WO2025061262A1

WO2025061262A1 - Methods for evaluating protein substrate gelling by microbial transglutaminase

Info

Publication number: WO2025061262A1
Application number: PCT/EP2023/075771
Authority: WO
Inventors: Caroline MOSBECH; Anders Lybecker CHRISTIANI; Gitte Budolfsen Lynglev; Mette HOLSE; Susanne SCHOESLER
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-09-19
Filing date: 2023-09-19
Publication date: 2025-03-27
Anticipated expiration: 2026-03-19

Abstract

The present invention relates to methods of pre-determining the ability of a plant-based substrate to form a self-standing gel by treatment with at least one microbial transglutaminase (MTGase) enzyme, and methods of producing a self-standing gel of such plant-based substrates, as well as uses of such plant-based substrates in the production of a self-standing gel or a plant-based food.

Description

Methods for Evaluating Protein Substrate Gelling by Microbial Transglutaminase

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Field of the Invention

The present invention relates to methods of pre-determining the ability of a plant-based substrate to form a self-standing gel by treatment with at least one microbial transglutaminase (MTGase) enzyme, and methods of producing a self-standing gel of such plant-based substrates, as well as uses of such plant-based substrates in the production of a self-standing gel or a plantbased food.

Background of the Invention

Replacing traditional meat products with plant proteins is one way to mitigate the climate crisis and offer a growing world population sustainably produced protein-rich foods. Legumes, such as soy and pulses, are attractive crops for the production of protein-rich foods. Other nonanimal protein sources are also being used in food production, e.g., from plants, algae or insects.

Texturized products, such as extruded products, are extensively used in the food industry. Extrusion is used primarily to give the food a specific texture and distinctive mouthfeel. The development of acceptable texture and flavor for meat analogues is one of the biggest challenges for food producers. However, besides the texturized products, plant-based food producers and researchers have also had a strong focus on the binding agents in plant-based meat. A glance at current meat analogues reveals that their characteristic attributes, i.e. , texture, flavor, color, etc., obviously depend on the ingredients used. A typical meat analogue contains water (50%-80%), textured vegetable proteins (10%-25%), nontextured proteins (4%-20%), flavorings (3%- 10%) , fat (0%-15%), binding agents (1 %-5%), and coloring agents (0%-0.5%) (See Egbert, R., Borders, C., 2006. Achieving success with meat analogs. Food Technology 60, 28-34).

It has been disclosed to manufacture plant-based meat substitutes by passing a mixture containing plant protein material and oat material and, optionally, a crosslinking enzyme and/or a protein deamidating enzyme at a temperature of 25-55°C through an extruder to make meat substitutes. Meat substitutes were produced, where transglutaminase was included using low temperature and high temperature extrusion and for each sensory property tested, and also for the overall quality rating, the low temperature samples gained better ratings than the corresponding high temperature samples (WO 2020/038541 ; Raisio Nutrition).

Extrusion has been employed for many years in the production of meat analogues for obtaining meat-like structures from plant protein. Even so, it is difficult to obtain extrudates of plant-based protein substrates with sufficiently good textural and functional properties for them to be used in meat analogues without having to add non-natural ingredients, such as methylcellulose. Methylcellulose is often used in the final product formulation and acts as a binder, ensuring that the product maintains its texture and shape. Methylcellulose has the ability to provide thermo-gelling upon heating, which keeps the product together and reduces water loss during cooking. However, a push from consumers for cleaner labels on food products has influenced food manufacturers to limit or completely eliminate the use of such ingredients in processed meat analogue products. Various natural binders like soy-, pea- and/or faba bean isolates have been suggested as alternatives and often in combination with cross-linker enzymes like transglutaminase. For example, Djoullah A., et al concluded, that the not only the composition of pea protein isolates, but also the structure and conformation of the protein plays a role in the enzymatic reaction with transglutaminase - yet no general guidance was provided (Djoullah et al. Gelation behaviors of denaturated pea albumin and globulin fractions during transglutaminase treatment, 2018, Food Hydrocolloids vol. 77 p. 636-645).

SUMMARY OF THE INVENTION

The present inventors have identified a measurable parameter that allows a quick predetermination of whether a plant-based protein substrate is able to provide a sufficiently resilient or self-standing gel by transglutaminase treatment, thereby circumventing the tedious trial-and- error step of having to actually produce the gels.

Accordingly, in a first aspect the present invention relates to methods of pre-determining the ability of a plant-based substrate to form a self-standing gel by treatment with at least one mature microbial transglutaminase (MTGase) enzyme, wherein the Distribution% of small proteins in the plant-based substrate is determined by size-exclusion chromatography (SEC HPLC), as exemplified herein, and wherein a Distribution% of small proteins below 33% positively indicates the ability of the plant-based substrate to form a self-standing gel by treatment with at least one mature microbial transglutaminase (MTGase) enzyme.

A second aspect of the invention relates to methods of producing a self-standing gel of one or more plant-based substrate by treatment with at least one mature microbial transglutaminase (MTGase) enzyme, wherein the one or more plant-based substrate has a Distribution% of small proteins below 33%, determined by size-exclusion chromatography (SEC HPLC), as exemplified herein.

A final aspect of the invention relates to uses of one or more plant-based substrate having a Distribution% of small proteins below 33%, determined by size-exclusion chromatography (SEC HPLC), as exemplified herein, in the production of a self-standing gel or a plant-based food, said use comprising an enzymatic treatment step with at least one mature microbial transglutaminase (MTGase) enzyme. Brief Description of the Figures

Figure 1 illustrates the calculation of Distribution% of Interval 1 of the plant-based substrate SPI 5 (soy protein isolate no. 5).

Figure 2 shows the collected data from analysis of 14 different soy protein isolates (SPI) from Examples 1 and 2; the bar chart illustrates the Distribution% of large proteins (upper bar. stripes) and small proteins (lower bar. solid grey) referring to the left y-axis. Black horizontal reference line at 33% on the left y-axis marks the maximum Distribution% of small proteins to achieve a self-standing gel with by transglutaminase treatment. A relative Distribution% of small proteins above 33% results in no gel formation upon Microbial Transglutaminase (MTGase) treatment. A black dotted vertical reference line separates the non-gelling SPIs (to the left) from the SPIs forming a self-standing gel (to the right) when treated with MTGase. Black point for each sample referring to the right y-axis is marking the gel hardness after MTGase treatment measured in triplicates. All self-standing gels exhibited gel hardness above 20 g.

Definitions

Enzyme: The terms “Microbial Transglutaminase” or “MTGase” are synonymous and interchangeable, and they mean a mature enzyme having transglutaminase activity (EC 2.3.2.13) that catalyzes the formation of a covalent bond between the y-carboxamide group of protein- or peptide-bound glutamine (acyl donors) and the free amine group of protein- or peptide-bound lysine (acyl acceptors), which is microbially produced and derived from a microbial source or donor if recombinantly produced. For purposes of the present invention, microbial transglutaminase activity is determined according to the procedure described in the Examples. In one aspect, a mature microbial transglutaminase enzyme of the present invention has at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the transglutaminase activity of the mature polypeptide of any SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5.

Legume: Legumes are plants in the Fabaceae family (or Leguminosae), or the fruit or seed of such a plant (also called a pulse, especially in the mature, dry condition). Well-known legumes include alfalfa, clover, beans, peas, chickpeas, lentils, lupins, mesquite, carob, soybeans, peanuts, and tamarind. Legumes produce a botanically unique type of fruit — a simple dry fruit that develops from a simple carpel and usually dehisces (opens along a seam) on two sides.

Pulse: The United Nations Food and Agriculture Organization (FAO) recognizes 11 types of pulses: dry beans, dry broad beans, dry peas, chickpeas, cow peas, pigeon peas, lentils, 15 Bambara beans, vetches, lupins and pulses NES (/.e. minor pulses, including Lablab, hyacinth bean (Lablab purpureus), Jack bean (Canavalia ensiformis), sword bean (Canavalia gladiata), Winged bean (Psophocarpus tetragonolobus), Velvet bean, cowitch (Mucuna pruriens var. utilis), Yam bean (Pachyrhizus erosus). Pulse and/or legume protein: The term 'pulse and/or legume protein means pulse protein and/or legume protein, a desirable constituent of pulse flour and/or legume flour; the term also includes processed and/or deflavoured pulse and/or legume flour, wherein the processed flour has a higher protein content than unprocessed flour. Processed or deflavoured pulse and/or legume flour may also be termed pulse and/or legume protein concentrate and/or isolate, respectively. Deflavoured pulse and/or legume flour or protein: In the context of the instant invention, the term 'deflavoured' means that the flour or protein component has been processed to reduce off-flavour, e.g., bitterness.

Expression: The term “expression” includes any step involved in the production of a transglutaminase polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

Mature polypeptide: The term “mature transglutaminase polypeptide” means a transglutaminase polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is the mature polypeptide in of any SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5. It is known in the art, that a host cell may produce a mixture of two of more different mature polypeptides (/.e., with different C-terminal and/or N-terminal amino acid residues) expressed from the same polynucleotide. The transglutaminase in SEQ ID NO:1 is produced in vivo in at least 3 different mature forms shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, wherein the N-terminal amino acid in the mature polypeptide differs by one residue in each sequence, respectively.

It is also known in the art that different host cells process polypeptides differently, and also that the pro-peptide may influence the N-terminal of the mature transglutaminase and, consequently, one host cell expressing a polynucleotide encoding a full-length polypeptide incl. signal- and pro-peptide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. The inventors expect that any number of differently processed mature transglutaminase polypeptides may be effective in the present invention; it is entirely trivial to test the suitability of any mature transglutaminase or the different mature forms thereof to identify one or more effective enzyme.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Variant: The term “variant” means a polypeptide having transglutaminase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

DETAILED DESCRIPTION OF THE INVENTION

Plant-based substrates of the invention

The examples of the instant invention were made with different soy protein isolates. Soy protein isolates are available from a number of commercial suppliers, for example, VEGACON® (Eurosoy GmBH, Germany), or the ProFam® product range (Archer Daniels Midland Company, USA). However, the inventors fully expect the method to work just as well with other plant-based substrates, preferably plant-based protein flour, concentrates or isolates, such as, pulse or legume protein isolates, e.g. the VITESSENCE® Pulse Pea Isolates product range (Ingredion, USA). Protein isolates from any food source are expected to be suitable for the instant invention, incl. from pulses and legumes.

In a preferred embodiment of the invention, the plant-based substrate comprises or consists of one or more plant protein flour, concentrate or isolate; preferably the the plant-based substrate comprises or consists of one or more protein isolate; preferably the plant-based substrate comprises or consists of one or more legume protein isolate or pulse protein isolate; even more preferably the plant-based substrate comprises or consists of one or more soy protein isolate.

Polypeptides Having transglutaminase Activity

In a preferred embodiment, the present invention relates to mature MTGase polypeptides having a sequence identity to the mature polypeptide of any of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have transglutaminase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

In another preferred embodiment, the mature MTGase polypeptide has been isolated. A mature MTGase polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or an allelic variant thereof; or is a fragment thereof having transglutaminase activity. In another aspect, the mature MTGase polypeptide comprises or consists of the mature polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

Yet another preferred embodiment of present invention relates to mature MTGase polypeptide variants of the SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature MTGase polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly. Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for transglutaminase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The mature MTGase polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The mature MTGase polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et a/., 1993, EMBO J. 12: 2575-2583; Dawson et a!., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.

Sources of Polypeptides Having transglutaminase Activity

A mature polypeptide having microbial transglutaminase activity of the present invention may be obtained from a microorganism of any genus. For purposes of the present invention, the terms “obtained from” or “derived from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the mature MTGase polypeptide obtained from a given source is secreted extracellularly.

The mature MTGase polypeptide may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having transglutaminase activity, or a Gramnegative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.

In one embodiment, the at least one mature MTGase polypeptide is a Bacillus alkalophilus, Bacillus altitudinis, Bacillus amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis polypeptide.

In another embodiment, the at least one mature MTGase polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.

In another embodiment, the at least one mature MTGase polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces mobaraensis, or Streptomyces lividans polypeptide. In yet another embodiment, the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis, Streptomyces caniferus or Streptoverticillium ladakanum, preferably the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis.

The at least one mature microbial transglutaminase (MTGase) polypeptide may be a fungal polypeptide. For example, the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.

In another embodiment, the at least one mature MTGase polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.

In another aspect, the at least one mature MTGase polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The at least one mature MTGase polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Testing gel strength

The resilience or gel-strength of a resulting gel after treating one or more plant-based substrate of the invention with a MTGase according to the aspects of the invention was determined as described in Example 1 below.

In a preferred embodiment, the self-standing gel exhibits a gel hardness above 20 g, determined using a 100 g load cell and an automated texture analyzer with a 6 mm diameter probe moved down from just above the self-standing gel until 5 mm into the sample with a speed of 0.135 mm/s, while continuously measuring the mechanical resistance in grams, as exemplified herein.

Determining Distribution% by SEC HPLC

The inventors utilized size exclusion high-performance liquid chromatography (SEC HPLC) to investigate the variation of protein sizes within the untreated SPIs as shown in Example 2. SEC HPLC was used to extract information about which protein sizes and their relative amount are essential to secure an adequate gel formation. It was discovered, that the Distribution% of small proteins, as determined by size-exclusion chromatography (SEC HPLC) could be used to make a reliable pre-determination of whether any SPI treated with MTGase resulted in a self- standing gel (gelling) or non-gelling; an observation that is readily applied broadly to other plantbased protein substrates.

In a preferred embodiment of the aspect of the invention, the Distribution% of small proteins in the plant-based substrate is determined by size-exclusion chromatography (SEC HPLC) by using the retention time of a standard Cytochrome C (12.38 kDa) to divide the resulting chromatogram into five intervals: Interval 1 with large proteins and Intervals 2-5 with small proteins, integrating the areas of Interval 1 and sum of Intervals 2-5, respectively, and calculating the Distribution% of small proteins (Figure 1).

Determining pH value of plant-based substrates

The inventors showed in Example 4, that the pH value of the plant-based substrates influenced their ability to form a gel by treatment with a mature microbial transglutaminase according to the invention.

Accordingly, a preferred embodiment of the invention relates to the first, second or final aspect, wherein a 6 % dry matter suspension of the plant-based substrate in de-ionized water incubated for 60 min at 25°C has a pH value higher than 7.26, preferably the suspension has a pH value in the range of 7.26 - 10.00, the suspension has a pH value in the range of 7.26 - 9.00, and most preferably the suspension has a pH value in the range of 7.26 - 8.52.

Methods of Production

Methods of producing a mature transglutaminase polypeptide of the present invention are well-known to the skilled person, typically, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide, OR comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates. The polypeptide may be detected using methods known in the art that are specific for the transglutaminase polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the present invention expressing the polypeptide is used as a source of the polypeptide.

EXAMPLES

Introduction:

The inventors have observed a wide quality variation within MTGase induced gelation of plant protein substrates for use in plant-based foods. In the examples below fourteen different commercial soy protein isolates (SPIs) have been evaluated. The aim was to define a way to distinguish between gelling and non-gelling plant protein substrates treated with MTGase.

One of the quality aspects of plant proteins is gel hardness that can be evaluated e.g. by a texture analysis or by visual inspection as described in Example 1. The observed significant variation in gel hardness is evident in Example 1.

The inventors then utilized size exclusion high-performance liquid chromatography (SEC HPLC) to investigate the variation of protein sizes within the untreated SPIs. In Example 2, SEC HPLC was used to extract information about which protein sizes and their relative amount are essential to secure an adequate gel formation. It was discovered, that the Distribution% of small proteins, as determined by size-exclusion chromatography (SEC HPLC) exemplified below as “Distribution% of interval 2-5”, could be used to make a reliable pre-determination of whether any SPI treated with MTGase resulted in a self-standing gel (gelling) or non-gelling; an observation that is readily applied broadly to other plant-based protein substrates.

MTGase activity assay:

Transglutaminase activity may be determined by any method known in the art. For example, analysis of transglutaminase activity may be done by quantitation of the released ammonia resulting from the formation of an isopeptide bond between a free amino group (6- aminohexanoic acid) and an acyl group from a glutamine (Z-GLN-GLY) like described below. Chemicals and enzymes used: 10 Z-GLN-GLY. Eg. Sigma C6154 6-aminohexanoic acid. Eg. Sigma 07260 L-Gluthatione reduced. Eg. Sigma G4251 a-Ketoglutarate. Eg. Sigma K3752 NADH 15 L-GLDH. Eg. Roche 107735 MOPS. Eg. Sigma M-1254. Transglutaminase standard Method: To 75 microliter of an enzyme solution, dissolved in 0.1 M MOPS/5 mM L-Gluthatione reduced pH 7.0, is added 50 microliter of 1% 6-aminohexanoic acid, and 75 microliter of 1% Z-GLN-GLY, and 75 microliter of (0.44 g/L NADH, 2.5 g/L o- Ketoglutarate in 0.1 M MOPS pH 7.0). The absorbance at 340 nm is followed by kinetic measurement for 5 min at 30 °C.

The enzyme activity is determined similar to a transglutaminase standard that has been aligned to be the transglutaminase Unit Definition (Folk, J. E. and Cole, P. W. (1966) Biochim. Biophys. Acta.241 , 5518-5525). MTGase activity expressed as TGHU(A).

Example 1. Assessment of hardness of gels prepared with and without MTGase from 14 different soy protein isolates

Soy protein isolates (SPIs) behave differently upon MTGase treatment. Some SPIs form a solid gel when treated with MTGase, while other SPIs do not form a gel. In this example, 14 different SPIs were treated with MTGase and gelling was evaluated in order to separate the substrates into two categories: self-standing gels and non-gelling.

10% w/w protein suspensions in MilliQ were prepared in beakers and stirred with a magnet. MTGase treatment was defined as adding enzyme to dedicated samples to a final concentration of 3.0 TGHU(A)/g protein and stirred. 2 mL sample was transferred in triplicates into flat bottom 24 well plate, Costar 3524, with 15mm well diameter and the plates were incubated for 1 h at 50 °C. Enzyme inactivation was performed by heating the plates to 95 °C for 20 min. The plates were stored overnight in the fridge.

Gelation of the protein samples was evaluated both on an automated texture analyzer and by visual assessment. The visual assessment was carried out by adding scores of 0-5 to each gel, where 4 and 5 was a self-standing gel. A “self-standing gel” was defined as a gel that can be moved out of the well and still maintain its shape and dimensions (± 10 %). By comparing the texture analysis results and the visual scores 4 and 5 it was concluded that self-standing gels all exhibited gel hardness above 20 g using the method described below.

The plates were taken out from the fridge 1 hour before texture analysis to adjust to room temperature. Hardness of the gels was measured using a 100 g loadcell (HT Sensor Technology Ltd., Xi'an, P.R.C.) connected to a HX711 breakout board (commercially available from many suppliers) with an Atmega 328P integrated circuit (OpenScale, SparkFun Electronics, Colorado, USA). A 3-D printed probe with fused deposition modeling in poly-lactic acid I polyhydroxyalkanoates (PLA/PHA) material (ColorFabb, DK Belfeld, The Netherlands) with a diameter of 6 mm was mounted on the loading cell. Upon measurement, the probe moved from just above the sample until it reached 5 mm down into the sample with a speed of 0.135 mm/s, while continuously measuring the mechanical resistance in grams. The maximum resistance detected was recorded as the hardness of the gel. A self-standing gel was defined as one that exhibited hardness above 20 g in this method.

Table 1: Gel hardness without/with MTGase and evaluation of self-standing gel after MTGase treatment (yes/no) of SPI 1-14.

Example 2. Assessment of protein molecular weight distribution by size-exclusion chromatography (SEC HPLC) related to MTGase gelling performance

It was hypothesized that a higher relative amount of smaller sized proteins present in the soy protein isolates would have a negative effect on gel hardness. Therefore, the influence of the amount of small proteins in the SPIs on the ability for MTGase to form a gel network was investigated. SEC HPLC was used as an analytical tool to separate dissolved macromolecules in the SPIs, mainly proteins, to assess the size distribution based on known standards which were included in the analysis.

The retention time of the commercially available standard Cytochrome C (12.38 kDa) (Table 2) was used to split the chromatogram into two, including proteins smaller and larger than approximately 12 kDa. In short, this was done by dividing the full chromatogram into intervals (Table 2), integrate the area of the selected intervals and calculate the relative percentage which the specific interval (or sum of intervals) constituted of the total area of the chromatogram.

In this example the following definitions are applied:

Large proteins: Proteins larger than 12 kDa (eluting before the 12.38 kDa standard).

Small proteins: Proteins smaller than 12 kDa (eluting after or at the same time as the 12.38 kDa standard).

Distribution%: Relative percentage of large and small proteins based on integrated area within the defined retention time intervals based on commercial standards listed in Table 2.

Distribution% of large proteins was calculated as the % of integrated area in interval 1 (retention time 6-9 min) relative to the total area of the chromatogram (Figure 1). Correspondingly, Distribution% of small proteins was calculated as the % of the sum of the integrated areas in the intervals between 10-42 min (Interval 2, 3, 4 and 5) relative to the total integrated area of the chromatogram (Figure 1).

Sample preparation:

For each SPI, 4 % w/w protein suspensions were prepared with MilliQ and incubated for 60 min at 25 °C in duplicates. Samples were centrifuged for 10 min at 10956 g and diluted 1.5 x with MilliQ. Samples were filtered through 0.22 pM Polytetrafluoroethylene filter and transferred to HPLC-vials.

The SEC HPLC measurements were made using a Superdex 30 Increase 10/300 GL column (Cytiva, Buckinghamshire, United Kingdom) with 15 m coil wire in front of the column to ensure sufficient back pressure. The solvent used was 15 % acetonitrile + 2 x PBS tablets at pH 7.4 (Millipore prod no.: 524650). 25 pL sample was injected and separated on a Thermo Fischer Scientific Vanquish HPLC-SEC system (Thermo Fischer Scientific, Waltham, Massachusetts, USA), consisting of a Vanquish Pump VC-P20-A, Vanquish Autosampler VC-A12-A and a Vanquish UV detector VC-D40-A at a flowrate of 0.8 mL min². Absorbance was measured at 214 nm. The system was calibrated with the following standards from SIGMA-ALDRICH: Cytochrome C (12.384 kDa) (CAS 9007-43-6), Vitamin B12 (1.355 kDa) (CAS 68-19-9) and Phe-Pro (0.262 Da) (CAS 7669-65-0) (SIGMA-ALDRICH, St. Louis, Missouri, USA) (see Table 2).

Table 2: The retention time intervals set to where commercially available standards were eluting.

Integration of chromatograms were made in Chromeleon software, version 7.2.10 ES (Thermo Fischer Scientific, Waltham, Massachusetts, USA). Integration was made on the duplicates for each SPI. Intervals (2, 3 and 4) were identified according to retention time intervals based on the standards used, as shown in Table 2. The Interval 1 was large proteins. The sum of Interval 2-5 was the small proteins (Table 3).

Table 3: %Distribution of retention time Interval 1-5 for SPI 1-14 and sum of %Distribution of Interval 2-5.

The Distribution% of large proteins and Distribution% of small proteins were compared to gel hardness of MTGase treated SPIs (Example 1).

The inventors found that the Distribution% of small proteins (as defined above) influenced the ability of MTGase to form a self-standing gel (hardness above 20 g) (Example 1). It was concluded that SPIs exhibiting Distribution% of small proteins above 33% did not form a selfstanding gel upon MTGase treatment. These results support the above-described hypothesis that small proteins disrupt the MTGase induced gel formation as displayed in Figure 2. This discovery enables efficient pre-determination of gelling or non-gelling plant-based substrates suitable for microbial transglutaminase induced gel formation for e.g. meat alternatives.

Example 3. Measurement of protein content of soy protein isolates

Protein content of the SPIs was determined to ensure that the included substrates have comparable protein content.

Protein content determination by combustion analysis was performed using a LEGO FP 628 Nitrogen Determinator (St. Joseph, Michigan, USA), where protein content is estimated using the Dumas combustion method. The analysis determines the nitrogen content of samples by detecting the content of nitrogen following complete combustion of the sample. The protein content is then estimated from the nitrogen content with a known nitrogen factor, in this case 6.25 for soy as per industry standard.

The following steps summarize the treatment of the sample in the LEGO FP 628 Nitrogen Determinator: A weighed, and encapsulated sample is rapidly combusted at 950 °C in pure oxygen at to remove atmospheric gas. The extracted sample is passed over a heated reduction tube, wherein N_XO_X is converted to N2. Thermal conductivity analysis is then used to determine the inert gas (% N₂) content of the sample. Finally, the N₂ content is converted to a protein content by multiplying the result with a known protein factor.

The system was calibrated with the following EDTA standard from LEGO Corporation (St. Joseph, Michigan, USA). The samples were measured in triplicates.

Sample preparation:

0.10 g of substrate was weighed into a tin foil cup and analyzed on a LECO FP 628 Nitrogen Determinator (LECO Corporation, St. Joseph, Michigan, USA).

Table 4: Percent protein of SPIs 1-14 measured by LECO.

Example 4. Measurement of pH of soy protein isolate suspensions

The pH of the soy protein isolates suspensions was measured to show the influence of pH on MTGase ability to form a self-standing gel.

Sample preparation:

A 6% dry matter SPI suspension was prepared with MilliQ and incubated for 60 min at 25°C. pH was measured using a HACH HQ11d pH-meter with a HACH pH probe (Loveland, Colorado, USA). The pH meter was calibrated using Hamilton DuraCal pH buffers of 4.01 pH, 7.01 pH and 10.01 (Reno, Nevada, USA).

A low pH (6.50-7.26) clearly influences MTGases ability to make a self-standing gel negatively, while high pH (7.26-8.52) improves MTGases ability to make a self-standing gel.

Table 5: pH values of SPI 1-14 sorted in ascending order.

Claims

1 . A method of pre-determining the ability of a plant-based substrate to form a self-standing gel by treatment with at least one mature microbial transglutaminase (MTGase) enzyme, wherein the Distribution% of small proteins in the plant-based substrate is determined by size-exclusion chromatography (SEC HPLC), as exemplified herein, and wherein a Distribution% of small proteins below 33% positively indicates the ability of the plant-based substrate to form a selfstanding gel by treatment with at least one mature microbial transglutaminase (MTGase) enzyme.

2. The method of claim 1 , wherein the plant-based substrate comprises or consists of one or more plant protein flour, concentrate or isolate; preferably the the plant-based substrate comprises or consists of one or more protein isolate; preferably the plant-based substrate comprises or consists of one or more legume protein isolate or pulse protein isolate; even more preferably the plant-based substrate comprises or consists of one or more soy protein isolate.

3. The method of claim 1 or 2, wherein the self-standing gel exhibits a gel hardness above 20 g, determined using a 100 g load cell and an automated texture analyzer with a 6 mm diameter probe moved down from just above the self-standing gel until 5 mm into the sample with a speed of 0.135 mm/s, while continuously measuring the mechanical resistance in grams, as exemplified herein.

4. The method of any of claims 1 - 3, wherein the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis, Streptomyces caniferus or Streptoverticillium ladakanum, preferably the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis.

5. The method of any of claims 1 - 4, wherein the Distri bution% of small proteins in the plantbased substrate is determined by size-exclusion chromatography (SEC HPLC) by using the retention time of a standard Cytochrome C (12.38 kDa) to divide the resulting chromatogram into five intervals: Interval 1 with large proteins and Intervals 2-5 with small proteins, integrating the areas of Interval 1 and sum of Intervals 2-5, respectively, and calculating the Distribution% of small proteins (Figure 1).

6. The method of any of claims 1 - 5, wherein a 6 % dry matter suspension of the plantbased substrate in de-ionized water incubated for 60 min at 25°C has a pH value higher than 7.26, preferably the suspension has a pH value in the range of 7.26 - 10.00, the suspension has a pH value in the range of 7.26 - 9.00, and most preferably the suspension has a pH value in the range of 7.26 - 8.52.

7. A method of producing a self-standing gel of one or more plant-based substrate by treatment with at least one mature microbial transglutaminase (MTGase) enzyme, wherein the one or more plant-based substrate has a Distribution% of small proteins below 33%, determined by size-exclusion chromatography (SEC HPLC), as exemplified herein.

8. The method of claim 7, wherein the plant-based substrate comprises or consists of one or more plant protein flour, concentrate or isolate; preferably the the plant-based substrate comprises or consists of one or more protein isolate; preferably the plant-based substrate comprises or consists of one or more legume protein isolate or pulse protein isolate; even more preferably the plant-based substrate comprises or consists of one or more soy protein isolate.

9. The method of claim 7 or 8, wherein the self-standing gel exhibits a gel hardness above 20 g, determined using a 100 g load cell and an automated texture analyzer with a 6 mm diameter probe moved down from just above the self-standing gel until 5 mm into the sample with a speed of 0.135 mm/s, while continuously measuring the mechanical resistance in grams, as exemplified herein.

10. The method of any of claims 7 - 9, wherein the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis, Streptomyces caniferus or Streptoverticillium ladakanum, preferably the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis.

11. The method of any of claims 7 - 10, wherein the Distribution% of small proteins in the plant-based substrate is determined by size-exclusion chromatography (SEC HPLC) by using the retention time of a standard Cytochrome C (12.38 kDa) to divide the resulting chromatogram into five intervals: Interval 1 with large proteins and Intervals 2-5 with small proteins, integrating the areas of Interval 1 and sum of Intervals 2-5, respectively, and calculating the Distribution% of small proteins (Figure 1).

12. The method of any of claims 7 - 11 , wherein a 6 % dry matter suspension of the plantbased substrate in de-ionized water incubated for 60 min at 25°C has a pH value higher than 7.26, preferably the suspension has a pH value in the range of 7.26 - 10.00, the suspension has a pH value in the range of 7.26 - 9.00, and most preferably the suspension has a pH value in the range of 7.26 - 8.52.

13. Use of one or more plant-based substrate having a Distribution% of small proteins below 33%, determined by size-exclusion chromatography (SEC HPLC), as exemplified herein, in the production of a self-standing gel or a plant-based food, said use comprising an enzymatic treatment step with at least one mature microbial transglutaminase (MTGase) enzyme.

14. The use of claim 13, wherein the plant-based substrate comprises or consists of one or more plant protein flour, concentrate or isolate; preferably the the plant-based substrate comprises or consists of one or more protein isolate; preferably the plant-based substrate comprises or consists of one or more legume protein isolate or pulse protein isolate; even more preferably the plant-based substrate comprises or consists of one or more soy protein isolate.

15. The use of claim 13 or 14, wherein the self-standing gel exhibits a gel hardness above 20 g, determined using a 100g loadcell and an automated texture analyzer with a 6mm diameter probe moved down from just above the self-standing gel until 5 mm into the sample with a speed of 0.135 mm/s, while continuously measuring the mechanical resistance in grams, as exemplified herein.

16. The use of any of claims 13 - 15, wherein the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis, Streptomyces caniferus or Streptoverticillium ladakanum, preferably the at least one mature microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptomyces mobaraensis.

17. The use of any of claims 13 - 16, wherein the Distribution% of small proteins in the plantbased substrate is determined by size-exclusion chromatography (SEC HPLC) by using the retention time of a standard Cytochrome C (12.38 kDa) to divide the resulting chromatogram into five intervals: Interval 1 with large proteins and Intervals 2-5 with small proteins, integrating the areas of Interval 1 and sum of Intervals 2-5, respectively, and calculating the Distribution% of small proteins (Figure 1).

18. The use of any of claims 1 - 5, wherein a 6 % dry matter suspension of the plant-based substrate in de-ionized water incubated for 60 min at 25°C has a pH value higher than 7.26, preferably the suspension has a pH value in the range of 7.26 - 10.00, the suspension has a pH value in the range of 7.26 - 9.00, and most preferably the suspension has a pH value in the range of 7.26 - 8.52.