyme having at least 85% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Even more preferably, the glucose oxidase is an enzyme having at least 90% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Even more preferably, the glucose oxidase is an enzyme having at least 95% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. Even more preferably, the glucose oxidase is an enzyme having at least 98% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. In yet more preferred embodiments, the glucose oxidase is an enzyme having at least 99% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof. In the most preferred embodiment, the glucose oxidase is the enzyme according to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof.

[0152] Preferably, the transglutaminase is derived from Streptomyces mobaraensis. In other preferred embodiments, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof. In an even more preferred embodiment, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1 or a transglutaminase active fragment thereof. In a most preferred embodiment, the transglutaminase is an enzyme according to SEQ ID NO: 1 or a transglutaminase active fragment thereof.

[0153] Preferably, the catalase is derived from Aspergillus niger. In other preferred embodiments, the catalase is an enzyme having at least 80% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. More preferably, the catalase is an enzyme having at least 85% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Even more preferably, the catalase is an enzyme having at least 90% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Even more preferably, the catalase is an enzyme having at least 95% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. In yet more preferred embodiments, the catalase is an enzyme having at least 98% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Even more preferably, the catalase is an enzyme having at least 99% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof. Most preferably, the catalase is an enzyme according to SEQ ID NO: 4, or a catalase-active fragment thereof.

[0154] Preferably, the food product is a meat substitute. Preferably, the meat substitute is a hot dog or pepperoni.

[0155] In another aspect of the present invention, an improved method for preparing a plant-based meat substitute is provided, the method having the following steps: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors and transglutaminase; b.) keeping the batter warm for less than 60 minutes; c.) molding the batter; and d.) baking the batter to provide the plant-based meat.

[0156] Preferably, the batter further comprises a texturized or structured protein derived from a plant. Preferably, the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat. More preferably, the plant protein is soy protein or pea protein.

[0157] Preferably, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Still more preferably, the transglutaminase is an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase active fragment thereof.

[0158] In yet more preferred embodiments, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Still more preferably, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. In a most preferred embodiment, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

[0159] Preferably, the incubation step lasts less than 30, 15, 10, 5, 2, or 1 minutes or is 0 minutes.

[0160] Preferably, the meat substitute is a hot dog, frankfurter, sausage, deli meat, bread, salami, meat loaf, pepperoni or barbecue. More preferably, the meat substitute is pepperoni.

[0161] Preferably, the transglutaminase is present in step a) in an amount of from about 5 to 500 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 10 to 400 mg / kg of vegetable protein. Still more preferably, the transglutaminase is present in an amount of from about 20 to 300 mg / kg of vegetable protein. In an even more preferred embodiment, the transglutaminase is present in an amount of from about 25 to 250 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 30 to 200 mg / kg of vegetable protein. In a most preferred embodiment, the transglutaminase is present in an amount of from about 50 to 150 mg / kg of vegetable protein.

[0162] Preferably, the plant-based meat has from 1% to 35% plant protein. More preferably, the plant-based meat has from 5% to 30% plant protein. Even more preferably, the plant-based meat has from 10% to 25% plant protein. More preferably, the plant-based meat has from 12% to 22% plant protein.

[0163] Preferably, baking step d) is performed at an initial temperature of 25°C-70°C for a period of 10 to 80 minutes, and then the temperature is increased until the meat substitute has an internal temperature of at least 90°C. More preferably, it is performed at this initial temperature for 20 to 60 minutes. More preferably, it is performed at this initial temperature for 30 to 45 minutes.

[0164] Preferably, the initial temperature is 35°C to 60°C. More preferably, the initial temperature is 45°C to 50°C.

[0165] In another aspect of the present invention, an improved method for preparing a plant-based meat substitute comprises the following steps: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloid, one or more meat flavors, and transglutaminase; b.) molding the batter; and c.) baking the batter to provide the plant-based meat. The improved method of the present invention does not have a holding step, thereby reducing complexity and lowering costs for manufacturers.

[0166] Preferably, the batter further comprises a texturized or structured protein derived from a plant. Preferably, the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat. More preferably, the plant protein is soy protein or pea protein.

[0167] Preferably, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Still more preferably, the transglutaminase is an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase active fragment thereof.

[0168] In yet more preferred embodiments, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Still more preferably, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. In a most preferred embodiment, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

[0169] Preferably, the meat substitute is a hot dog, frankfurter, sausage, deli meat, bread, salami, meat loaf, pepperoni or barbecue. More preferably, the meat substitute is pepperoni.

[0170] Preferably, the transglutaminase is present in step a) in an amount of from about 5 to 500 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 10 to 400 mg / kg of vegetable protein. Still more preferably, the transglutaminase is present in an amount of from about 20 to 300 mg / kg of vegetable protein. In an even more preferred embodiment, the transglutaminase is present in an amount of from about 25 to 250 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 30 to 200 mg / kg of vegetable protein. In a most preferred embodiment, the transglutaminase is present in an amount of from about 50 to 150 mg / kg of vegetable protein.

[0171] Preferably, the plant-based meat has from 1% to 35% plant protein. More preferably, the plant-based meat has from 5% to 30% plant protein. Even more preferably, the plant-based meat has from 10% to 25% plant protein. More preferably, the plant-based meat has from 12% to 22% plant protein.

[0172] Preferably, baking step d) is performed at an initial temperature of 25°C-70°C for a period of 10 to 80 minutes, and then the temperature is increased until the meat substitute has an internal temperature of at least 90°C. More preferably, it is performed at this initial temperature for 20 to 60 minutes. More preferably, it is performed at this initial temperature for 30 to 45 minutes.

[0173] Preferably, the initial temperature is 35°C to 60°C. More preferably, the initial temperature is 45°C to 50°C.

[0174] In another aspect of the present invention, an improved method for preparing a plant-based meat substitute is provided, wherein there is no holding step, the steps comprising the steps of: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors and transglutaminase; b.) molding the batter; and c.) baking the batter to provide the plant-based meat.

[0175] Preferably, the batter further comprises a texturized or structured protein derived from a plant. Preferably, the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat. More preferably, the plant protein is soy protein or pea protein.

[0176] Preferably, the transglutaminase is an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Still more preferably, the transglutaminase is an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Even more preferably, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. More preferably, the transglutaminase is an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase active fragment thereof.

[0177] In yet more preferred embodiments, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. Still more preferably, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof. In a most preferred embodiment, the transglutaminase is an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

[0178] Preferably, the meat substitute is a hot dog, frankfurter, sausage, deli meat, bread, salami, meat loaf, pepperoni or barbecue. More preferably, the meat substitute is pepperoni.

[0179] Preferably, the transglutaminase is present in step a) in an amount of from about 5 to 500 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 10 to 400 mg / kg of vegetable protein. Still more preferably, the transglutaminase is present in an amount of from about 20 to 300 mg / kg of vegetable protein. In an even more preferred embodiment, the transglutaminase is present in an amount of from about 25 to 250 mg / kg of vegetable protein. More preferably, the transglutaminase is present in an amount of from about 30 to 200 mg / kg of vegetable protein. In a most preferred embodiment, the transglutaminase is present in an amount of from about 50 to 150 mg / kg of vegetable protein.

[0180] Preferably, the plant-based meat has from 1% to 35% plant protein. More preferably, the plant-based meat has from 5% to 30% plant protein. Even more preferably, the plant-based meat has from 10% to 25% plant protein. More preferably, the plant-based meat has from 12% to 22% plant protein.

[0181] Preferably, baking step d) is performed at an initial temperature of 25°C-70°C for a period of 10 to 80 minutes, and then the temperature is increased until the meat substitute has an internal temperature of at least 90°C. More preferably, it is performed at this initial temperature for 20 to 60 minutes. More preferably, it is performed at this initial temperature for 30 to 45 minutes.

[0182] Preferably, the initial temperature is 35°C to 60°C. More preferably, the initial temperature is 45°C to 50°C.

[0183] Examples

[0184] Materials and Methods

[0185] Reagent-grade chemicals (including N-acetylcysteine (NAC), o-phthalaldehyde (OPA), sodium tetraborate decahydrate, and hydrochloric acid (HCl, 37%)) were purchased from Sigma-Aldrich (St. Louis, MO). Soy protein powder SUPRO EX45 (90% protein, dry basis) was obtained from IFF (New Century, KS). Transglutaminase (TG) was purchased from Modernist Pantry (Eliot, ME), while glucose oxidase (GOX) and FoodPro CAT (catalase) were obtained from IFF (New Century, KS).

[0186] Example 1:

[0187] Biochemical assays: Assessment of TG activity in combination with GOX and catalase using the OPA assay

[0188] TG catalyzes the formation of an isopeptide bond between the γ-carboxamide group of glutamine and the ε-amino group of a lysine residue, thereby releasing ammonia (NH3) ( M et al., 2015). TG activity was assessed using the OPA assay. In this assay, ammonia released from the TG-catalyzed reaction is derivatized with NAC and OPA in the presence of TCEP to form fluorescent isoindole adducts (Robert-Peillard, F. et al., 2017). Based on this concept, the effects of GOX and catalase on TG activity were evaluated as follows. First, a protein solution containing 3% SUPRO EX45 was prepared in purified water. The protein was then treated with 3% TG (w / w protein), and / or GOX (0.05%, 0.1%, or 0.2% of the total solution), and catalase (0.1% or 0.2% of the total solution). Dextrose (0.4% of the total solution) was added as a substrate for GOX. Negative and positive controls were also prepared, with the negative control containing no enzyme and the positive control containing only TG without GOX and catalase.

[0189] Similarly, samples containing only GOX and catalase (no TG) were prepared to adjust for potential background interference that could interfere with fluorescence readings. The samples were then incubated at 40°C for 1 hour, and the enzyme was inactivated at 95°C for 10 minutes. The solution was then centrifuged, and the supernatant of each solution was used for OPA determination. For OPA determination, a working solution containing 200mM NAC (in 1M HCl), 20mM TCEP (in 1M HCl), and 200mM OPA (in ethanol) was prepared in 0.1M sodium borate buffer (pH 10.5). Subsequently, 5 μL of each supernatant from the test sample was reacted with 50 μL of working solution in a 96-well plate. The samples were then mixed and incubated at room temperature for 5 min, and then fluorescence was read. TG activity was then determined based on fluorescence intensity, with the excitation wavelength and emission wavelength set to λex=415nm and λem=485nm, respectively.

[0190] Results of Example 1:

[0191] like Figure 1 As shown, ammonia release (TG activity) appears to decrease with increasing levels of GOX compared to a control containing only TG. GOX promotes the oxidation of glucose to hydrogen peroxide and D-gluconolactone (Wong, CM et al., 2008). Although the mechanism by which GOX may affect TG activity is unclear, one potential explanation is that hydrogen peroxide released by GOX activity may oxidize cysteine residues in TG and reduce its activity, as TG is known to be highly sensitive to oxidation (Stamnaes, J. et al., 2010). Interestingly, this effect appears to be offset when catalase is added at levels of 0.1% and 0.2%. This effect may be due to the conversion of excess hydrogen peroxide to water through the action of catalase, while a small amount of hydrogen peroxide may also promote other reactions leading to ammonia release. However, even in the presence of catalase, higher levels of GOX (0.2% of the total solution) still reduced TG activity. Based on this study, the combination of lower levels of GOX and catalase may enhance TG activity.

[0192] Example 2:

[0193] Model system: evaluating the effects of TG activity in combination with GOX and catalase on the textural properties of protein gels

[0194] While biochemical assays provide insights into the extent of TG activity through the amount of ammonia released, it is difficult to determine protein cross-linking and texture modification in plant-based proteins using this assay. Therefore, we developed a model system to evaluate the effect of TG on the overall texture properties of protein gels. To prepare the protein gel, 12% soy protein (SUPRO EX 45) was mixed with purified water under vacuum using a STEPHAN mixer. 80 g of protein gel was then aliquoted into small glass jars, to which different enzyme combinations were added. TG (4% w / w of protein) and dextrose (0.4% of the total solution) were added to each jar, along with different levels of GOX and catalase, as described in Tables 2 and 3. After all enzymes and dextrose were added, the samples were manually mixed for approximately 1 min and then incubated at room temperature for approximately 15 hours before texture analysis.

[0195] Table 2: List of variables used to evaluate the effect of TG in combination with GOX and catalase

[0196]

[0197] Additionally, the following sample groups were evaluated separately, where texture was analyzed at both the 8-hour and 24-hour incubation periods.

[0198] Table 3: List of variables with and without catalase.

[0199]

[0200] *Note: TG was added at 4% (w / w) of protein in all samples.

[0201] Texture Profile Analysis:

[0202] Carry out texture analysis to assess the influence of the strength / hardness of the gel prepared using soy protein by different enzyme combinations. In this test, the resistance to stress is measured by using TA-XT PLUS texture analyzer (Texture Technologies Corp, Scarsdale, New York). Texture is measured using the TA-23 cylindrical probe (12.7mm in diameter) with Exponent software. Each jar equipped with protein gel is placed below the probe, applying a 5g trigger force to reach a fracture distance of 20mm. Because these samples are compressed into gel and permanent deformation occurs, each sample is only compressed once, and the probe advances at 0.5mm / sec. Then the results are reported as gel hardness (power, g).

[0203] Results of Example 2:

[0204] As observed in biochemical assays, increasing GOX levels resulted in a significant decrease in gel strength or firmness ( Figure 2 ). However, this effect appeared to be counteracted when catalase was added at levels of 0.1% and 0.2%. Although higher levels of GOX (0.2% of the total solution) appeared to reduce gel strength even in the presence of catalase, the gel strength was comparable to, if not higher than, that of gels containing TG alone. This study confirmed that the increased ammonia release observed in the biochemical assay did lead to higher protein cross-linking, resulting in tighter gels. Furthermore, while catalase may have used excess hydrogen peroxide, small amounts of hydrogen peroxide may have promoted the formation of additional cross-links by acting on cysteine residues or other reactive sites to create disulfide bonds (Vemulapalli, V. and Hoseney, RC 1998).

[0205] like Figure 3 As shown, an additional variable was incorporated, in which one sample used only TG and catalase (no GOX). The results showed that TG and catalase performed similarly to the control (TG only), and neither enzyme resulted in an increase in gel strength when used alone, unless GOX was added. This effect was observed at both 8 and 24 hour incubation times. This finding further confirmed that the improvement in gel strength was a result of the addition of all three enzymes.

[0206] Example 3:

[0207] Pilot application trial: Texture improvement of plant-based hot dog samples prepared using TG in combination with GOX and catalase

[0208] As highlighted above, the use of TG in combination with GOX and catalase was evaluated in biochemical assays and model systems. The next step is to evaluate this effect in pilot application trials of meat substitute products. In this test, a modified hot dog recipe was used that consisted of plant-based proteins without the addition of other ingredients (such as seasonings, pigments, fats, and antimicrobials) that might play a role in modifying the functionality of the protein and / or the activity of the enzymes. The recipes with different enzyme combinations are provided in Table 4.

[0209] Table 4: Plant-based hot dog recipes used to evaluate the effects of combining TG with GOX and catalase

[0210]

[0211] *Note: This recipe does not contain any flavoring, coloring, or fat source.

[0212] The four tests evaluated in this trial include:

[0213] Test 1 (T1): TG only (0.4%) - control

[0214] Test 2 (T2): TG (0.4%) + GOX (0.025%) + catalase (0.01%)

[0215] Test 3 (T3): TG (0.4%) + GOX (0.025%)

[0216] Test 4 (T4): TG (0.4%) + catalase (0.01%)

[0217] Preparation of plant-based hot dogs:

[0218] Plant-based hot dog samples were prepared using a STEPHAN mixer according to the recipe presented in Table 3. First, 300 g of hydration water was reserved to dissolve the enzyme. The remaining water was then added to the STEPHAN mixer, followed by the addition of SUPRO EX45. The protein was then thoroughly mixed and hydrated under vacuum for approximately 2-3 minutes. The enzyme and dextrose were then dissolved in 300 g of water and added to the STEPHAN mixer along with the protein solution. During this step, vacuum was not introduced during mixing because it is important to ensure that GOX has access to sufficient oxygen to catalyze the oxidation of glucose to hydrogen peroxide and D-gluconolactone (hydrolyzed to gluconic acid). After the protein and enzyme were thoroughly mixed for approximately 3-4 minutes, the protein gel was transferred to a vacuum bag and filled into a plastic casing. The sample was then cooked in a Rational oven, with a cooking cycle consisting of a 40-minute hold at 40°C and a final temperature increase of 95°C. After the sample was cooked, it was stored in a refrigerator (4°C) overnight before texture analysis.

[0219] Texture Analysis:

[0220] Similar to the model system samples, the hardness of the hot dog samples was evaluated using a TA-XT2i texture analyzer (Texture Technologies Corp, Scarsdale, New York). The probe used in this test was a TA-30 cylindrical probe (76.2 mm in diameter). The hot dog samples were cut into 3 cm pieces and each test was repeated 10 times. Each hot dog sample was placed under the probe and a 10 g trigger force was applied to achieve a 10 mm break distance. The samples were compressed twice to evaluate hardness and other texture properties, such as resilience, adhesion, cohesion, chewiness, elasticity, gummy properties, etc. For the purposes of this study, only hardness, chewiness, and gummy properties were evaluated to determine the effect of the enzyme combination on the hot dog samples.

[0221] Pilot application test results:

[0222] The samples were stored in a refrigerator (3°C-4°C) overnight before texture analysis. Figure 5 As shown, T2 containing all three enzymes performed best in terms of gel hardness, cohesiveness and chewiness. This finding is very consistent with the results from the above-mentioned biochemical assays and model system work. Among these samples, T3 was found to have the lowest gel hardness, while T4 performed similarly to the control (T1). Adding GOX without adding catalase seemed to have a negative impact on the hardness of the hot dog samples. Combined with the results of the biochemical assays and model systems, these findings support the use of TG in combination with GOX and catalase to improve the firmness of soy-based protein formulas, which can be used for different meat substitute applications.

[0223] Example 4: TG dose response

[0224] Materials and Methods

[0225] Soy protein isolate and structured soy protein (90% and 77% protein, respectively, on a dry basis) were obtained from IFF (New Century, Kansas). Hydrocolloids from IFF, including carrageenan, methylcellulose, and modified corn starch, were used. Other dry ingredients, including flavors (vegetarian burger flavor, vegetarian beef bouillon, natural pepperoni flavor, frankfurter and bolognese flavor, and beef or pork flavor), and colors (chili red, tomato extract 2LWS, red raspberry, malt extract, and caramel color), as well as antimicrobial agents, were also obtained from IFF.

[0226] Transglutaminase (TG) (SEQ ID NO: 1) was purchased from Modernist Pantry (Elliot, ME).

[0227] Pilot application trial: Evaluation of different doses of triglycerides in plant-based deli meat formulations

[0228] In this example, the effect of TG at certain levels on the textural properties of a plant-based deli meat formula was evaluated. The plant-based deli meat formula consisted of extruded structured soy protein added at 21.9% w / w. However, it is known that the enzyme TG does not work well on extruded proteins because the process modifies the protein to the extent that the enzyme may not be able to access its side chains. Soy protein isolate, which acts as an excellent substrate for TG, was added to the formula at 10% w / w. TG was added at 0.05%, 0.15%, 0.3%, 0.45% and 0.6% of the formula. Other dry ingredients, including seasonings, flavors and colors, were added at the levels described in Table 5.

[0229] Table 5: Evaluation of different doses of TG 1 The Impact of Plant-Based Deli Meat Recipes

[0230]

[0231] 1Samples T1-T5 represent different enzyme dosages, where T1 = 0.05% TG, T2 = 0.15% TG, T3 = 0.3% TG, T4 = 0.45% TG, T5 = 0.60% TG

[0232] Processing conditions:

[0233] Structured soy protein and hydration water are added to a Hobart mixer and mixed at low speed for 1 minute. Then add soy protein isolate and enzyme TG and mix for another 3-5 minutes. After the protein sample is fully hydrated, add other dry ingredients (including seasonings, flavors, pigments) and the remaining water and mix until completely homogenized for about 2-3 minutes. Then add molten coconut fat and mix for another 3-4 minutes until it is completely incorporated into the matrix. The resulting batter is transferred to a plastic bag, vacuum sealed to remove bubbles, and then filled. The batter is poured into plastic casings and kept warm at 4°C for 22 hours, or placed directly in an oven after filling. Use the step-by-step cooking cycle shown in Table 6 to bake the plant-based deli meat samples. The final 90°C (internal temperature) ensures enzyme inactivation. After the sample is cooked, it is placed in ice water for about 30 minutes and then sliced in a refrigerator (4°C) overnight.

[0234] Table 6: Programmed step-by-step cooking cycles for plant-based deli meat samples containing TG

[0235] Cooking Oven Temperature Profiles Working time (minutes) Temperature Step 1 40 55℃ Step 2 30 65℃ Step 3 20 75℃ Step 4 20 85℃ Step 5 20 98°C (to internal temperature 90°C) Step 6 10 Cold shower for 10 minutes

[0236] Texture Analysis:

[0237] The texture properties of deli meat samples were evaluated using a TA-XT2i texture analyzer (Texture Technologies Corp, Scarsdale, NY). The probe used in this test was a TA-30 cylindrical probe (76.2 mm diameter). The samples were cut into 20 mm cubes at 4°C-5°C, and each test was repeated 10 times. Each deli meat sample was placed under the probe and the texture profile was analyzed. The texture measurement conditions were set as follows: pre-test, test, and post-test speeds were set to 2 mm / s, 1 mm / s, and 5 mm / s, respectively. The trigger force was set to 10 g, the distance was 6 mm, and the relaxation time was 5 seconds. The samples were evaluated for differences in hardness, chewiness, and gumminess.

[0238] result:

[0239] Samples containing lower levels of TG (0.05% and 0.15%) were softer and did not slice as well as the other samples. Texture analysis showed that hardness, chewiness, and gumminess increased with increasing TG levels, up to a maximum of 0.45%. However, 0.60% enzyme had a negative impact on textural properties, suggesting a potential overdose effect. This is consistent with results observed in model system experiments (not shown), where excessive cross-linking due to high levels of TG caused syneresis and negatively impacted overall textural properties. Based on this experiment, it is recommended that the ideal TG usage level in similar plant-based meat alternative formulations is 0.3%-0.5%.

[0240] Example 5:

[0241] Pilot Application Trial: Determining the Effect of Holding Time on the Texture Development of Plant-Based Deli Meats Prepared Using TG

[0242] The purpose of this study was to evaluate the effect of holding time before cooking on TG activity to develop desirable texture properties in plant-based deli meat products. This example demonstrates that no holding time is required and that this cooking cycle allows the enzymes to function as desired.

[0243] The samples studied included 4 different time points and negative controls:

[0244] T1 - Incubate at 4°C for 22 hours

[0245] T2 – no incubation at 4°C

[0246] T3 – Negative control sample without enzyme

[0247] Similar to the plant-based deli meat recipe described in Example 4, 21.9% structured soy protein and 10% soy protein isolate were used. TG was added at 0.3% w / w of the recipe. Processing conditions were identical to those in Example 4, with the textured and powdered proteins first hydrated with TG before adding the other dry ingredients. After the sample was fully homogenized, it was vacuum-packed and stuffed into specialized casings, which were then held overnight before cooking. The cooking cycle was identical to that in Example 4 (Table 6).

[0248] Table 7: Plant-based deli meat samples 1 Recipe

[0249] Enzymes - T1 and T2* T3-Negative Control-No Enzyme Element ％ ％ Water, protein hydration 43.9 43.9 Structured soy protein 21.9 21.9 Transglutaminase (TG) 0.3 0 soy protein isolate 10 10 Vegetarian burger flavor 2 2 Vegetarian Beef Broth 1.3 1.3 Salt 0.5 0.5 caramel coloring 0.2 0.2 DI water, for hydration of dry ingredients 13.9 14.2 coconut oil, melted 6 6 total 100 100

[0250] 1 Samples T1 and T2 represent 22 hours of heat preservation and no heat preservation, respectively

[0251] result:

[0252] During sectioning (cold), the negative control was noted to be brittle, rough in texture, and difficult to section. The samples containing the enzyme were smoother and had less fat on the outer surface, indicating better emulsification. The enzyme samples also showed greater flexibility and elasticity. Texture Analysis ( Figure 7 ) showed that the hardness, gummyness, and chewiness of the negative control were significantly lower than those of the enzyme-treated samples. However, the difference in holding time did not significantly affect the protein binding properties of the enzyme or the texture of the cooked meat samples, indicating that under the processing conditions of the present invention, holding time before cooking is not necessary to achieve the positive effect of transglutaminase activity. Therefore, the necessary enzyme activity occurs during cooking.

[0253] Example 6:

[0254] Pilot application trial: Evaluation of the effect of TG activity on the textural properties of plant-based pepperoni compared to hydrocolloids ring

[0255] In this example, the effect of TG in a soy-based pepperoni formulation was evaluated and compared to a control sample containing hydrocolloids (carrageenan and methylcellulose). TG was added at 0.4% (w / w) of the formulation, while the hydrocolloids were added at 5.5% (w / w) of the formulation.

[0256] Table 8: Soy Protein Pepperoni Recipe

[0257] With hydrocolloid With TG Element ％ ％ water 46.67 40 Structured soy protein 23.33 20 Coconut oil - melted 12 12 Carrageenan and methylcellulose 5.5 0 Natural Pepperoni Flavor 2 2 Malt extract powder 0.1 0.1 Chili red 0.55 0.55 Tomato extract 0.4 0.4 Water (for dispersing dry ingredients) 6.7 11.8 Salt 1.5 1.5 antimicrobial agents 1.25 1.25 soy protein isolate 0 10 TG enzyme 0 0.4 total 100 100

[0258] Processing conditions:

[0259] The structured soy protein was hydrated in a Hobart mixer for 2-3 minutes. Subsequently, hydrocolloid was added to the control sample, while soy protein isolate and TG were added to the enzyme sample and mixed for another 2-3 minutes. After the protein was fully hydrated, the other dry ingredients (including pigments, seasoning blends, antimicrobial agents and salt) were added and the melted coconut fat was added after thorough mixing. While the dry ingredients were being mixed, coconut oil was slowly added to the mixture. The protein dough was then mixed for about 3-4 minutes or until fully emulsified (no visible oil droplets). The sample was removed from the mixer, vacuum sealed, poured into plastic casings, and then placed in an oven. The cooking cycles used in this test are shown in Table 5. After cooking, the sample was placed in ice water for about 30 minutes and then sliced after being placed in a refrigerator (4°C) overnight. Texture analysis was performed as shown in Example 4.

[0260] Table 9: Oven Cooking Temperature Profile for Plant-Based Pepperoni Samples

[0261] Cooking Oven Temperature Profiles Working time (minutes) Temperature Step 1 40 40℃ Step 2 20 65℃ Step 3 20 75℃ Step 5 20 98°C (to internal temperature 90°C)

[0262] result:

[0263] The pepperoni samples were first sliced (1.8-2 mm) to assess their mechanical sliceability and sensory attributes. During the slicing process, it was noted that the samples containing TG sliced more easily than the hydrocolloid samples. Additionally, the TG samples were better able to incorporate oil into the protein dough compared to the control. During the sensory evaluation, the samples were either tasted as is or baked on cheese pizzas. Notably, the TG samples exhibited the desired meat-like qualities after cooking on pizza. The TG pepperoni slices exhibited cupping or curling and oil exudation similar to meat-based pepperoni, which was not observed with the hydrocolloid samples. Additionally, the flavor from the TG samples appeared more balanced and did not overwhelm the palate.

[0264] Texture analysis results ( Figure 8 ) showed that the TG samples were firmer than the control, which could explain their better sliceability. The TG samples also showed higher gumminess and chewiness. This finding is consistent with the sensory attributes observed during tasting.

[0265] Example 7:

[0266] Pilot application trial: Evaluation of the effect of TG activity on plant-based hot dogs compared to plant-based hot dogs prepared using hydrocolloids Influence of texture properties

[0267] In this example, the effect of TG on the textural properties of plant-based hot dog samples was evaluated and compared to hot dog samples prepared using hydrocolloids (carrageenan and methylcellulose). The formulation compositions are shown in Table 10.

[0268] Table 10: Plant-based hot dog recipes using hydrocolloids compared to TG

[0269]

[0270] Processing conditions:

[0271] For the hydrocolloid sample, cold water, soy protein isolate, and hydrocolloid were added to a Stephan vacuum mixer. The sample was then mixed at low speed for 2 minutes and then at high speed for 2-3 minutes to ensure that the protein was fully hydrated. For the sample containing TG, the protein was first hydrated for 3-4 minutes, and then the enzyme was added after the protein was fully hydrated. The remaining dry ingredients were added and mixed for about 3-4 minutes. A vacuum was then applied and mixed for an additional 2 minutes before adding the melted coconut oil. The hot dog batter was then thoroughly mixed under vacuum until all the oil was fully absorbed and emulsified. The batter was then transferred to a plastic bag, vacuum-sealed, stuffed into casings, and cooked according to the cooking cycle shown in Table 11.

[0272] Table 11: Cooking Oven Temperature Profile for Hot Dog Recipe

[0273]

[0274] Texture Analysis:

[0275] Texture analysis was performed using cold samples (3°C-5°C) and re-cooked samples. The re-cooked samples were placed in boiling water until the internal temperature reached approximately 74°C. The probe used for texture analysis in this test was a TA-30 cylindrical probe. The hot dog samples were cut into 3 cm pieces and each test was repeated 10 times. Each hot dog sample was placed under the probe and the following measurement conditions were used: the pre-test, test and post-test speeds were set to 2 mm / s, 1 mm / s and 5 mm / s, respectively. The trigger force was set to 10 g, the distance was 10 mm, and the relaxation time was 5 seconds. The samples were compressed twice to evaluate hardness, chewiness and gumminess.

[0276] result:

[0277] For the cold PB hot dogs, the firmness of the samples containing TG and hydrocolloid was similar. The TG sample was chewier and more chewy. This was consistent with the sensory observations. During the tasting, the sample containing hydrocolloid was noted to be mushy and did not have as much bite as the TG sample. For the re-cooked hot dogs, the sample containing hydrocolloid was slightly firmer, but the chewiness and chewiness of the TG sample were higher. The sensory attributes of the re-cooked samples were similar to those of the cold samples, with the hydrocolloid sample appearing to have less bite than the control. In addition, the overall flavor and mouthfeel of the TG sample were noted to be superior to the hydrocolloid sample.

[0278] Example 8:

[0279] Pilot Application Trial: Evaluating TG Activity at Different Protein Levels in Plant-Based Hot Dog Recipes

[0280] The objective of this example was to determine the ideal substrate protein range required for TG to develop the desired texture properties in a plant-based hot dog formulation. The samples evaluated in this study were:

[0281] T1: Soy protein isolate with 18% protein

[0282] T2: Soy protein isolate with 15% protein

[0283] T3: Soy protein isolate with 12% protein

[0284] T4: Soy protein isolate with 10% protein

[0285] Processing conditions and cooking cycle were the same as in Example 7.

[0286] Table 12: Plant-based hot dog recipes prepared using TG with different protein levels

[0287]

[0288] result:

[0289] Sample 4 (T4), containing 10% protein, did not form a gel after cooking, so its texture measurements were not included in the results. Texture analysis showed that firmness, chewiness, and gumminess increased with increasing protein levels in both cold and refrigerated samples. Sensory evaluation determined that while the 12% protein sample formed a gel structure, it was not firm enough to exhibit meat-like texture characteristics. Samples containing 15% to 18% protein exhibited desirable texture characteristics in terms of firmness / hardness and bite.

[0290] Example 9

[0291] Cloning and expression of transglutaminase

[0292] The transglutaminase gene was identified from a public database (NCBI). The SEQ ID NO. for each gene is listed in Table 12. Typically, genes encoding transglutaminases (without signal peptides) are codon-optimized based on the codon preference of Bacillus subtilis and cloned into the p2JM vector (Vogtentanz, Protein Expr Purif. [Protein Expression and Purification] 55:40-52, 2007). The AprE promoter and signal peptide are used for the transcription and secretion of transglutaminases. The constructed vector is subjected to rolling circle amplification and transformed into a suitable Bacillus subtilis host. Transformants are screened on LB plates supplemented with 5ppm chloramphenicol. Single colonies are inoculated into 20mL LB medium with 5ppm chloramphenicol and subsequently used to inoculate a culture medium for cell growth and protein expression. The clarified culture supernatant is harvested by centrifugation and concentrated for protein purification. All transglutaminases are purified using standard procedures known in the art, including hydrophobic interaction chromatography, ion / cation exchange chromatography, and size exclusion chromatography. Fractions containing the target protein were identified by SDS-PAGE and the following activity assay. Purified protein samples were stored in 40% glycerol at -20°C until use.

[0293] Table 12. Sequence listing of transglutaminases.

[0294]

[0295] Example 10

[0296] Transglutaminase-induced protein gelation assay

[0297] The purpose of this determination is to study the performance of TG enzyme samples in protein gelation reaction under different conditions by measuring gel strength with a texture analyzer. By adding 60g soy protein isolate to 440g Milli-Q water, the slurry is fully mixed with an electric stirrer and kept at room temperature for at least 2 hours to fully hydrate the protein isolate to prepare soy protein slurry (12% w / w). Weigh 20g protein slurry into an RVA container. Add an appropriate amount of TG enzyme sample, and fully mix the enzyme solution and protein slurry with RVA at 40°C and 960rpm for 2min. Seal the RVA container with food preservative film. Incubate at 40°C for 4 hours in a shaking table. At the end of the incubation, measure the gel strength with a texture analyzer equipped with a P / 5 probe (4mm DIA stainless steel cylinder). The maximum force used for puncture is recorded as the rupture force, and the force at 4mm depth is recorded as the gel strength. Penetrate the protein gel to the maximum target depth of 10mm at a constant speed of 1mm / s and record the maximum force. Gel strength and rupture force were determined based on the average of three replicates (Table 13).

[0298] Table 13. Protein gelling properties of transglutaminases.

[0299]

[0300]

[0301] Although the foregoing invention has been described in detail by way of illustration and example for the purpose of clarity of understanding, certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference were individually incorporated by reference. To the extent that any reference (including website or accession number) may change over time, the effective version refers to the version effective on the date of filing this application. Unless otherwise apparent from the context, any step, element, aspect, embodiment feature may be used in combination with any other.

[0302] References:

[0303] 1. M., Migge, A., Hertel, TC, & Pietzsch, M. (2015). Afluorescence-based array screen for transglutaminase substrates. ChemBioChem, 16(8), 1169-1174.

[0304] 2.Robert-Peillard,F.,Barco,E.P.,Ciulu,M.,Demelas,C.,Théraulaz,F.,Boudenne,J.L.,&Coulomb,B.(2017).High throughput determination of ammonium andprimary amine compounds in environmental and food samples.MicrochemicalJournal,133,216-221.

[0305] 3.Wong,C.M.,Wong,K.H.,&Chen,X.D.(2008).Glucose oxidase:naturaloccurrence,function,properties and industrial applications.Appliedmicrobiology and biotechnology,78(6),927-938.

[0306] 4.Stamnaes,J.,Pinkas,D.M.,Fleckenstein,B.,Khosla,C.,&Sollid,L.M.(2010).Redox regulation of transglutaminase 2activity.Journal of BiologicalChemistry,285(33),25402-25409.

[0307] 5.Vemulapalli,V.,&Hoseney,R.C.(1998).Glucose oxidase effects ongluten and water solubles.Cereal Chemistry,75(6),859-862。

Claims

1. A method for producing a food containing plant protein, the method comprising: Transglutaminase, sugar oxidoreductase and catalase are added to food raw materials containing plant protein.

2. The method of claim 1, wherein the plant protein is soy, pea, soybean, fava bean, gluten or oat.

3. The method of claim 2, wherein the vegetable protein is soy protein.

4. The method of any one of the preceding claims, wherein the oxidoreductase is a hexose oxidase.

5. The method of claim 4, wherein the hexose oxidase is derived from Hansenula polymorpha.

6. The method of claim 4, wherein the hexose oxidase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

7. The method of claim 6, wherein the hexose oxidase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

8. The method of claim 7, wherein the hexose oxidase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

9. The method of claim 8, wherein the hexose oxidase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

10. The method of claim 9, wherein the hexose oxidase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

11. The method of claim 10, wherein the hexose oxidase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

12. The method of claim 11, wherein the hexose oxidase comprises an enzyme according to SEQ ID NO: 3 or a hexose oxidase-active fragment thereof.

13. The method of any one of claims 1 to 3, wherein the oxidoreductase is glucose oxidase.

14. The method of claim 13, wherein the glucose oxidase is derived from Aspergillus niger.

15. The method of claim 13, wherein the glucose oxidase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 2 or a glucose oxidase-active fragment thereof.

16. The method of claim 15, wherein the glucose oxidase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 2 or a glucose oxidase-active fragment thereof.

17. The method of claim 16, wherein the glucose oxidase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 2 or a glucose oxidase-active fragment thereof.

18. The method of claim 17, wherein the glucose oxidase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof.

19. The method of claim 18, wherein the glucose oxidase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof.

20. The method of claim 19, wherein the glucose oxidase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 2, or a glucose oxidase-active fragment thereof.

21. The method of claim 20, wherein the glucose oxidase comprises an enzyme according to SEQ ID NO: 2 or a glucose oxidase-active fragment thereof.

22. The method of any one of the preceding claims, wherein the transglutaminase is derived from Streptomyces mobaraensis.

23. The method of any one of the preceding claims, wherein the transglutaminase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 1, or a transglutaminase-active fragment thereof.

24. The method of claim 23, wherein the transglutaminase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

25. The method of claim 24, wherein the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

26. The method of claim 25, wherein the transglutaminase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

27. The method of claim 26, wherein the transglutaminase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

28. The method of claim 27, wherein the transglutaminase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

29. The method of claim 28, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1 or a transglutaminase-active fragment thereof.

30. The method of any one of the preceding claims, wherein the catalase is derived from Aspergillus niger.

31. The method of claim 30, wherein the catalase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof.

32. The method of claim 31, wherein the catalase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof.

33. The method of claim 32, wherein the catalase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof.

34. The method of claim 33, wherein the catalase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof.

35. The method of claim 34, wherein the catalase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof.

36. The method of claim 35, wherein the catalase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 4, or a catalase-active fragment thereof.

37. The method of claim 36, wherein the catalase comprises the enzyme according to SEQ ID NO: 4 or a catalase-active fragment thereof.

38. The method of any preceding claim, wherein the food product is a meat substitute.

39. The method of claim 38, wherein the meat substitute is a hot dog.

40. The method of claim 38, wherein the meat substitute is pepperoni.

41. An improved method for preparing a plant-based meat substitute, comprising: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors, and transglutaminase; b.) keeping the batter warm for less than 60 minutes; c.) molding the batter; and d.) baking the batter to provide the plant-based meat.

42. The method of claim 41, wherein the batter further comprises a texturized or structured protein derived from a plant.

43. The method of claim 41 or 42, wherein the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat.

44. The method of claim 43, wherein the plant protein is soy protein or pea protein.

45. The method of any one of claims 41 to 44, wherein the transglutaminase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

46. The method of claim 45, wherein the transglutaminase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

47. The method of claim 46, wherein the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

48. The method of claim 47, wherein the transglutaminase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

49. The method of claim 48, wherein the transglutaminase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

50. The method of claim 48, wherein the transglutaminase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

51. The method of claim 50, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

52. The method of claim 51, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO:

15.

53. The method of any one of claims 41 to 52, wherein the incubation step lasts less than 30 minutes.

54. The method of claim 53, wherein the incubation step lasts less than 15 minutes.

55. The method of claim 54, wherein the incubation step lasts for less than 10 minutes.

56. The method of claim 55, wherein the incubation step lasts less than 5 minutes.

57. The method of claim 56, wherein the incubation step lasts less than 2 minutes.

58. The method of claim 57, wherein the incubation step lasts for less than 1 minute.

59. The method of claim 58, wherein the incubation step is 0 minutes.

60. The method of any one of claims 41 to 59, wherein the meat substitute is a hot dog, frankfurter, sausage, deli meat, bread, salami, meat loaf, pepperoni, or barbecue.

61. The method of claim 60, wherein the meat substitute is pepperoni.

62. The method of any one of claims 41 to 61, wherein the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein.

63. The method of claim 62, wherein the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein.

64. The method of claim 63, wherein the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein.

65. The method of claim 63, wherein the transglutaminase is present in an amount from about 25 to 250 mg / kg plant protein.

66. The method of claim 65, wherein the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein.

67. The method of claim 66, wherein the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

68. The method of any one of claims 41 to 67, wherein the plant-based meat has from 1% to 35% plant protein.

69. The method of claim 68, wherein the plant-based meat has from 5% to 30% plant protein.

70. The method of claim 69, wherein the plant-based meat has from 10% to 25% plant protein.

71. The method of claim 70, wherein the plant-based meat has from 12% to 22% plant protein.

72. The method of any one of claims 41 to 71, wherein the baking step d) is carried out at an initial temperature of 25-70°C for a period of 10 to 80 minutes and then increasing the temperature until the meat substitute has an internal temperature of at least 90°C.

73. The method of claim 72, wherein the initial temperature is maintained for 20 to 60 minutes.

74. The method of claim 73, wherein the initial temperature is maintained for 30 to 45 minutes.

75. The method of any one of claims 72 to 74, wherein the initial temperature is 35°C to 60°C.

76. The method of claim 75, wherein the initial temperature is 45°C to 50°C.

77. An improved method for preparing a plant-based meat substitute, comprising the steps of: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors, and transglutaminase; b.) molding the batter; and c.) baking the batter to provide the plant-based meat.

78. The method of claim 77, wherein the batter further comprises a texturized or structured protein derived from a plant.

79. The method of claim 77 or 78, wherein the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat.

80. The method of claim 79, wherein the plant protein is soy protein or pea protein.

81. The method of any one of claims 77 to 80, wherein the transglutaminase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

82. The method of claim 81, wherein the transglutaminase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

83. The method of claim 82, wherein the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

84. The method of claim 83, wherein the transglutaminase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

85. The method of claim 84, wherein the transglutaminase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

86. The method of claim 85, wherein the transglutaminase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

87. The method of claim 86, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

88. The method of claim 87, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO:

15.

89. The method of any one of claims 77 to 88, wherein the meat substitute is a hot dog, frankfurter, sausage, deli meat, bread, salami, meat loaf, pepperoni, or barbecue.

90. The method of claim 89, wherein the meat substitute is pepperoni.

91. The method of any one of claims 77 to 90, wherein the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein.

92. The method of claim 91, wherein the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein.

93. The method of claim 92, wherein the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein.

94. The method of claim 93, wherein the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein.

95. The method of claim 94, wherein the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein.

96. The method of claim 95, wherein the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

97. The method of any one of claims 77 to 96, wherein the plant-based meat has from 1% to 35% plant protein.

98. The method of claim 97, wherein the plant-based meat has from 5% to 30% plant protein.

99. The method of claim 98, wherein the plant-based meat has from 10% to 25% plant protein.

100. The method of claim 99, wherein the plant-based meat has from 12% to 22% plant protein.

101. The method of any one of claims 77 to 100, wherein the baking step c) is performed at an initial temperature of 25-70°C for a period of 10 to 80 minutes and then increasing the temperature until the meat substitute has an internal temperature of at least 90°C.

102. The method of claim 101, wherein the initial temperature is maintained for 20 to 60 minutes.

103. The method of claim 102, wherein the initial temperature is maintained for 30 to 45 minutes.

104. The method of any one of claims 101-103, wherein the initial temperature is 35°C to 60°C.

105. The method of claim 104, wherein the initial temperature is 45°C to 50°C.

106. An improved method for preparing a plant-based meat substitute, wherein the method comprises the steps of: a.) mixing a batter comprising water, vegetable protein, coconut oil, hydrocolloid, one or more meat flavors, and transglutaminase; b.) molding the batter; and c.) baking the batter to provide the plant-based meat.

107. The method of claim 106, wherein the batter further comprises a texturized or structured protein derived from a plant.

108. The method of claim 106 or 107, wherein the plant protein is chickpea, soy, pea, soybean, fava bean, gluten or oat.

109. The method of claim 108, wherein the plant protein is soy protein or pea protein.

110. The method of any one of claims 106-109, wherein the transglutaminase comprises an enzyme having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

111. The method of claim 110, wherein the transglutaminase comprises an enzyme having at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

112. The method of claim 111, wherein the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

113. The method of claim 112, wherein the transglutaminase comprises an enzyme having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

114. The method of claim 113, wherein the transglutaminase comprises an enzyme having at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

115. The method of claim 114, wherein the transglutaminase comprises an enzyme having at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

116. The method of claim 115, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15, or a transglutaminase-active fragment thereof.

117. The method of claim 116, wherein the transglutaminase comprises an enzyme according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO:

15.

118. The method of any one of claims 106-117, wherein the meat substitute is a hot dog, frankfurter, sausage, deli meat, bread, salami, meat loaf, pepperoni, or barbecue.

119. The method of claim 118, wherein the meat substitute is pepperoni.

120. The method of any one of claims 106-119, wherein the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein.

121. The method of claim 120, wherein the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein.

122. The method of claim 121, wherein the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein.

123. The method of claim 122, wherein the transglutaminase is present in an amount from about 25 to 250 mg / kg plant protein.

124. The method of claim 123, wherein the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein.

125. The method of claim 124, wherein the transglutaminase is present in an amount from about 50 to 150 mg / kg plant protein.

126. The method of any one of claims 106-125, wherein the plant-based meat has from 1% to 35% plant protein.

127. The method of claim 126, wherein the plant-based meat has from 5% to 30% plant protein.

128. The method of claim 127, wherein the plant-based meat has from 10% to 25% plant protein.

129. The method of claim 99, wherein the plant-based meat has from 12% to 22% plant protein.

130. The method of any one of claims 77-100, wherein the baking step c) is performed at an initial temperature of 25-70°C for a period of 10 to 80 minutes and then increasing the temperature until the meat substitute has an internal temperature of at least 90°C.

131. The method of claim 130, wherein the initial temperature is maintained for 20 to 60 minutes.

132. The method of claim 131 , wherein the initial temperature is maintained for 30 to 45 minutes.

133. The method of any one of claims 130-132, wherein the initial temperature is 35°C to 60°C.

134. The method of claim 134, wherein the initial temperature is 45°C to 50°C.

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