Methods for producing plant based food alternatives

Abstract

The present invention relates to a method or treating plant proteins with enzymes to produce a plant-based food, including plant-based meat alternatives. More particularly, the method relates to use of transglutaminase, a sugar oxidase, and catalase to treat plant-based proteins.

Description

[0001] TITLE

[0002] METHODS FOR PRODUCING PLANT BASED FOOD ALTERNATIVES

[0003] TECHNICAL FIELD

[0004] The present invention relates to methods for producing plant-based food alternatives. More particularly, the present invention relates to use of enzymes to treat plant proteins to generate a plant-based food matrix.

[0005] BACKGROUND

[0006] As concerns of high meat consumption surge due to potential risks to the environment, animal welfare, and human health, consumers are increasingly looking for alternative products that could serve as meat substitutes. Furthermore, the projected population growth poses an additional challenge for the sustainability of the food system. These conditions have urged the scientific community and the food industry to come up with meat alternatives that are produced from grains and pulses to meet the rising demand for nonmeat-based meals. While these plant-based meat alternatives are becoming more available, there is still a significant gap in emulating the texture and sensory properties of meat.

[0007] To address these gaps, several food technologies have been introduced. One such technology is the use of the enzyme, transglutaminase, to generate meat-like structure by facilitating plant-protein cross-linking. Transglutaminase catalyzes cross-linking by the process of acyl transfer reaction between the y-carboxamide group of glutamine residues and the s-amino group of lysine residues, resulting in the formation of intermolecular and intramolecular covalently linked iso-peptide bonds. This cross-linking between proteins enables the modification of protein functionality including gelling property, solubility, and emulsifying capacity.

[0008] The meat-like gel structure generated by treating plant proteins with transglutaminase is only one aspect of creating genuine plant-based meat alternatives that are acceptable to consumers. Plant-based meat alternatives must also have an appropriate 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, preventing, or inhibiting microbial growth. Removal of excess oxygen also prevents undesired food browning. In the bakery industry, glucose oxidase is used to modify wheat flour proteins for improved crumb texture and dough strength. And it has been suggested that glucose oxidase can be used to improve plant-based meat texture. However, it has been observed that glucose oxidase can negatively impact transglutaminase and the resultant meatlike gel’s produced thereby.

[0009] There is a continuing need for ways to improve plant-based meat texture and shelf-life without negatively impacting gel strength.

[0010] SUMMARY OF THE INVENTION

[0011] In an aspect of the present invention, a method is presented for producing a plant proteincontaining food, the method having the step of adding a transglutaminase, a sugar oxidoreductase and a catalase to a food raw material containing a plant protein.

[0012] Optionally, the plant protein is soy, pea, soy, fava, gluten or oat. Optionally, the plant protein is soy protein.

[0013] Optionally, the oxidoreductase is a hexose oxidase. Optionally, the hexose oxidase is derived from Hansenida polymorpha. Optionally, the hexose oxidase is an enzyme having at least 80% sequence identity to SEQ ID NO:3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 85% sequence identity to SEQ ID NO:3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 95% sequence identity to SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 98% sequence identity to SEQ ID NO: 3 or a hexose oxidase active fragment thereof. Optionally, the hexose oxidase is an enzyme having at least 99% sequence identity to 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] Optionally, the food is a meat alternative. Optionally, the meat alternative is a hot dog or a pepperoni slice.

[0018] In another aspect of the present invention, an improved method for preparing a plant-based meat alternative is presented, having the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) holding said batter for less than 60 minutes; c.) moulding the batter; and d.) baking the batter to provide the plant-based meat.

[0019] Optionally, the batter further has textured or structured protein derived from plants. Optionally, the plant protein is chickpea, soy, pea, soy, fava, gluten or oat. Optionally, the plant protein is soy protein or pea protein.

[0020] 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.

[0021] 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.

[0022] Optionally, the holding step is less than 30, 15, 10, 5, 2, or 1 minute or is 0 minutes. Optionally, the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni or roast. Optionally, the meat alternative is a pepperoni.

[0023] Optionally, the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

[0024] 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.

[0025] Optionally, baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C. Optionally, the initial temperature is conducted for 20 to 60 minutes. Optionally, the initial temperature is conducted for 30 to 45 minutes.

[0026] Optionally, the initial temperature is 35 to 60°C. Optionally, the initial temperature is 45 to 50°C.

[0027] In another aspect of the present invention, an improved method for preparing a plantbased meat alternative, having only the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) moulding the batter; and c.) baking the batter to provide the plant-based meat. The instant improved method does not have a holding step, resulting in decreased complexity and lowered cost for the manufacturer.

[0028] Optionally, the batter further has textured or structured protein derived from plants.

[0029] Optionally, the plant protein is chickpea, soy, pea, soy, fava, gluten, or oat.

[0030] Optionally, the plant protein is soy protein or pea protein.

[0031] 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 N0:6, SEQ ID N0:9, 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.

[0032] 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 N0:15.

[0033] Optionally, the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni, or roast. Optionally, the meat alternative is a pepperoni.

[0034] Optionally, the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

[0035] 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. Optionally, baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C. Optionally, the initial temperature is conducted for 20 to 60 minutes. Optionally, the initial temperature is conducted for 30 to 45 minutes.

[0036] Optionally, the initial temperature is 35 to 60°C. Optionally, the initial temperature is 45 to 50°C.

[0037] In another aspect of the present invention, an improved method for preparing a plantbased meat alternative in which there is no holding step is provided having the steps of comprising the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) moulding the batter; and c.) baking the batter to provide the plant-based meat.

[0038] Optionally, the batter further has textured or structured protein derived from plants. Optionally, the plant protein is chickpea, soy, pea, soy, fava, gluten, or oat. Optionally, the plant protein is soy protein or pea protein.

[0039] 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. 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 N0:6, 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 N0:I5.

[0040] Optionally, the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni, or roast. Optionally, the meat alternative is a pepperoni.

[0041] Optionally, the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein. Optionally, the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

[0042] 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.

[0043] Optionally, baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C. Optionally, the initial temperature is conducted for 20 to 60 minutes. Optionally, the initial temperature is conducted for 30 to 45 minutes.

[0044] Optionally, the initial temperature is 35 to 60°C. Optionally, the initial temperature is 45 to 50°C. BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0045] SEQ ID NO:1 is the amino acid sequence of the transglutaminase mature protein.

[0046] SEQ ID N0:2 is the amino acid sequence of the glucose oxidase (GOX) mature protein.

[0047] SEQ ID NO:3 is the amino acid sequence of the hexose oxidase (HOX) mature protein.

[0048] SEQ ID NO:4 is the amino acid sequence of the catalase mature protein.

[0049] SEQ ID NO:5 is the amino acid sequence of CRC24210 SciTG2 precursor protein.

[0050] SEQ ID NO:6 is the amino acid sequence of CRC24210 SciTG2 predicted mature protein.

[0051] SEQ ID NO:7 is the nucleic acid sequence of CRC24210 SciTG2 synthetic / optimized full length DNA.

[0052] SEQ ID NO:8 is the protein sequence of CRC26523 SnoTG2 precursor protein.

[0053] SEQ ID NO:9 is the protein sequence of CRC26523 SnoTG2 predicted mature protein.

[0054] SEQ ID NO: 10 is the nucleic acid sequence of CRC26523 SnoTG2 synthetic / optimized full length DNA.

[0055] SEQ ID NO: 11 is the protein sequence of CRC26802 SroTGl precursor protein.

[0056] SEQ ID NO: 12 is the protein sequence of CRC26802 SroTGl predicted mature protein.

[0057] SEQ ID NO: 13 is the nucleic acid sequence of CRC26802 SroTGl synthetic / optimized full length DNA.

[0058] SEQ ID NO: 14 is the protein sequence of CRC26807 SsyTGl precursor protein.

[0059] SEQ ID NO: 15 is the protein sequence of CRC26807 SsyTGl predicted mature protein.

[0060] SEQ ID NO: 16 is the nucleic acid sequence of CRC26807 SsyTGl synthetic / optimized full length DNA.

[0061] BRIEF DESCRITPTION OF THE FIGURES

[0062] FIG. 1 shows transglutaminase activity on the y-axis as reflected by ammonia production using OPA assay in protein solutions containing different levels of GOX and catalase (x-axis).

[0063] FIG. 2 shows a compression test to determine the effect of TG in combination with GOX and catalase at different levels in soy-based protein gels.

[0064] FIG. 3 shows a compression test to determine the effect of TG in combination with GOX and catalase in soy -based protein gels with 8 or 24hr incubation period.

[0065] FIG. 4 shows soy-based hot dog samples containing TG and different levels of GOX and catalase. 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).

[0066] FIG. 5 shows texture analysis of plant-based hot dog samples containing TG and different 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).

[0067] FIG. 6 shows texture analysis of plant-based deli meat samples using TG held at different time points prior to cooking.

[0068] FIG. 7 shows texture analysis of plant-based deli meat samples using TG comparing different hold times.

[0069] FIG. 8 shows texture evaluation of plant-based pepperoni samples prepared using hydrocolloids as compared to those prepared using TG.

[0070] FIG. 9A&B show texture analysis of plant-based hot dogs samples prepared using hydrocolloids as compared to those prepared using TG. Cold (A) and recooked (B) samples.

[0071] FIG. 10A&B shows texture analysis of plant-based hot dogs samples prepared using TG with different levels of protein. Cold (A) and recooked (B) samples.

[0072] DETAILED DESCRIPTION OF THE INVENTION

[0073] Definitions

[0074] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N— >C).

[0075] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may 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 may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0076] The term “plant protein” means substrate protein from plants such as for example soy protein isolate(s) which transglutaminase is able to act on, which excludes textured or structured protein derived from plants which is denatured and does not serve as a substrate for transglutaminase.

[0077] The units of measurement for Hardness is gram-force (g). Gumminess is a product of hardness and cohesiveness. Chewiness is a product of hardness, cohesiveness, and springiness. Cohesiveness is the area of work during the second compression divided by the area of the work during the first compression. Springiness is the ratio of the product’s original height.

[0078] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention encompasses variants, homologues, derivatives, and fragments thereof. The term "variant" is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence.

[0079] For example, a variant may include substitutions, insertions, deletions, truncations, transversions and / or inversions at one or more position(s) relative to a wild-type sequence. Variants can be made using methods known in the art for example site scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis and directed- evolution as well as using recombinant methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences may readily be synthesized using methods known in the art.

[0080] In some aspects, the variant is a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence. For example, the variant may be a natural genetic variant.

[0081] In some aspects, the variant is an engineered variant. For example, the variant may be engineered by recombinant methods.

[0082] The protein sequences of the instant invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. 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.

[0083] Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other as set forth in Table 1.

[0084] Table 1.

[0085] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e., like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e., from one class 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), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0086] Replacements may also be made by synthetic amino acids (e.g. unnatural amino acids) include; alpha* and alpha-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*, B-alanine*, L-a- amino butyric acid*, L-g-amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic 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 (l,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L- diaminopropionic acid* and L-Phe (4-benzyl)*.

[0087] The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilized to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

[0088] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue’s nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0089] The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or poly lysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.

[0090] The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl 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, Academic Press. Each of these general texts is herein incorporated by reference.

[0091] As used herein, “percent (%) sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See

[0092] Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the

[0093] CLUSTAL W algorithm are:

[0094] Gap opening penalty: 10.0

[0095] Gap extension penalty: 0.05

[0096] Protein weight matrix: BLOSUM series

[0097] DNA weight matrix: IUB

[0098] Delay divergent sequences %: 40

[0099] Gap separation distance: 8

[0100] DNA transitions weight: 0.50

[0101] List hydrophilic residues: GPSNDQEKR

[0102] Use negative matrix: OFF

[0103] Toggle Residue specific penalties: ON

[0104] Toggle hydrophilic penalties: ON

[0105] Toggle end gap separation penalty: OFF

[0106] Deletions are counted as non-identical residues, compared to a reference sequence.

[0107] Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues x 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to a mature polypeptide.

[0108] All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

[0109] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0110] Production of enzymes

[0111] The enzymes of the present invention can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g. , a whole-cell broth) having an enzyme can be obtained following secretion of the enzyme into the cell medium. Optionally, the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme. Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei. Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis, as well as Streptomyces, E. coli.

[0112] Vectors

[0113] A DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding an enzyme can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0114] The vector may be any vector that can be transformed into and replicated within a 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 propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional enzyme. Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, at www.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011) Applied Environ. Microbiol. Th 3916-22. pJG153can be modified with routine skill to comprise and express a nucleic acid encoding an enzyme.

[0115] A nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding an enzyme, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris A0X1 or A0X2 promoters, cbhl is an endogenous, inducible promoter from Trichoderma reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl ) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0116] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the enzyme gene to be expressed or from a different Genus or species. A signal sequence and a promoter sequence comprising a 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 cbhl signal sequence that is operably linked to a cbhl promoter.

[0117] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, poly adenylation sequences operably linked to the DNA sequence encoding a variant enzyme. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0118] The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBUO, pE194, pAMBl, and pIJ702.

[0119] The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xx.vC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91 / 17243.

[0120] Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of enzyme for subsequent enrichment or purification. Extracellular secretion of enzyme into the culture medium can also be used to make a cultured cell material comprising the isolated enzyme. The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the enzyme is operably linked to the control sequences in proper manner with respect to expression.

[0121] The procedures used to ligate the DNA construct encoding an enzyme, the promoter, terminator, and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art {see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nded., Cold Spring Harbor, 1989, and 3rded., 2001).

[0122] Transformation and Culture of Host Cells

[0123] An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an enzyme according to the instant invention. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells. Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streplomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis', Lactobacillus sp. including Lactobacillus reuteri', Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.

[0124] A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species.

[0125] Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type enzyme. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties. It may be advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2, egll, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0126] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.

[0127] The preparation of Trichoderma sp. for transformation, for example, may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56. The mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually, the concentration of these stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solution of sorbitol can be used in the suspension medium.

[0128] Uptake of DNA into the host Trichoderma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50 mM CaCh is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0129] Usually, transformation of Trichoderma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 10sto 107 / mL, particularly 2xlO6 / mL. A volume of 100 pL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCh) may be mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725.

[0130] As used herein, Protein Identification (“JGI PID”) numbers for native Trichoderma genes reference 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 cd., Nucleic Acids Res 2012 Jan;40(Database issue):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 the nucleotide accession numbers GL985056.1 through GL985132.1. Expression

[0131] A method of producing an enzyme of the instant invention may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0132] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0133] An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an enzyme. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

[0134] An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

[0135] The polynucleotide encoding an enzyme in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.

[0136] Host cells may be cultured under suitable conditions that allow expression of an enzyme. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system.

[0137] An expression host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that 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 production of the desired enzyme. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g. , from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of an enzyme.

[0138] Methods for Enriching and Purifying enzymes

[0139] Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare an enzyme polypeptide-containing solution.

[0140] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

[0141] It is desirable to concentrate an enzyme polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the enriched or purified enzyme precipitate.

[0142] The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and / or ultrafiltration.

[0143] The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated enzyme polypeptide-containing solution is at a desired level.

[0144] Concentration may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent. Metal halide precipitation agents 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 precipitation agent, sodium chloride, can also be used as a preservative.

[0145] The metal halide precipitation agent is used in an amount effective to precipitate an enzyme. The selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.

[0146] Generally, at least about 5% w / v (weight / volume) to about 25% w / v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w / v. Generally, no more than about 25% w / v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w / v. The optimal concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific enzyme polypeptide and on its concentration in the concentrated enzyme solution.

[0147] Another alternative way to precipitate the enzyme is to use organic compounds. Exemplary organic compound precipitating 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 agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.

[0148] Generally, the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitation agents can be, for example, linear or branched alkyl esters of 4- hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds. Exemplary organic compounds are linear alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used. Additional organic compounds also include but are not limited to 4- hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both preservative agents. For further descriptions, see, e.g. , U.S. Patent No. 5,281,526.

[0149] Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, enzyme concentration, precipitation agent concentration, and time of incubation.

[0150] The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing.

[0151] Generally, at least about 0.01% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually at least about 0.02% w / v. Generally, no more than about 0.3% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually no more than about 0.2% w / v.

[0152] The concentrated polypeptide solution, containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be enriched or purified. Generally, the pH is adjusted at a level near the isoelectric point of the enzyme. The pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pl) up to about 2.5 pH units above the isoelectric point.

[0153] The incubation time necessary to obtain an enriched or purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less about 10 hours and in most cases even about 6 hours.

[0154] Generally, the temperature during incubation is between about 4°C and about 50°C. Usually, the method is carried out 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 according to the solution conditions and the enzyme or precipitation agent(s) used. The overall recovery of enriched or purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound. The agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.

[0155] After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enriched or purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.

[0156] During fermentation, an enzyme polypeptide accumulates in the culture broth. For the isolation, enrichment, or purification of 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 broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column and eluted to recover the enzyme-active fraction. For further enrichment or purification, a conventional procedure such as ion exchange chromatography may be used.

[0157] Enriched or purified enzymes can be made into a final product that is either liquid

[0158] (solution, slurry) or solid (granular, powder).

[0159] Description of the Preferred Embodiments In an aspect of the present invention, a method is presented for producing a plant proteincontaining food, the method having the step of adding a transglutaminase, a sugar oxidoreductase and a catalase to a food raw material containing a plant protein.

[0160] Preferably, the plant protein is soy, pea, soy, fava, gluten or oat. More preferably, the plant protein is soy protein.

[0161] 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. Still 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 the most preferred embodiments, the hexose oxidase comprises an enzyme according to SEQ ID NO: 3 or a hexose oxidase active fragment thereof.

[0162] In other preferred embodiments, the oxidoreductase is a 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. Still 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. Yet 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. Still 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 embodiments, the glucose oxidase is an enzyme according to SEQ ID NO:2 or a glucose oxidase active fragment thereof.

[0163] 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. Still more preferably, the transglutaminase is an enzyme having at least 85% sequence identity to SEQ ID NO:1 or a transglutaminase active fragment thereof. Y et more preferably, the transglutaminase comprises an enzyme having at least 90% sequence identity to SEQ ID NO: 1 or a transglutaminase active fragment thereof. Still more preferably, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO:1 or a transglutaminase active fragment thereof. Yet 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 still more preferred embodiments, the transglutaminase is an enzyme having at least 99% sequence identity to SEQ ID NO: 1 or a transglutaminase active fragment thereof. In the most preferred embodiments, the transglutaminase is an enzyme according to SEQ ID NO:1 or a transglutaminase active fragment thereof.

[0164] 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. Still more preferably, the catalase is an enzyme having at least 90% sequence identity to SEQ ID NO:4 or a catalase active fragment thereof. Yet 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 still 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. Yet 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.

[0165] Preferably, the food is a meat alternative. Preferably, the meat alternative is a hot dog or a pepperoni.

[0166] In another aspect of the present invention, an improved method for preparing a plantbased meat alternative is presented, having the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) holding said batter for less than 60 minutes; c.) moulding the batter; and d.) baking the batter to provide the plant-based meat.

[0167] Preferably, the batter further has textured or structured protein derived from plants.

[0168] Preferably, the plant protein is chickpea, soy, pea, soy, fava, gluten, or oat. More preferably, the plant protein is soy protein or pea protein.

[0169] 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. Y et more preferably, the transglutaminase is an enzyme having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID N0:9, 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.

[0170] 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 the most preferred embodiments, 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.

[0171] Preferably, the holding step is less than 30, 15, 10, 5, 2 or 1 minute or is 0 minutes.

[0172] Preferably, the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni, or roast. More preferably, the meat alternative is a pepperoni.

[0173] Preferably, the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein. Still more preferably, the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein. In yet more preferred embodiments, the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein. In the most preferred embodiments, the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

[0174] 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. Still 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. Preferably, baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C. More preferably, the initial temperature is conducted for 20 to 60 minutes. More preferably, the initial temperature is conducted for 30 to 45 minutes.

[0175] Preferably, the initial temperature is 35 to 60°C. More preferably, the initial temperature is 45 to 50°C.

[0176] In another aspect of the present invention, an improved method for preparing a plantbased meat alternative, having only the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) moulding the batter; and c.) baking the batter to provide the plant-based meat. The instant improved method does not have a holding step, resulting in decreased complexity and lowered cost for the manufacturer.

[0177] Preferably, the batter further has textured or structured protein derived from plants. Preferably, the plant protein is chickpea, soy, pea, soy, fava, gluten, or oat. More preferably, the plant protein is soy protein or pea protein.

[0178] 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. Y et 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.

[0179] 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 the most preferred embodiments, 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.

[0180] Preferably, the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni, or roast. More preferably, the meat alternative is a pepperoni.

[0181] Preferably, the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein. Still more preferably, the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein. In yet more preferred embodiments, the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein. In the most preferred embodiments, the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

[0182] 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. Still 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.

[0183] Preferably, baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C. More preferably, the initial temperature is conducted for 20 to 60 minutes. More preferably, the initial temperature is conducted for 30 to 45 minutes.

[0184] Preferably, the initial temperature is 35 to 60°C. More preferably, the initial temperature is 45 to 50°C.

[0185] In another aspect of the present invention, an improved method for preparing a plant- based meat alternative in which there is no holding step is provided having the steps of comprising the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) moulding the batter; and c.) baking the batter to provide the plant-based meat.

[0186] Preferably, the batter further has textured or structured protein derived from plants. Preferably, the plant protein is chickpea, soy, pea, soy, fava, gluten, or oat. More preferably, the plant protein is soy protein or pea protein.

[0187] 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. Y et 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.

[0188] 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 the most preferred embodiments, 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.

[0189] Preferably, the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni, or roast. More preferably, the meat alternative is a pepperoni.

[0190] Preferably, the transglutaminase is present in step a) in an amount from about 5 to 500 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 10 to 400 mg / kg of plant protein. Still more preferably, the transglutaminase is present in an amount from about 20 to 300 mg / kg of plant protein. In yet more preferred embodiments, the transglutaminase is present in an amount from about 25 to 250 mg / kg of plant protein. More preferably, the transglutaminase is present in an amount from about 30 to 200 mg / kg of plant protein. In the most preferred embodiments, the transglutaminase is present in an amount from about 50 to 150 mg / kg of plant protein.

[0191] 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. Still 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.

[0192] Preferably, baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C. More preferably, the initial temperature is conducted for 20 to 60 minutes. More preferably, the initial temperature is conducted for 30 to 45 minutes.

[0193] Preferably, the initial temperature is 35 to 60°C. More preferably, the initial temperature is 45 to 50°C.

[0194] EXAMPLES Materials and Methods

[0195] Reagent grade chemicals including N-Acetyl Cysteine (NAC), o-Phthalaldehyde (OPA), Sodium Tetraborate decahydrate, hydrochloric acid (HC1, 37%) were purchased from Sigma- Aldrich (St. Louis, MO). Soy protein powder, SUPRO EX45, consisting of 90% protein, dry basis, was obtained from IFF (New Century, KS). The enzyme transglutaminase (TG) was purchased from Modernist Pantry (Eliot, ME) whereas Glucose Oxidase (GOX) and FoodPro CAT (catalase) were obtained from IFF (New Century, KS).

[0196] Example 1:

[0197] Biochemical assays: Evaluation of TG activity in combination with GOX and catalase using OPA assay

[0198] TG catalyzes the formation of isopeptide bonds between y-carboxamide group of glutamine and the e-amine group of lysine residues thereby releasing ammonia (NH3) (Malesevic,M et al., 2015). OPA assay was used to evaluate TG activity. In this assay, ammonia released from TG catalyzed reaction is derivatized with NAC and OPA in the presence of TCEP into a fluorescent isoindol adduct (Robert-Peillard, F., et al., 2017). Based on this concept, the effect of GOX and catalase on TG activity was evaluated as follows. First, a protein solution containing 3% SUPRO EX45 was prepared using purified water. The protein was then treated with 3% TG (w / w of protein), and / or GOX (0.05, 0.1, or 0.2% of total solution), and catalase (0.1 or 0.2% of total solution). Dextrose (0.4% of total solution) was added as a substrate for GOX. Negative and positive controls were also prepared in which the negative control did not contain any enzymes and the positive control contained TG only without the GOX and catalase.

[0199] Similarly, samples containing only GOX and catalase (without TG) were prepared to adjust for potential background interference that may interfere with the fluorescence reading. The samples were then incubated at 40°C for Ihr and enzymes were deactivated at 95°C for 10 min. The solutions were then centrifuged, and the supernatant of each solution was used for OPA assay. For the OPA assay, a working solution containing 200mM NAC (in 1 M HC1), 20 mM TCEP (in 1 M HC1), and 200 mM OPA (in ethanol) was prepared in 0.1 M sodium borate buffer (pH 10.5). Subsequently, 5 pL of each supernatant from the test samples was reacted with 50 ,u L of the working solution in a 96 well plate. The samples were then mixed and incubated at room temperature for 5 min prior to reading fluorescence. TG activity was then determined based fluorescence intensity with excitation and emission wavelengths set at Xex = 415 nm and Zem = 485 nm respectively.

[0200] Results for example 1:

[0201] As shown in FIG. 1 , the ammonia release (TG activity) seemed to be reduced with increased level of GOX as compared to the control containing only TG. GOX facilitates the oxidation of glucose to hydrogen peroxide and D-gluconolactone (Wong, C. M., et al., 2008). While the mechanism by which GOX could affect TG activity is yet unclear, a potential explanation could be that the hydrogen peroxide release from GOX activity could oxidize cysteine residues of TG and reduce its activity as it is known to be highly susceptible to oxidation (Stamnaes, J., et al., 2010). Interestingly, this effect seems to be counteracted when catalase was added both at 0.1 and 0.2% level. This effect may be due to the excess hydrogen peroxide being converted to water by the action of catalase while a small portion of the hydrogen peroxide may also facilitate other reactions that could result in the release of ammonia. However, higher level of GOX (0.2% of total solution) reduced TG activity despite the presence of catalase. Based on this study, a combination of GOX and catalase at lower levels may enhance TG activity.

[0202] Example 2:

[0203] Model systems: Evaluation of TG activity in combination with GOX and catalase on the textural properties of protein gels

[0204] While the biochemical assays provide insight into the extent of TG activity through the amount of ammonia released, it is, however, difficult to determine protein cross-linking and texture modifications in plant-based proteins using this assay. As a result, we have developed a model system to evaluating the effect of TG on the overall texture properties of protein gels. To prepare the protein gels, 12% soy protein (SUPRO EX 45) was mixed with purified water under vacuum using a STEPHAN mixer. 80 g of the protein gel was then aliquoted into small glass jars in which different enzyme combinations were added. TG (4% w / w of protein) and dextrose (0.4% of total solution) were both added to each jar along with varying levels of GOX and catalase as described in Tables 2 and 3. Once all the enzymes and dextrose were added, the samples were manually mixed for - 1 min and subsequently incubated for approximately 15 hours at room temperature prior to texture analysis.

[0205] Table 2: List of variables used to evaluate the effect of TG in combination with GOX and catalase

[0206] Additionally, the set of samples listed below were evaluated separately where texture was analyzed at 8 and 24hr incubation period.

[0207] Table 3: List of variables for catalase and no catalase.

[0208] *Note: TG was added at 4% of protein (w / w) in all the samples. Texture Profile Analysis:

[0209] Texture analysis was carried out to evaluate the effect of different enzyme combinations on the strength / hardness of the gel prepared using soy protein. In this test, the gel strength is determined by measuring the mechanical resistance to stress using TA-XT PLUS Texture Analyzer (Texture Technologies Corp, Scarsdale, New York). A TA-23, cylinder probe (12.7 mm diameter) with Exponent Software was used to measure texture. Each jar containing the protein gel was placed underneath the probe and trigger force of 5 g was applied to reach a break distance of 20 mm. As these samples are compressed to permanent deformation of the gel, each sample is only compressed once with the probe traveling at 0.5 mm / sec. The results are then reported as gel hardness (force, g).

[0210] Results for example 2:

[0211] As observed in the biochemical assay, increased level of GOX resulted in significantly decreased gel strength or firmness (FIG. 2). However, this effect seems to be offset when catalase was added both at 0.1 and 0.2% level. While higher level of GOX (0.2% of total solution) seem to reduce gel strength even with the presence of catalase, the gel strength was still comparable if not higher than the gel containing only TG. This study confirmed that the increased ammonia release observed in the biochemical assays did in fact result in higher protein crosslinking resulting in firmer gels. Furthermore, while the excess hydrogen peroxide may have been used by the catalase, small amounts of the hydrogen peroxide could facilitate the formation of additional crosslinking by acting on cysteine residues or other reaction sites to generate disulfide bonds (Vemulapalli, V., & Hoseney, R. C. 1998).

[0212] As shown in FIG. 3, an additional variable was incorporated in which only TG and catalase (no GOX) were used in one of the samples. The results show that TG and catalase performed similar to the control (TG only) and the two enzymes alone do not result in increased gel strength unless GOX is added. This effect was observed both at 8 and 24 hr. of incubation time. This finding further confirms that gel strength improvement is a result of the addition of all three enzymes.

[0213] Example 3:

[0214] Pilot application trials: Texture modifications of plant-based hot dog samples prepared using TG in combination with GOX and catalase

[0215] As highlighted above, the use of TG in combination with GOX and catalase was evaluated in biochemical assays and model systems. The next step was to evaluate this effect in pilot application trials for meat alternative products. In this test, a modified hot dog formulation consisting of plant-based proteins was used without the addition of other ingredients such as seasonings, color, fat, and antimicrobials that may play a role in modifying the functionality of the protein and / or the activity of the enzymes. The formulations with different enzymes combinations are provided in Table 4.

[0216] Table 4: Plant-based hot dog formulation to evaluate the effect of TG in combination with

[0217] GOX and catalase

[0218] *Note: This formulation does not contain ingredients such as seasonings, color and source of fat The four tests evaluated in this trial include:

[0219] Test 1 (Tl): TG only (0.4%) - control

[0220] Test 2 (T2): TG (0.4%) + GOX (0.025%) + Catalase (0.01%)

[0221] Test 3 (T3): TG (0.4%) + GOX (0.025%) Test 4 (T4): TG (0.4%) + Catalase (0.01%)

[0222] Preparation of the plant based hot dogs:

[0223] The plant-based hot dog samples were prepared using a STEPHAN mixer according to the formulation presented in Table 3. First, 300 g of the hydration water was set aside to dissolve the enzymes. The reminder of water was then added into the STEPHAN mixer followed by SUPRO EX45. The protein is then thoroughly mixed and hydrated for prox. 2-3 min under vacuum. The enzymes and dextrose were then dissolved in 300 g of water and added into the STEPHAN mixer with the protein solution. In this step, vacuum was not introduced during mixing as it was important to ensure there was enough oxygen accessible for GOX to catalyzes the oxidation of glucose to hydrogen peroxide and D-gluconolactone which gets hydrolyzed to gluonic acid. Once the protein was thoroughly mixed with the enzymes for approx. 3-4 min, the protein gel was then transferred into a vacuum bag and stuffed into plastic casings. The samples were then cooked in a Rational Oven with a cooking cycle consisting of a hold time at 40°C for 40 min and a temperature ramp to final temperature of 95 °C. Once the samples are cooked, they are stored in a refrigerator overnight (4 °C) prior to texture analysis.

[0224] Texture analysis:

[0225] Similar to the model system samples, the hardness of hot dog samples was evaluated using TA-XT2i Texture Analyzer (Texture Technologies Corp, Scarsdale, New York). The probe used in this test was TA-30 cylinder probe (76.2 mm diameter). The hot dog samples were sliced into 3 cm sizes with 10 replicates for each test. Each hot dog sample was placed underneath the probe and a trigger force of 10 g was applied to reach a break distance of 10 mm. The samples are compressed twice to evaluate hardness as well as other texture properties such as resilience, adhesiveness, cohesion, chewiness, springiness, gumminess etc. For the purpose of this study, only hardness, chewiness, and gumminess were evaluated to determine the effect of the enzyme combination on the hot dog samples.

[0226] Results for pilot application trials:

[0227] The samples were stored in a cool room (3 - 4 °C) overnight prior to texture analysis. As shown in the FIG. 5, T2, which contains all three enzymes performed best in terms of gel hardness, gumminess, and chewiness. This finding was well aligned with the results from biochemical assay and model system work described above. Among the samples, T3 was found to be the lowest in gel hardness whereas T4 performed similar to the control (Tl).

[0228] The addition of GOX without catalase seems to have a negative impact on hardness of the hot dog samples. These findings in combination with the biochemical assays and model system, support the use of TG in combination with GOX and catalase for improved firmness in soybased protein formulations that could be used in different meat alternative applications.

[0229] Example 4: TG Dose response

[0230] Materials and Methods

[0231] Soy protein isolates and structured soy protein, consisting of 90 and 77 % protein respectively, dry basis, was obtained from IFF (New Century, KS). Hydrocolloids from IFF including carrageenan, methylcellulose, and modified corn starch were used. Other dry ingredients including flavors (Vegan Burger type flavor, vegetarian beef bouillon, natural pepperoni flavor, Frank and Bologna, beef, or pork type flavor) and colors (paprika, Tomato Extract 2 LWS, Red Raspberry, malt extract, caramel color) as well as antimicrobials were also obtained from IFF. The enzyme transglutaminase (TG) (SEQ ID NO: 1) was purchased from Modernist Pantry (Eliot, ME).

[0232] Pilot application trials: Evaluation of different doses of TG in plant-based deli meat formulation

[0233] In this example, the effect of TG at levels on the texture properties of a plant-based deli meat formulation is evaluated. The plant-based deli meat formulation consisted of an extruded, structured soy protein added at 21.9%, w / w. However, it is known that the enzyme TG does not work well on extruded protein as the process modifies the protein to such an extent that the side chains are likely not accessible. Soy protein isolate, which serves as an excellent substrate for TG, was added into the formulation at 10%, w / w. TG was added at 0.05, 0.15, 0.3, 0.45, and 0.6% of the formulation. Other dry ingredients including seasonings, flavor and color are added at levels described in Table 5.

[0234] Table 5: Plant-based deli meat formulation used to evaluate the effect of different doses of

[0235] TG1

[0236] ’The samples T1-T5 indicate different doses of enzyme where T1 = 0.05% TG, T2 = 0.15%

[0237] TG, T3 = 0.3% TG, T4 = 0.45% TG, T5 = 0.60%TG

[0238] Processing conditions: The structured soy protein was added into a Hobart Mixer with the hydration water and mixed at low speed for 1 min. The soy protein isolates were then added along with the enzyme, TG and mixed for an additional 3 - 5 min. Once the protein samples were fully hydrated, the other dry ingredients including the seasonings, flavor, color as well as the rest of the water were added and mixed until fully homogenized for approximately 2 - 3 min. The melted coconut fat was then added and mixed until fully incorporated into the matrix for an additional 3 - 4 min. The resulting batter was transferred to a plastic bag and vacuum- sealed to remove air bubbles prior to stuffing. Batter was stuffed into plastic casings and held at 4 °C for 22 hr. or directly placed in the oven after stuffing. A stepwise cooking cycle shown in Table 6 was used to bake the plant-based deli meat samples. The final 90°C (internal temperature) ensures the deactivation of the enzyme. Once the samples were cooked, they were placed in ice water for approx. 30 min and placed in a refrigerator (4 °C) overnight before slicing.

[0239] Table 6: Programmed stepwise cooking cycle for plant-based deli meat sample containing TG

[0240] Texture analysis:

[0241] The texture properties of the deli meat samples were evaluated using TA-XT2i Texture Analyzer (Texture Technologies Corp, Scarsdale, New York). The probe used in this test was TA 30-cylinder probe (76.2 mm diameter). The samples were sliced into 20 mm sized cubes at 4 - 5 °C with 10 replicates for each test. Each deli meat sample was placed underneath the probe and subjected to a texture profile analysis. The texture measurement conditions were set as follows: pre-test, test and post-test speed were set at 2 mm / sec, 1 mm / sec and 5 mm / sec respectively. The trigger force was set at 10 g with distance of 6 mm and relaxation time of 5 sec. The samples were evaluated for differences in hardness, chewiness, and gumminess.

[0242] Results:

[0243] The sample 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 higher level of TG up to 0,45%. However, 0.60% of the enzyme had a negative impact on the texture properties, indicating a potential overdose effect. This agrees with results observed in model system experiments (not shown) where too much cross-linking, due to high levels of TG, resulted in syneresis and negatively impacted the overall texture properties. Based on this experiment, it was recommended that TG usage level of 0.3 - 0.5% may be ideal in similar plant-based meat alternative formulations.

[0244] Example 5:

[0245] Pilot application trials: Determining the effect of hold time on the texture development of plant-based deli meat prepared using TG

[0246] The objective of this study was to evaluate the effect of hold-time prior to cooking on the activity of TG to develop the desired texture properties of plant-based deli meat products. The present example demonstrates hold-time is not required and the cook-cycle allows the enzyme to work as needed.

[0247] The samples studied included 4 different timepoints and a negative control:

[0248] T1 - Hold 22 hr. at 4°C

[0249] T2 - No hold at 4°C

[0250] T3 - Negative control sample without enzyme Similar to the plant-based deli meat formulation shown in Example 4, the structured soy protein was used at 21.9%, soy protein isolates were used at 10%. The TG was added at 0.3%, w / w of the formulation. The processing conditions were the same as in Example 4 where the textured protein and powdered protein were hydrated first along with the TG prior to adding the other dry ingredients. Once the samples were fully homogenized, vacuumed and stuffed into the specific casings, they were held overnight prior to cooking. The cookcycle was the same as example 4 (table 6).

[0251] Table 7: Formulation of plant-based deli meat samples1

[0252] 'The samples T1 and T2 indicate hold time of 22 hr and no hold respectively

[0253] Results:

[0254] During slicing (cold), it was noted that the negative control was crumbly, had a rough texture and was difficult to slice. The enzyme containing samples were smoother and there was less fat cap on the outer surface, indicating better emulsification. The enzyme samples also showed greater flexibility and elasticity. The texture analysis (Figure 7) showed that the negative control had substantially lower hardness, gumminess, and chewiness than the enzyme-treated samples. However, differences in hold-times did not significantly affect the protein-binding properties of the enzyme and texture of the deli meat samples, showing that under the present processing conditions a hold time prior to cooking is not essential for obtaining the positive effects of transglutaminase activity. Hence, the necessary enzyme activity happens during the cooking process.

[0255] Example 6:

[0256] Pilot application trials: Evaluation of TG activity on the texture properties of plant-based pepperoni as compared to hydrocolloids

[0257] In this example, the effect of TG in soy-based pepperoni formulation was evaluated and compared against a control sample containing the hydrocolloids - carrageenan and methylcellulose. The TG was added at 0.4% (w / w) whereas the hydrocolloid was added at 5.5% (w / w) of the formulation.

[0258] Table 8: Soy protein pepperoni formula

[0259] Processing conditions:

[0260] The structured soy protein was hydrated in Hobart mixer for 2 - 3 min. Next, the hydrocolloid was added to the control sample, whereas soy protein isolates and TG were added to the enzyme sample and mixed for an additional 2 - 3 min. Once the proteins were fully hydrated. The other dry ingredients including the colors, seasoning blends, antimicrobial and salt were added and mixed thoroughly prior to adding the melted coconut fat. The coconut oil was slowly added to the mixture as the dry ingredients were mixing. The protein dough was then mixed for approximately 3 - 4 min or until fully emulsified (no visible oil droplets). Samples were removed from the mixer, vacuum sealed, stuffed into plastic casings, and placed in the oven. The cook-cycle used in this trial is shown in table 5. After cooking, the samples were placed in ice water for approx. 30 min and in a refrigerator (4 °C) overnight before slicing. Texture analysis was carried out as indicated in Example 4.

[0261] Table 9: Oven cooking temperature profile for plant-based pepperoni samples Results:

[0262] The pepperoni samples were first sliced (1.8 - 2 mm) to evaluate the mechanical sliceablity as well as the sensory attributes of the samples. During the slicing process, it was noted that the sample containing TG sliced better than the hydrocolloid sample. Additionally, the TG sample incorporated the oil well into the protein dough as compared to the control. During sensory evaluation, the samples were tasted as-is or baked on a cheesepizza. It was noted that the TG sample showed desirable meat-like qualities once cooked on a pizza. The TG pepperoni slices showed cupping or curling and oiling out similar to meatbased pepperoni, which was not observed in the hydrocolloid sample. Additionally, the flavor from the TG sample appeared to be well-rounded and did not overcoat the palate.

[0263] The texture analysis results (figure 8) show that the TG sample was firmer than the control, which can explain the better sliceability of the sample. The gumminess and chewiness were also higher in the TG sample. This finding was in alignment with the sensory attributes observed during tasting.

[0264] Example 7:

[0265] Pilot application trials: Evaluation of TG activity on the texture properties of plant-based hot dogs as compared to those prepared using hydrocolloids

[0266] In this example, the effect of using TG on the texture properties of plant-based hot dog samples were evaluated and compared against those prepared using hydrocolloids (carrageen and methylcellulose). The composition of the formulation is indicated in table 10.

[0267] Table 10: Plant-based hot dog formulation using hydrocolloid vs TG

[0268] Processing conditions:

[0269] For the hydrocolloid sample, the cold water, soy protein isolates and the hydrocolloids were added into a Stephan Vacuum mixer. Then, the sample was mixed for 2 min on low speed and additional 2 - 3 min on high speed to ensure the protein was fully hydrated. For the sample containing TG, the protein was hydrated first for 3 - 4 min and the enzyme was added once the protein was fully hydrated. The rest of the dry ingredients were added and mixed for approximately 3 - 4 min. Vacuum was then applied and mixed for an additional 2 min prior to adding the melted coconut oil. The hot dog batter was then mixed thoroughly under vacuum until all the oil is fully absorbed and emulsified. The batter was then transferred into a plastic bag, sealed under vacuum, stuffed into a casing, and cooked according to the cook-cycle shown in table 11.

[0270] Table 11: Oven cooking temperature profile for hot dog recipe

[0271] Texture analysis:

[0272] The texture analysis was carried out both with cold (3 - 5 °C) and recooked samples. The recooked samples were placed in boiling water until the internal temperature reached approximately 74 °C. The probe used for texture analysis in this test was TA-30-cylinder probe. The hot dog samples were sliced into 3 cm pieces with 10 replicates for each test. Each hot dog sample was placed underneath the probe and the following measurement conditions were used: pre-test, test and post-test speed were set at 2 mm / sec, 1 mm / sec and 5 mm / sec respectively. The trigger force was set at 10 g with distance of 10 mm and relaxation time of 5 sec. The samples are compressed twice to evaluate hardness chewiness, and gumminess.

[0273] Results:

[0274] For the cold PB hot dogs, the samples containing TG and hydrocolloid were similar in hardness. The chewiness and gumminess were higher in the TG samples. This was in agreement with the sensory observations. During tasting, it was noted that the sample containing hydrocolloids was pasty and did not have as much bite as the TG samples. For the recooked hot-dogs, the sample containing hydrocolloids was slightly firmer, yet the chewiness and gumminess were higher in the TG samples. The sensory attributes of the recooked samples were similar to the cold samples in which the hydrocolloid sample appeared to have less bite than the control. Additionally, it was noted that the overall flavor and mouth feel of the TG sample was better than the hydrocolloid sample. Example 8:

[0275] Pilot application trials: Evaluation of TG activity at different protein levels in plant-based hot dog formulation

[0276] The purpose of this example was to determine the ideal substrate protein range needed for the TG to develop desirable texture properties in plant-based hot dog formulations. The samples evaluated in this study were:

[0277] Tl : soy protein isolates with 18% protein

[0278] T2: soy protein isolates with 15% protein

[0279] T3: soy protein isolates with 12% protein

[0280] T4: soy protein isolates with 10% protein

[0281] The processing conditions and cook-cycle were the same as example 7.

[0282] Table 12: Plant-based hot dog formulation prepared using TG with different levels of protein

[0283] Results: Sample 4 (T4) containing 10% protein did not form a gel after cooking and thus texture measurements were not included in the results. The texture analysis results showed that hardness, chewiness, and gumminess increased with increased level of protein both for the cold and recooked samples. It was determined through sensory evaluation that, while the 12% protein sample formed a gel structure, it was not firm enough to have meat-like texture characteristics. The samples containing 15 - 18% had desirable texture in terms of firmness / hardness, and bite.

[0284] EXAMPLE 9

[0285] Cloning and expression of transglutaminase

[0286] The transglutaminase genes were identified from public databases (NCBI). The SEQ ID NO. of each gene is listed in Table 12. Generally, the gene encoding transglutaminase (without signal peptide) were codon-optimized based on Bacillus subtilis codon preference and cloned into p2JM vector (Vogtentanz, Protein Expr Purif. 55:40-52, 2007). AprE promoter and signal peptide were used for transcription and secretion of transglutaminase. The constructed vector was subjected to rolling-circle amplification and transformed into a suitable Bacillus subtilis host. The transformants were screened on LB plate supplementing 5 ppm chloramphenicol. Single colonies were inoculated into 20 mL LB medium with 5 ppm chloramphenicol and subsequently used to inoculate culture media for cell growth and protein expression. Clarified culture supernatant was harvested by centrifugation, concentrated, and used for protein purification. All transglutaminases were purified using standard procedures known in the art, including hydrophobic interaction chromatography, ion / cation exchange chromatography and size-exclusion chromatography. The fractions containing the protein of interest were identified by SDS-PAGE and activity assay described below. Sample of purified protein was stored in 40% glycerol at -20 °C until usage. Table 12. Sequence list of transglutaminases.

[0287] SEQ ID

[0288] Gene name CRC No. Description

[0289] No.

[0290] SciTG2 CRC24210-WT Precursor PRT 5

[0291] SciTG2 CRC24210-WT Predicted Mature PRT 6

[0292] SciTG2 CRC24210-WT Synthetic / optimized full length DNA 7

[0293] SnoTG2 CRC26523-WT Precursor PRT 8

[0294] SnoTG2 CRC26523-WT Predicted Mature PRT 9

[0295] SnoTG2 CRC26523-WT Synthetic / optimized full length DNA 10

[0296] SroTGl CRC26802-WT Precursor PRT 11

[0297] SroTGl CRC26802-WT Predicted Mature PRT 12

[0298] SroTGl CRC26802-WT Synthetic / optimized full length DNA 13

[0299] SsyTGl CRC26807-WT Precursor PRT 14

[0300] SsyTGl CRC26807-WT Predicted Mature PRT 15

[0301] SsyTGl CRC26807-WT Synthetic / optimized full length DNA 16

[0302] Ajinomoto

[0303] CRC17097-WT Mature PRT 1

[0304] TG

[0305] Example 10

[0306] Transglutaminase-induce Protein Gelation Assay

[0307] The objective of this assay is to investigate the performance of TGase samples in protein gelation reactions under different conditions by measuring the gel strength with a Texture analyzer. Soy protein slurry (12% w / w) was prepared by adding 60 g of soy protein isolates to 440 g of Milli-Q water, mixing the slurry well with an electric whisk, and keeping it at room temperature for at least 2 hours for full hydration of protein isolates. 20 g of the protein slurry was weighed into an RVA container. A suitable amount of TGase sample was added and mix the enzyme solution and protein slurry well with RVA at 40 °C, 960 rpm for 2 min. Seal the RVA container with food wrap. Incubate in the shaker at 40 °C for 4 hours. At the end of incubation, measure the gel strength with a texture analyzer equipped with a P / 5 probe (4mm DIA CYLINDER STAINLESS). The maximum force used for puncture was recorded as the rupture force, while the force at the depth of 4mm was recorded as gel strength. The protein gel was penetrated at a constant speed of 1 mm / s to a maximum target depth of 10 mm and the maximum force was recorded. The gel strength and rupture force were determined based on the average of three replicates (Table 13).

[0308] Table 13. Protein gelation performance of transglutaminase.

[0309] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, certain changes and modifications can be practiced 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 was individually incorporated by reference. To the extent the content of any citation, including website or accession number may change with time, the version in effect at the filing date of this application is meant. Unless otherwise apparent from the context any step, element, aspect, feature of embodiment can be used in combination with any other.

[0310] References:

[0311] 1. Malesevic, M., Migge, A., Hertel, T. C., & Pietzsch, M. (2015). A fluorescence-based array screen for transglutaminase substrates. ChemBioChem, 16(8), 1169-1174.

[0312] 2. Robert-Peillard, F., Barco, E. P., Ciulu, M., Demelas, C., Theraulaz, F., Boudenne, J. L., & Coulomb, B. (2017). High throughput determination of ammonium and primary amine compounds in environmental and food samples. Microchemical Journal, 133, 216-221.

[0313] 3. Wong, C. M., Wong, K. H., & Chen, X. D. (2008). Glucose oxidase: natural occurrence, function, properties and industrial applications. Applied microbiology and biotechnology, 78(6), 927-938.

[0314] 4. Stamnaes, J., Pinkas, D. M., Fleckenstein, B., Khosla, C., & Sollid, L. M. (2010). Redox regulation of transglutaminase 2 activity. Journal of Biological Chemistry, 285(33), 25402- 25409.

[0315] 5. Vemulapalli, V., & Hoseney, R. C. (1998). Glucose oxidase effects on gluten and water solubles. Cereal Chemistry, 75(6), 859-862.

Claims

What is claimed is:

1. A method for producing a plant protein-containing food, the method comprising: adding a transglutaminase, a sugar oxidoreductase and a catalase to a food raw material comprising a plant protein.

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

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

4. The method of any 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 claims 1 to 3 wherein the oxidoreductase is a 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 of the preceding claims wherein the transglutaminase is derived from Streptomyces mobaraensis.

23. The method of any 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 of the previous 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 an enzyme according to SEQID N0:4 or a catalase active fragment thereof.

38. The method of any of the preceding claims wherein the food is a meat alternative.

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

40. The method of claim 38 wherein the meat alternative is a pepperoni.

41. An improved method for preparing a plant-based meat alternative, comprising: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) holding said batter for less than 60 minutes; c.) moulding the batter; and d.) baking the batter to provide the plant-based meat.

42. The method of claim 41 wherein the batter further comprises textured or structured protein derived from plants.

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

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

45. The method of any 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 of claims 41 to 52 wherein the holding step is for less than 30 minutes.

54. The method of claim 53 wherein the holding step is for less than 15 minutes.

55. The method of claim 54 wherein the holding step is for less than 10 minutes.

56. The method of claim 55 wherein the holding step is for less than 5 minutes.

57. The method of claim 56 wherein the holding step is for less than 2 minutes.

58. The method of claim 57 wherein the holding step is for less than 1 minute.

59. The method of claim 58 wherein the holding step is for 0 minutes.

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

61. The method of claim 60 wherein the meat alternative is a pepperoni.

62. The method of any 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 of 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 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 of claims 41 to 71 wherein the baking step d) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C.

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

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

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

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

77. An improved method for preparing a plant-based meat alternative, consisting of the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) moulding the batter; and c.) baking the batter to provide the plant-based meat.

78. The method of claim 77 wherein the batter further comprises textured or structured protein derived from plants.

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

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

81. The method of any 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 of claims 77 to 88 wherein the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni or roast.

90. The method of claim 89 wherein the meat alternative is a pepperoni.

91. The method of any 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 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 of claims 77 to 100 wherein the baking step c) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C.

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

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

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

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

106. An improved method for preparing a plant-based meat alternative in which there is no holding step, comprising the steps of: a.) mixing a batter comprising water, plant protein, coconut oil, hydrocolloids, one or more meat flavours and a transglutaminase; b.) moulding the batter; and c.) baking the batter to provide the plant-based meat.

107. The method of claim 106 wherein the batter further comprises textured or structured protein derived from plants.

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

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

110. The method of any 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.1 1 . The method of claim 11 1 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 of claims 106-117 wherein the meat alternative is a hot dog, frankfurter, sausage, deli meat, loaf, salami, meat loaf, pepperoni or roast.1 19. The method of claim 1 18 wherein the meat alternative is a pepperoni.

120. The method of any 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 of 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 of plant protein.

126. The method of any 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 of claims 77-100 wherein the baking step c) is conducted at an initial temperature of 25 - 70°C for a period of 10 to 80 minutes, followed by a temperature ramp until the meat alternative has an internal temperature of at least 90 °C.

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

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

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

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

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