Method for producing hybrid meat

Abstract

The present invention relates to a method of producing a hybrid meat product comprising textured plant-based protein and animal-derived or cultured meat, which comprises treatment with a transglutaminase.

Description

[0001] HYBRID MEAT

[0002] Reference to sequence listing

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a method of producing a hybrid meat product comprising textured plant-based protein and animal-derived or cultured meat, which comprises treatment with a transglutaminase.

[0006] BACKGROUND OF THE INVENTION

[0007] Replacing traditional meat products with plant protein is one way to mitigate the climate crisis and offer a growing world population sustainably produced protein-rich food. Food production as it appears for now is responsible for a significant portion of the human impact on climate and the environment. Given that the United Nations estimates the human population will reach 9.7 billion in 2050, there is a general agreement that achieving global food security sustainably will not be easy. Livestock accounts for about 15% of the greenhouse gas emission and further livestock takes up 80% of all farmlands for e.g. feed production.

[0008] Legumes, such as soy and pea, are attractive crops for the production of protein-rich food. Other non-animal protein sources are also being used in food production, e.g., from other plants, cultivated meat, filamentous fungi, algae or insects. Initiatives across the globe have been taken to introduce plant-based meat alternatives. It is difficult to achieve though the same taste, juiciness, and texture as is known from traditional animal-derived meat products.

[0009] Cultivated meat obtained by cellular agriculture aims to replicate the organoleptic properties of conventional meat, such as taste, texture, and nutritional content, making it a good substitute for traditional animal-derived meat products (Lew, E.T., Yuen, J.S.K., Zhang, K.L. et al. Chemical and sensory analyses of cultivated pork fat tissue as a flavor enhancer for meat alternatives. Sc / Rep 14, 17643 (2024). https: / / doi.org / 10.1038 / s41598-024-68247-4). Research indicates that cultivated meat can achieve sensory characteristics very similar to conventional meat, which is crucial for consumer acceptance. For instance, studies have shown that the flavor and aroma of cultivated meat can be enhanced to closely resemble those of conventional meat (Lee, M., Park, S., Choi, B. et al. Cultured meat with enriched organoleptic properties by regulating cell differentiation. Nat Commun 15, 77 (2024). https: / / doi.org / 10.1038 / s41467-023-44359-9). Likewise, the nutritional profile can match conventional meat (FDA Cell Culture Consultation No. 001, DOSSIER IN SUPPORT OF THE SAFETY OF GOOD MEAT CULTURED CHICKEN AS A HUMAN FOOD INGREDIENT, March 4, 2022, https: / / www.fda.gov / food / human-food-made-cultured-animal-cells / inventory-completed-pre-market-consultations-human-food-made-cultured-animal-cells).

[0010] Although there is a general interest in lowering meat consumption, it is not an easy task to make the world population change its diet. To achieve a partial substitution of animal-derived protein in the diet with more sustainable plant protein, hybrid foods which blend animal-derived ingredients with plant-based ones could be a useful tool in diet change, helping to bridge the gap between the two markets. Hybrid meat offerings could be considered as a sort of transition product; a food that still tastes, looks and has the texture of meat, but at the same time has an increased content of plant-based ingredients. Such hybrid products can further be associated with positive health claims such as ‘one of your five a day’ or ‘a source of fibre’. Hybrid meat products might be suitable for those consumers who want to try something different and / or lower their meat consumption but are not yet ready to move to meat-free alternatives (Simona Grassa, Hybrid foods: the best of both worlds, New Food Magazine, issue 5, 2020, https: / / www.newfoodmagazine.com / arti-cle / 124250 / hybrid-foods-the-best-of-both-worlds / ).

[0011] So far, hybrid meat products have not had much success on the market. One likely explanation being that these products are still lacking behind traditional animal-derived meat products when it comes to properties such as taste, juiciness, and texture.

[0012] Hybrid meat has been discussed for some time in the literature including efforts to improve the texture.

[0013] In US 2010 / 0166940 A1, it has been discovered that producing an animal meat composition or a simulated animal meat composition which contains no animal meat under conditions of low pH results in a meat composition with improved meat-like qualities. Disclosed is a process for producing a structured plant protein product comprising combining a plant protein material with a pH-lowering agent and extruding the mixture to form a structured plant protein product comprising protein fibers that are substantially aligned. Also disclosed is a process for producing an animal meat composition comprising combining animal meat, a structured plant protein product comprising protein fibers that are substantially aligned, the structured plant protein product comprising an extrudate of plant material, and a pH-lowering agent, and extruding the mixture. The patent application teaches that a variety of ingredients that contain protein may be utilized in an extrusion process to produce structured plant protein products suitable for use in animal meat compositions and simulated animal meat compositions which contain no animal meat. Such ingredients include both plant protein and protein derived from animals such as dairy protein and egg protein, i.e. animal-derived protein which is not animal meat as such. It is envisioned that if the protein-containing starting material is gluten-free, an edible crosslink agent may be utilized to facilitate filament formation during the extrusion process. Nonlimiting examples of suitable crosslink agentsinclude Konjac glucomannan (KGM) flour, edible crosslink agents such as transglutaminase, beta glucan, calcium salts, and magnesium salts.

[0014] US 2008 / 0268112 A1 discloses a ground meat composition comprising structured plant protein products having protein fibers that are substantially aligned, animal meat and a color composition. Also disclosed is a simulated ground meat composition that contains no animal meat, the simulated ground meat composition comprising a structured plant protein product comprising protein fibers that are substantially aligned, the structured plant protein product comprising an extrudate of plant material, and a color composition. The patent application teaches that a variety of ingredients that contain protein may be utilized in a thermal plastic extrusion process to produce structured protein products suitable for use in the ground meat simulated meat compositions that contain no animal meat. Such ingredients include both plant protein and protein derived from animals such as dairy protein, egg protein, meat protein or protein ingredients, i.e. animal-derived protein which is not animal meat as such. It is envisioned that if the protein-containing starting material is gluten-free, an edible crosslinking agent may be utilized to facilitate filament formation during the extrusion process. Nonlimiting examples of suitable crosslinking agents include Konjac glucomannan (KGM) flour, BetaGlucan, transglutaminase, calcium salts, and magnesium salts.

[0015] Santos et al., 2023, Effect of Transglutaminase Treatment on the Structure and Sensory Properties of Rice- or Soy-Based Hybrid Sausages, Foods 2023, 12, 4226, https: / / doi.org / 10.3390 / foods12234226, have investigated the effect of transglutaminase in hybrid emulsified sausages where transglutaminase is used to treat an emulsion made from ground meat, concentrated soy or rice protein, a vegetable fat blend and other ingredients. Santos et al. report a reduction in the heat treatment yield, particularly of the hybrid emulsified sausages made with rice protein, as well as a substantial increase of texture parameters. Santos et al. conclude that the intrinsic compatibility of the selected plant protein and the meat matrix needs to be considered. In conclusion, transglutaminase offers a promising strategy for improving the textural and sensory attributes of hybrid meat products, although the degree of success may vary depending on the type of plant protein used.

[0016] In addition to traditional emulsified sausages as exemplified in Santos et al., 2023, where an emulsion is made from finely milled concentrates of plant protein, ground meat and a vegetable blend, there is a desire to also develop hybrid meat products having a more meat-like consistency such as hybrid burger patties, hybrid meat balls, hybrid schnitzels, or hybrid coarse-type sausages which are not made from an emulsification, but are merely sausages having a good meat-like bite often perceived as more high-quality.

[0017] Extrusion is a thermo-mechanical texturization process used in the food industry to create various textures and shapes of food products by forcing a mixture through a specially designed die under controlled conditions of temperature, pressure, and shear. This process is widely used forproducing snacks, cereals, pasta, and textured plant protein. To produce textured plant protein, the protein is mixed with water and perhaps other ingredients and the mixture is fed into an extruder, which is a machine consisting of a barrel and a screw (or multiple screws) that moves the mixture forward. As the mixture moves through the extruder, it is subjected to high temperatures and shear forces, causing the ingredients to cook and interact with each other. The cooked mixture is forced through a die at the end of the extruder, shaping it into the desired form. The extrudate exits the die and is cooled and cut into pieces of the desired size.

[0018] High-moisture (HM) extrusion) and low-moisture (LM) extrusion are two types of extrusion processes that differ primarily in their moisture content and the resulting texture of the final product. High-moisture extrusion typically involves a higher moisture content, usually ranging from 40% to 70%, and it generally involves lower temperatures and pressures compared to low-moisture extrusion to maintain the higher moisture content. High-moisture extrusion produces a fibrous, meatlike texture. Low-moisture extrusion involves a lower moisture content, typically less than 30%, and it requires higher temperatures and pressures to cook and shape the low-moisture mixture. Low-moisture extrusion produces a more porous and less fibrous texture. The result is often a crunchy or chewy product that requires rehydration before use. LM extruded vegetable protein (also called TVP which is short for texturized vegetable protein) may be formed into various shapes (chunks, flakes, nuggets, grains, and strips) and sizes.

[0019] Shear cell technology is a different process which can also be used to produce textured plant protein. Plant protein, such as from soy, wheat gluten or pea, is mixed with water and perhaps other ingredients and the mixture is subjected to shear forces within a shear cell device. This device typically consists of two concentric cylinders or plates that rotate relative to each other. The applied shear forces align the protein fibers, creating a fibrous structure similar to muscle tissue in animal meat. The mixture is often heated to set the protein structure and then cooled to stabilize the formed fibers. Shear cell technology typically operates with a relatively high moisture content, similar to high-moisture extrusion. The exact moisture content can vary depending on the specific application and desired texture of the final product. The moisture content in shear cell technology generally ranges from 50% to 80%.

[0020] Use of low-moisture extrudate (LME) or high-moisture extrudate (HME) in hybrid meat products has been reported in several studies.

[0021] Baune M-C, Broucke K, Ebert S, Gibis M, Weiss J, Enneking U, Profeta A, Terjung N and Heinz V (2023) Meat hybrids - An assessment of sensorial aspects, consumer acceptance, and nutritional properties, Front. Nutr. 10:1101479, doi: 10.3389 / fnut.2023.1101479, report that data demonstrate that meat hybrids with a high substantial meat substitution level often fail in the market. But at the same time show that a meat hybrid with a relatively high share of 30% plant-basedproteins with peas as a protein source and TVP as a processing method can still attract consumers.

[0022] It is an object of the present invention to provide improved hybrid meat products having a structure resembling traditional high-quality meat products to be able to better meet the quality requirements from consumers wishing to reduce consumption of animal-derived meat.

[0023] SUMMARY OF THE INVENTION

[0024] The invention provides improved hybrid meat products having a high proportion of protein from textured plant-based protein such as TVP (texturized vegetable protein). Such hybrid meat products have a structure which is closer to traditional high-quality meat products if compared with hybrid meat products which are based on a protein-comprising emulsion where the major part of the plant protein is plant protein concentrate or isolate in powdered form.

[0025] Textured plant-based protein such as TVP is not expected to be a good substrate for enzymes as it is specifically processed to be less soluble and therefore less accessible for the enzyme. Surprisingly the inventors found that transglutaminase can improve hybrid products having a high content of textured plant-based protein such as TVP, e.g., by reducing the cooking loss, increasing the hardness, increasing the chewiness, increasing the cohesiveness, increasing the resilience and / or increasing the springiness.

[0026] Due to the similarities between animal-based meat and meat obtained by cellular agriculture, it can be reasonably assumed that with respect to the present invention, there will be no difference in hybrid products prepared with either of animal-derived or cultivated meat combined with textured plant protein. Further, cultivated meat is expected to improve the nutritional profile in a hybrid product to the same degree as animal-derived meat.

[0027] The invention provides a method for producing a hybrid meat product, which method comprises: a) providing a hydrated textured plant-based protein source having a water content of 50-90% (w / w) by optionally hydrating textured plant-based protein in water;

[0028] b) providing finely chopped or minced animal-derived or cultivated meat;

[0029] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0030] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c) and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0031] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging; wherein transglutaminase is added before, during or after any of steps a), b), c), and / or d).The transglutaminase may be preferentially added during the optional hydration in step a), to the binder of step c) during or after its preparation, and / or during or after the mixing of step d).

[0032] Therefore, in one embodiment, the invention provides a method for producing a hybrid meat product, which method comprises:

[0033] a) providing a hydrated textured plant-based protein source having a water content of 50-90% (w / w);

[0034] b) providing finely chopped or minced animal-derived or cultivated meat;

[0035] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0036] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), transglutaminase, and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein; e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging. In another embodiment, the invention provides a method for producing a hybrid meat product, which method comprises:

[0037] a) hydrating a textured plant-based protein source to obtain a hydrated textured plant-based protein source having a water content of 50-90% (w / w), wherein a transglutaminase is added during the hydration;

[0038] b) providing finely chopped or minced animal-derived or cultivated meat;

[0039] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0040] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0041] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging. In yet another embodiment, the invention provides a method for producing a hybrid meat product, which method comprises:

[0042] a) providing a hydrated textured plant-based protein source having a water content of 50-90% (w / w);

[0043] b) providing finely chopped or minced animal-derived or cultivated meat;

[0044] c) providing a binder comprising water, oil, a non-textured plant-based protein source and transglutaminase;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), the binder of step c) and optionally other ingredients, to arrive at a mixture comprising 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein;

[0045] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging. The invention further provides a hybrid meat product comprising 20-80% (w / w) textured plantbased protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein, and also comprising transglutaminase.

[0046] BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 shows the mean hardness for the hybrid sausages N1, N2 and N3 obtained from the texture analyser (Example 5).

[0048] Figure 2 shows the mean cohesiveness for the hybrid sausages N1, N2 and N3 obtained from the texture analyser (Example 5).

[0049] Figure 3 shows the mean adhesiveness for the hybrid sausages N1, N2 and N3 obtained from the texture analyser (Example 5).

[0050] Figure 4 shows the mean chewiness for the hybrid sausages N1, N2 and N3 obtained from the texture analyser (Example 5).

[0051] Figure 5 shows the mean resilience for the hybrid sausages N1, N2 and N3 obtained from the texture analyser (Example 5).

[0052] Figure 6 shows the mean springiness for the hybrid sausages N1, N2 and N3 obtained from the texture analyser (Example 5).

[0053] Sequences:

[0054] SEQ ID NO: 1 is transglutaminase from Streptoverticillium mobaraensis including a signal peptide and a propeptide.

[0055] SEQ I D NO: 2, 3 and 4 are mature forms of the transglutaminase of SEQ I D NO: 1 without signal peptide and propeptide. These sequences vary slightly only with respect to the length at the N-terminal.

[0056] SEQ ID NO: 5 is transglutaminase from Streptoverticillium ladakanum including a signal peptide and a propeptide. Its mature part (amino acids 78-410) has a high sequence identity to SEQ ID NO: 2, 3 and 4 (>93% identity).Definitions:

[0057] AlphaFold structure calculation: AlphaFold version 2 (AlphaFold2, AF2) is a computational method for calculating the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., 2021, Nature 596: 583-589). Three-dimensional structures of millions of polypeptides deposited in the UniProt database have been calculated and deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 algorithm (Varadi et al., 2021, Nucleic Acids Res. 50(D1):D439-D444). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

[0058] In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and calculating structures of new polypeptides at source code repositories such as Github.com under deepmind / alphafold / , using notebooks / AlphaFold.ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github.com under sokryp-ton / ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).

[0059] AlphaFold 2 produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction.

[0060] Enzyme: Transglutaminase means 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. A transglutaminase in the context of the present invention is preferably a microbial transglutaminase. For purposes of the present invention, microbial transglutaminase activity is determined according to the procedure described in the Examples. In one aspect, a 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. The terms “Microbial Transglutaminase” or “MTGase” are synonymous and interchangeable.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, faba beans, 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.

[0061] Textured plant protein is defined herein to cover broadly high-moisture extrudates, low-moisture extrudates and textured plant protein produced using shear cell technology or similar technology where shear forces are used to texturize plant protein creating a fibrous meat-like structure. TVP: Low-moisture extrudate (LME) is sometimes referred to as TVP which is short for texturized vegetable protein.

[0062] Cultivated meat: The term “cultivated meat” means cultivated meat obtained by cellular agriculture.

[0063] 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.

[0064] 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).

[0065] Mature polypeptide: The term “mature transglutaminase” 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 of any SEQ ID NO:1 or SEQ ID NO:5. In another aspect, the mature polypeptide is the polypeptide of any SEQ ID NO:2, SEQ ID NO: 3 and / or SEQ ID NO:4. 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 or two residues 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 propeptide 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 enzymes.

[0066] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0067] 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 etal., 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:

[0068] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment) Structural Similarity: For purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”. A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g., X-ray crystallography or calculated using in silico methods such as AlphaFold 2 (vide supra). The structural similarity between three-dimensional structures may then be determined by the TM-score, which is calculated using the following general formula (Zhang & Skolnick, 2004, Proteins 57:702-710):

[0069] TM -score

[0070]

[0071] where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, dj is the distance between pair / of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition. For the purposes of the present invention, LN is the length of the reference polypeptide:

[0072] TM score >

[0073]

[0074] A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, 1998, Protein Eng., 11:739-747), DALI (Holm and Sander, 1995, Trends Biochem. Sci., 20:478-480), or TM-align (Zhang and Skolnick, 2005, Nucleic Acids Res. 33(7):2302-2309).

[0075] For the purposes of the present invention, TM-align is applied. For convenience, TM-score is integrated in the TM-align software, which is available from the author’s website (zhang-group.org / TM-score / ). The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:

[0076] TMalign <query.pdb> <reference.pdb> -L <length of reference> where <query.pdb> is the name of the PDB file containing coordinates of the query polypeptide, <reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0077] The maximal TM-score is 1, e.g., 1.0, corresponding to identical three-dimensional structures.

[0078] 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.

[0079] 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.

[0080] DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention relates to a method for producing a hybrid meat product, which method comprises:

[0082] a) providing a hydrated textured plant-based protein source having a water content of 50-90% (w / w) by optionally hydrating textured plant-based protein in water;

[0083] b) providing finely chopped or minced animal-derived or cultivated meat;

[0084] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c) and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0085] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging; wherein transglutaminase is added before, during or after any of steps a), b), c), and / or d).

[0086] Preferably, the hybrid meat product comprises 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein. In one embodiment, in step d), a mixture comprising 40-80% (w / w) textured plant-based protein out of total protein and 20-60% (w / w) animal-derived or cultivated meat protein out of total protein is arrived at, and the hybrid meat product comprises 40-80% (w / w) textured plant-based protein out of total protein and 20-60% (w / w) animal-derived or cultivated meat protein out of total protein. In another embodiment, in step d), a mixture comprising 45-80% (w / w) textured plant-based protein out of total protein and 20-55% (w / w) animal-derived or cultivated meat protein out of total protein is arrived at, and the hybrid meat product comprises 45-80% (w / w) textured plant-based protein out of total protein and 20-55% (w / w) animal-derived or cultivated meat protein out of total protein.

[0087] The textured plant-based protein source is preferably obtained using extrusion or shear cell technology, more preferably extrusion.

[0088] Preferably, the textured plant-based protein source is an extruded plant-based protein source. Such textured plant-based protein source may be a plant-based low-moisture extrudate or a plantbased high-moisture extrudate, preferably a plant-based low-moisture extrudate, more preferably a low-moisture extrudate having a moisture content of less than 30%.

[0089] A plant-based low-moisture extrudate is sometimes referred to as a texturized vegetable protein or TVP.

[0090] In a preferred embodiment, the textured plant-based protein source is TVP.

[0091] The textured plant-based protein may be textured protein from any plant.

[0092] The textured plant-based protein may be obtained e.g. from legumes, such as from pulses, e.g. peas, lentils, chickpea, or from oil crops, e.g. soybean, peanuts; from seeds, e.g. hemp, sunflower, flax, sesame, chia; from cereals, e.g. wheat, oats, corn; from pseudocereals, e.g. quinoa; from grasses or pasture legumes, e.g. alfalfa, clover; from any of rapeseed, canola, nuts, vegetables, fruits, mushrooms, cottonseed; or any combination of any of these.In a preferred embodiment, the textured plant-based protein is from a legume. In a more preferred embodiment, the textured plant-based protein is from soy, pea, faba bean, chickpea, lentil, and / or wheat, preferably from soy and / or pea.

[0093] In a preferred embodiment, the textured plant-based protein source is TVP of soy protein and / or a TVP of pea protein.

[0094] In a preferred embodiment, the textured plant-based protein source is TVP made from soy protein concentrate or isolate and / or pea protein concentrate or isolate, preferably TVP made from soy protein concentrate and / or pea protein concentrate.

[0095] In case of using as the textured plant-based protein source a high-moisture extrudate of a plantbased protein or a plant-based protein source which has been textured using shear cell technology, a hydration step is generally not needed.

[0096] The transglutaminase is added before, during or after any of steps a), b), c), and / or d).

[0097] The transglutaminase may be added before, during or after step a) such as before or during hydration, e.g., the transglutaminase may be added to the hydration water before the hydration water is mixed with the textured plant-based protein source, or the transglutaminase may be added after the hydration water has been mixed with the textured plant-based protein source. In these cases, the transglutaminase can work on the hydrated textured plant-based protein during the hydration step. Or the transglutaminase may be added to the hydrated textured plant-based protein source, e.g., after hydration, or if the hydrated textured plant-based protein source is a high-moisture extrudate of a plant-based protein or a plant-based protein source which has been textured using shear cell technology.

[0098] The transglutaminase may be added to the finely chopped or minced animal-derived or cultivated meat of step b).

[0099] The transglutaminase may be added to the binder of optional step c). It may be added before, during or after the preparation of the binder.

[0100] The transglutaminase may be added before, during or after the mixing of step d).

[0101] Transglutaminase may be added at several steps of the method. Transglutaminase may be added, e.g., both during hydration in step a) and during mixing in step d).

[0102] Preferably, the transglutaminase is added before or during the optional hydration in step a), to the binder of optional step c) during or after its preparation, and / or in step d) before or during the mixing.

[0103] In a first preferred embodiment, the transglutaminase is added in step d) before or during the mixing. This embodiment provides a method for producing a hybrid meat product, which method comprises:a) providing a hydrated textured plant-based protein source having a water content of 50-90% (w / w);

[0104] b) providing finely chopped or minced animal-derived or cultivated meat;

[0105] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0106] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), transglutaminase, and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein; and

[0107] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

[0108] In a second preferred embodiment, the transglutaminase is added before or during hydration in step a). This embodiment provides a method for producing a hybrid meat product, which method comprises:

[0109] a) hydrating a textured plant-based protein source to obtain a hydrated textured plant-based protein source having a water content of 50-90% (w / w), wherein a transglutaminase is added before or during the hydration;

[0110] b) providing finely chopped or minced animal-derived or cultivated meat;

[0111] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0112] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0113] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging. Preferably, the textured plant-based protein source is hydrated in water for 10 minutes to 24 hours.

[0114] In a third preferred embodiment, the transglutaminase is added to the binder of step c). This embodiment provides a method for producing a hybrid meat product, which method comprises: a) providing a hydrated textured plant-based protein source having a water content of 50-90% (w / w);

[0115] b) providing finely chopped or minced animal-derived or cultivated meat;

[0116] c) providing a binder comprising water, oil, a non-textured plant-based protein source and transglutaminase;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), the binder of step c) and optionally other ingredients, to arrive at a mixture comprising 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein;

[0117] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging. In some embodiments, the hybrid meat product comprises 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein.

[0118] In one embodiment, in step d), a mixture comprising 40-75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plant-based protein out of total protein, and 20-55%(w / w) animal-derived or cultivated meat protein out of total protein is arrived at, and the hybrid meat product comprises 40-75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plant-based protein out of total protein, and 20-55% (w / w) animal-derived or cultivated meat protein out of total protein.

[0119] In another embodiment, in step d), a mixture comprising 45-75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plant-based protein out of total protein, and 20-50% (w / w) animal-derived or cultivated meat protein out of total protein is arrived at, and the hybrid meat product comprises 40-75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plant-based protein out of total protein, and 20-50% (w / w) animal-derived or cultivated meat protein out of total protein

[0120] The non-textured plant-based protein source is preferably an isolate or a concentrate of a legume protein, more preferably an isolate.

[0121] The non-textured plant-based protein source is preferably an isolate or a concentrate, more preferably an isolate, of protein from soy, pea, faba bean, chickpea and / or lentils, preferably of protein from soy and / or pea, more preferably of protein from soy.

[0122] The binder preferably comprises 60-80% water, 10-20% isolate of a legume protein, 10-20% of a plant oil, and transglutaminase.

[0123] In a preferred embodiment, 5-20% (w / w), preferably 5-15%, of the total protein in the mixture of step d) is from the binder.

[0124] The animal-derived or cultivated meat to be used in the method of the invention is preferably animal-derived, more preferably from beef, pork, chicken, turkey, fish and / or seafood. In a preferred embodiment, the animal-derived meat is not organ meat.Preferably, the hybrid meat product obtained by the method of the invention is a hybrid burger patty, a hybrid sausage, a hybrid nugget, a hybrid schnitzel, a hybrid meatball, a hybrid deli cut, a hybrid fish product, or a hybrid minced meat, more preferably a hybrid burger patty or a hybrid sausage, even more preferably a hybrid burger patty or a hybrid coarse-type sausage, such as a hybrid burger patty or a hybrid coarse-type non-emulsified sausage.

[0125] In a preferred embodiment, the hybrid meat product is a hybrid burger patty.

[0126] In a preferred embodiment, the hybrid meat product obtained by the method of the invention has a total protein content of 10-25% (w / w).

[0127] Preferably, the mixture of the hydrated textured plant-based protein source, the animal-derived or cultivated meat and optionally other ingredients is not an emulsion. Preferably, the hybrid meat product is not an emulsified hybrid meat product.

[0128] In a preferred embodiment, the hybrid meat product does not comprise methylcellulose.

[0129] For the avoidance of any doubt, the transglutaminase has transglutaminase activity.

[0130] Preferably, the transglutaminase is a polypeptide having transglutaminase activity selected from the group consisting of:

[0131] (i) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of a mature polypeptide of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2, (ii) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2, (iii) a polypeptide having a sequence identity to a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably to a mature polypeptide of SEQ ID NO: 1, 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%,

[0132] (iv) a polypeptide having a sequence identity to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably to SEQ ID NO: 3, of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 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%,

[0133] (v) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably SEQ ID NO: 1, and (vi) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of any of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably SEQ ID NO: 3. Preferably, the transglutaminase has a sequence which comprises or consists of a mature polypeptide sequence of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably SEQ ID NO: 1. Preferably, the transglutaminase has a sequence which comprises or consists of the sequence of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably SEQ ID NO: 3.

[0134] Preferably, a mature polypeptide sequence of SEQ ID NO: 1 is amino acids 73-405, amino acids 75-405 and / or amino acids 76-405 of SEQ ID NO: 1, i.e. the polypeptides having an amino acid sequences as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. When the transglutaminase of SEQ ID NO:1 is produced in vivo, at least these 3 different mature forms are produced. More preferably, a mature polypeptide sequence of SEQ ID NO: 1 is amino acids 75-405 of SEQ ID NO: 1, i.e. the polypeptide having an amino acid sequences as shown in SEQ ID NO: 3.

[0135] Preferably, a mature polypeptide sequence of SEQ ID NO: 5 is amino acids 78-410 of SEQ ID NO: 5.

[0136] Preferably, the transglutaminase is a microbial transglutaminase, more preferably a microbial transglutaminase derived from Streptoverticillium mobaraensis, Streptoverticillium caniferus or Streptoverticillium ladakanum, preferably from Streptoverticillium mobaraensis.

[0137] Preferably, the transglutaminase is a mature transglutaminase.

[0138] Preferably, the transglutaminase is an isolated transglutaminase.

[0139] In a preferred embodiment, the transglutaminase is added at a concentration of 0.1-100 TGHll(A) of transglutaminase per g of total protein, preferably 0.5-50, more preferably 1-25 TGHll(A) per g of total protein.

[0140] The present invention also relates to a hybrid meat product having been produced by the method of the invention.

[0141] The present invention also relates to a hybrid meat product comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein, where at least part of the protein is cross-linked.

[0142] Preferably, the cross-linked protein of such hybrid meat product is transglutaminase-treated protein.The present invention also relates to a hybrid meat product comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein, and also comprising transglutaminase.

[0143] Preferably, the hybrid meat product of the invention comprises 40-80% (w / w), more preferably 45-80%, plant-based protein out of total protein.

[0144] More preferably, the hybrid meat product of the invention comprises 40-80% (w / w), more preferably 45-80%, textured plant-based protein out of total protein.

[0145] Preferably, the hybrid meat product of the invention comprises 20-60% (w / w), preferably 20-55%, animal-derived or cultivated meat protein out of total protein.

[0146] In some embodiments, the hybrid meat product of the invention comprises 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein.

[0147] Preferably, the hybrid meat product of the invention is a hybrid burger patty, a hybrid sausage, a hybrid nugget, a hybrid schnitzel, a hybrid meatball, a hybrid deli cut, a hybrid fish product, or a hybrid minced meat, more preferably a hybrid burger patty or a hybrid sausage, such as a hybrid burger patty or a hybrid coarse-type sausage, even more preferably a hybrid burger patty or a hybrid coarse-type non-emulsified sausage, such as a hybrid burger patty.

[0148] The hybrid meat product of the invention preferably has a total protein content of 10-25% (w / w). In a preferred embodiment, the textured plant-based protein in the hybrid meat product of the invention is extruded.

[0149] In another preferred embodiment, the textured plant-based protein in the hybrid meat product of the invention is low-moisture extruded plant protein, preferably low-moisture extruded plant protein having a moisture content of less than 30%.

[0150] In a preferred embodiment, the textured plant-based protein in the hybrid meat product of the invention is protein from TVP, preferably from hydrated TVP.

[0151] Preferably, the textured plant-based protein in the hybrid meat product of the invention is from a legume.

[0152] More preferably, the textured plant-based protein in the hybrid meat product of the invention is from soy, pea, faba bean, chickpea, lentil, and / or wheat, preferably from soy and / or pea.

[0153] Preferably, the animal-derived or cultivated meat protein in the hybrid meat product of the invention is animal-derived, more the animal-derived or cultivated meat protein preferably from beef, pork, chicken, fish and / or seafood.

[0154] Preferably, the hybrid meat product of the invention comprises 0.1-100 TGHll(A) of transglutaminase per g of total protein, preferably 0.5-50, more preferably 1-25 TGHll(A) per g of total protein.In some embodiments, the transglutaminase in the hybrid meat product of the invention is a polypeptide having transglutaminase activity selected from the group consisting of:

[0155] (i) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of a mature polypeptide of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2, (ii) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2, (iii) a polypeptide having a sequence identity to a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably to a mature polypeptide of SEQ ID NO: 1, 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%,

[0156] (iv) a polypeptide having a sequence identity to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably to SEQ ID NO: 3, 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%,

[0157] (v) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably SEQ ID NO: 1, and (vi) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of any of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably SEQ ID NO: 3. In one preferred embodiment, the transglutaminase in the hybrid meat product of the invention has a sequence which comprises or consists of a mature polypeptide sequence of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably SEQ ID NO: 1.

[0158] In another preferred embodiment, the transglutaminase in the hybrid meat product of the invention has a sequence which comprises or consists of the sequence of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably SEQ ID NO: 3.

[0159] Preferably, the transglutaminase in the hybrid meat product of the invention is a microbial transglutaminase, more preferably a microbial transglutaminase derived from Streptoverticilliummobaraensis, Streptoverticillium caniferus or Streptoverticillium ladakanum, preferably from Streptoverticillium mobaraensis.

[0160] Preferably, the hybrid meat product of the invention is not a hybrid emulsified meat product. Preferably, the hybrid meat product of the invention does not comprise methylcellulose.

[0161] The hybrid meat product of the invention or which has been produced according to the method of the invention will preferably have a reduced cooking loss of at least 5%, such as at least 10%, compared to a hybrid meat product obtained using the same method but without addition of a transglutaminase.

[0162] The skilled person will know how to determine the cooking loss. It may preferably be determined in line with what is described in the Examples, in particular Example 2.

[0163] The hybrid meat product of the invention or which has been produced according to the method of the invention, preferably after cooking, will preferably have a hardness, preferably a hardness which is measured instrumentally, which is at least 10% increased, preferably at least 20% increased, such as at least 30%, at least 40% or at least 50% increased, compared to a hybrid meat product obtained using the same method but without addition of a transglutaminase.

[0164] The skilled person will know how to determine the hardness. Hardness, in particular instrumentally determined hardness, may preferably be determined in line with what is described in the Examples. Instrumentally determined means that it is determined using an instrument as opposed to sensorically determined hardness, using, e.g., a panel of sensorically trained individuals. The hybrid meat product of the invention or which has been produced according to the method of the invention, preferably after cooking, will preferably have a chewiness, preferably a chewiness which is measured instrumentally, which is at least 20% increased, such as at least 30 increased, compared to a hybrid meat product obtained using the same method but without addition of a transglutaminase.

[0165] The skilled person will know howto determine the chewiness. Chewiness, in particular instrumentally determined chewiness, may preferably be determined in line with what is described in the Examples.

[0166] The hybrid meat product of the invention or which has been produced according to the method of the invention, preferably after cooking, will preferably have a resilience, preferably a resilience which is measured instrumentally, which is at least 25% increased, compared to a hybrid meat product obtained using the same method but without addition of a transglutaminase.

[0167] The skilled person will know how to determine the resilience. Resilience, in particular instrumentally determined resilience, may preferably be determined in line with what is described in the Examples.The hybrid meat product of the invention or which has been produced according to the method of the invention, preferably after cooking, will preferably have a cohesiveness, preferably a cohesiveness which is measured instrumentally, which is at least 5% increased, such as at least 10% increased, compared to a hybrid meat product obtained using the same method but without addition of a transglutaminase.

[0168] The skilled person will know how to determine the cohesiveness. Cohesiveness, in particular instrumentally determined cohesiveness, may preferably be determined in line with what is described in the Examples.

[0169] Disclosed herein is also hybrid meat products where plant protein such as legume protein is combined with protein from filamentous fungi, algae or insects, as well as precision-fermented proteins and / or 3D printed protein networks. Disclosed is also hybrid meat products where animal-derived or cultivated meat is combined with protein from filamentous fungi, algae or insects, as well as precision-fermented proteins and / or 3D printed protein networks.

[0170] Transglutaminase

[0171] In a preferred embodiment, the present invention relates to use of transglutaminase polypeptides having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2.

[0172] In another preferred embodiment, the present invention relates to use of transglutaminase polypeptides having a sequence identity to a 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%. In another preferred embodiment, the transglutaminase polypeptide has been isolated.

[0173] A mature transglutaminase polypeptide of the present invention preferably comprises or consists of the amino acid sequence of a mature transglutaminase 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 or an allelic variant thereof; or is a fragment thereof having transglutaminase activity. In another aspect, the mature transglutaminase polypeptide comprises or consists of a mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0174] Yet another preferred embodiment of present invention relates to use of transglutaminase polypeptide variants of 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 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 transglutaminase 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.

[0175] 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 aminoterminal 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.

[0176] 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.

[0177] 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., Low-man 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).

[0178] 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.

[0179] The transglutaminase 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.

[0180] The transglutaminase 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 al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0181] 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. 76: 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; Carteret al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0182] Sources of transglutaminase polypeptidesA transglutaminase polypeptide to be used according to 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 MTGase polypeptide obtained from a given source is secreted extracellularly.

[0183] The 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 Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.

[0184] In one embodiment, the 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 li-cheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis polypeptide.

[0185] In another embodiment, the MTGase polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide. In another embodiment, the MTGase polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces mobaraensis, or Streptomyces lividans polypeptide.

[0186] In a preferred embodiment, the microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptoverticillium mobaraensis, Streptoverticillium caniferus or Streptoverticillium ladakanum, preferably the microbial transglutaminase (MTGase) enzyme comprises or consists of a mature microbial transglutaminase (MTGase) enzyme derived from Streptoverticillium mobaraensis.

[0187] The microbial transglutaminase (MTGase) polypeptide may be a fungal polypeptide. For example, the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccha-romyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceri-poriopsis, 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, Phan-erochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Tricho-derma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide.

[0188] In another embodiment, the MTGase polypeptide is a Saccharomyces carlsbergensis, Saccharo-myces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluy-veri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.

[0189] In another aspect, the MTGase polypeptide is an Acremonium cellulolyticus, Aspergillus acule-atus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium panni-cola, 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 mi-crospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

[0190] 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.

[0191] 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 Mikroorganis-men und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). The 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 beisolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

[0192] Methods of Production

[0193] Methods of producing a 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 expressing the transglutaminase polypeptide to be used according to the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.PREFERRED EMBODIMENTS

[0198] 1. A method for producing a hybrid meat product, which method comprises:

[0199] a) providing a hydrated textured plant-based protein source having a water content of 50- 90% (w / w) by optionally hydrating textured plant-based protein in water;

[0200] b) providing finely chopped or minced animal-derived or cultivated meat;

[0201] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0202] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0203] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging;

[0204] wherein transglutaminase is added before, during or after any of steps a), b), c), and / or d).

[0205] 2. The method of embodiment 1, wherein the transglutaminase is added before or during the optional hydration in step a), to the binder of optional step c) during or after its preparation, and / or in step d) before or during the mixing.

[0206] 3. A method for producing a hybrid meat product, which method comprises:

[0207] a) providing a hydrated textured plant-based protein source having a water content of 50- 90% (w / w);

[0208] b) providing finely chopped or minced animal-derived or cultivated meat;

[0209] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0210] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), transglutaminase, and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0211] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

[0212] 4. A method for producing a hybrid meat product, which method comprises:a) hydrating a textured plant-based protein source to obtain a hydrated textured plant-based protein source having a water content of 50-90% (w / w), wherein a transglutaminase is added before or during the hydration;

[0213] b) providing finely chopped or minced animal-derived or cultivated meat;

[0214] c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;

[0215] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;

[0216] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

[0217] 5. The method of the preceding embodiment, wherein the textured plant-based protein source is hydrated in water for 10 minutes to 24 hours.

[0218] 6. The method of any of the preceding embodiments, wherein the hybrid meat product comprises 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein.

[0219] 7. The method of any of the preceding embodiments, where in step d), a mixture comprising 40-80% (w / w) textured plant-based protein out of total protein and 20-60% (w / w) animal- derived or cultivated meat protein out of total protein is arrived at, and wherein the hybrid meat product comprises 40-80% (w / w) textured plant-based protein out of total protein and 20-60% (w / w) animal-derived or cultivated meat protein out of total protein.

[0220] 8. The method of any of the preceding embodiments, where in step d), a mixture comprising 45-80% (w / w) textured plant-based protein out of total protein and 20-55% (w / w) animal- derived or cultivated meat protein out of total protein is arrived at, and wherein the hybrid meat product comprises 45-80% (w / w) textured plant-based protein out of total protein and 20-55% (w / w) animal-derived or cultivated meat protein out of total protein.

[0221] 9. A method for producing a hybrid meat product, which method comprises:

[0222] a) providing a hydrated textured plant-based protein source having a water content of 50- 90% (w / w);

[0223] b) providing finely chopped or minced animal-derived or cultivated meat;c) providing a binder comprising water, oil, a non-textured plant-based protein source and transglutaminase;

[0224] d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein;

[0225] e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

[0226] 10. The method of any of the preceding embodiments, where the binder comprises 60-80% water, 10-20% isolate of a legume protein and 10-20% of a plant oil.

[0227] 11. The method of any of the preceding embodiments, where 5-20% (w / w), preferably 5-15%, of the total protein in the mixture of step d) is from the binder.

[0228] 12. The method of any of the preceding embodiments, wherein the hybrid meat product comprises 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non- textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein.

[0229] 13. The method of any of the preceding embodiments, where in step d), a mixture comprising 40-75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plant-based protein out of total protein, and 20-55%(w / w) animal-derived or cultivated meat protein out of total protein is arrived at, and wherein the hybrid meat product comprises 40- 75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plantbased protein out of total protein, and 20-55% (w / w) animal-derived or cultivated meat protein out of total protein.

[0230] 14. The method of any of the preceding embodiments, where in step d), a mixture comprising 45-75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plant-based protein out of total protein, and 20-50% (w / w) animal-derived or cultivated meat protein out of total protein is arrived at, and wherein the hybrid meat product comprises 40- 75% (w / w) textured plant-based protein out of total protein, 5-35% (w / w) non-textured plantbased protein out of total protein, and 20-50% (w / w) animal-derived or cultivated meat protein out of total protein.15. The method of any of the preceding embodiments, where the textured plant-based protein source is obtained using extrusion or shear cell technology, preferably using extrusion.

[0231] 16. The method of any of the preceding embodiments, where the textured plant-based protein source is an extruded plant-based protein source.

[0232] 17. The method of any of the preceding embodiments, where the textured plant-based protein source is a low-moisture extrudate, preferably a low-moisture extrudate having a moisture content of less than 30%.

[0233] 18. The method of any of the preceding embodiments, where the textured plant-based protein source is TVP.

[0234] 19. The method of the preceding embodiment, where TVP means texturized vegetable protein.

[0235] 20. The method of any of the preceding embodiments, where the textured plant-based protein is from a legume.

[0236] 21. The method of any of the preceding embodiments, where the textured plant-based protein is from soy, pea, faba bean, chickpea, lentil, and / or wheat, preferably from soy and / or pea.

[0237] 22. The method of any of the preceding embodiments, where the textured plant-based protein source is TVP of soy protein and / or TVP of pea protein.

[0238] 23. The method of any of the preceding embodiments, where the textured plant-based protein source is TVP made from soy protein concentrate or isolate and / or pea protein concentrate or isolate, preferably TVP made from soy protein concentrate and / or pea protein concentrate.

[0239] 24. The method of any of the preceding embodiments, where the animal-derived or cultivated meat protein is animal-derived, preferably from beef, pork, chicken, turkey, fish and / or seafood.

[0240] 25. The method of any of the preceding embodiments, where the non-textured plant-based protein source is an isolate or a concentrate of a legume protein, preferably an isolate.The method of any of the preceding embodiments, where the non-textured plant-based protein source is an isolate or a concentrate, preferably an isolate, of protein from soy, pea, faba bean, chickpea and / or lentils, more preferably of protein from soy and / or pea, even more preferably of protein from soy.

[0241] The method of any of the preceding embodiments, wherein the transglutaminase is a polypeptide having transglutaminase activity selected from the group consisting of:

[0242] (i) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of a mature polypeptide of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2,

[0243] (ii) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2,

[0244] (iii) a polypeptide having a sequence identity to a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably to a mature polypeptide of SEQ ID NO: 1, 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%,

[0245] (iv) a polypeptide having a sequence identity to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably to SEQ ID NO: 3, 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%, (v) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably SEQ ID NO: 1, and(vi) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of any of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably SEQ ID NO: 3.

[0246] 28. The method of any of the preceding embodiments, wherein the transglutaminase has a sequence which comprises or consists of a mature polypeptide sequence of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably SEQ ID NO: 1.

[0247] 29. The method of any of the preceding embodiments, wherein the transglutaminase has a sequence which comprises or consists of the sequence of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably SEQ ID NO: 3.

[0248] 30. The method of any of the preceding embodiments wherein the transglutaminase is a microbial transglutaminase, preferably a microbial transglutaminase derived from Streptoverticillium mobaraensis, Streptoverticillium caniferus or Streptoverticillium ladakanum, preferably from Streptoverticillium mobaraensis.

[0249] 31. The method of any of the preceding embodiments, wherein the transglutaminase is a mature transglutaminase.

[0250] 32. The method of any of the preceding embodiments, wherein the transglutaminase is an isolated transglutaminase.

[0251] 33. The method of any of the preceding embodiments, where the transglutaminase is added at a concentration of 0.1-100 TGHll(A) of transglutaminase per g of total protein, preferably 0.5- 50, more preferably 1-25 TGHll(A) per g of total protein.

[0252] 34. The method of any of the preceding embodiments, wherein the hybrid meat product is a hybrid burger patty, a hybrid sausage, a hybrid nugget, a hybrid schnitzel, a hybrid meatball, a hybrid deli cut, a hybrid fish product, ora hybrid minced meat, preferably wherein the hybrid meat product is a hybrid burger patty or a hybrid sausage, more preferably wherein the hybrid meat product is a hybrid burger patty or a hybrid coarse-type sausage, even more preferably wherein the hybrid meat product is a hybrid burger patty or a hybrid coarse-type nonemulsified sausage, most preferably wherein the hybrid meat product is a hybrid burger patty.35. The method of any of the preceding embodiments, wherein the hybrid meat product has a total protein content of 10-25% (w / w).

[0253] 36. The method of any of the preceding embodiments, wherein the mixture of step d) is not an emulsion.

[0254] 37. The method of any of the preceding embodiments, wherein the hybrid meat product is not a hybrid emulsified meat product.

[0255] 38. The method of any of the preceding embodiments, wherein the hybrid meat product does not comprise methylcellulose.

[0256] 39. A hybrid meat product produced by any of the preceding embodiments.

[0257] 40. A hybrid meat product comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein, where at least part of the protein is cross-linked.

[0258] 41. The hybrid meat product of the preceding embodiment, where the cross-linked protein is transglutaminase-treated protein.

[0259] 42. A hybrid meat product comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein, and also comprising transglutaminase.

[0260] 43. The hybrid meat product of any of the three preceding embodiments, which comprises 40- 80% (w / w), preferably 45-80%, plant-based protein out of total protein.

[0261] 44. The hybrid meat product of any of the four preceding embodiments, which comprises 40-80% (w / w), preferably 45-80%, textured plant-based protein out of total protein.

[0262] 45. The hybrid meat product of any of the five preceding embodiments, which comprises 20-60% (w / w), preferably 20-55%, animal-derived or cultivated meat protein out of total protein.The hybrid meat product of any of the six preceding embodiments, which comprises 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein.

[0263] The hybrid meat product of any of the seven preceding embodiments, which is a hybrid burger patty, a hybrid sausage, a hybrid nugget, a hybrid schnitzel, a hybrid meatball, a hybrid deli cut, a hybrid fish product, or a hybrid minced meat, preferably a hybrid burger patty or a hybrid sausage, more preferably a hybrid burger patty or a hybrid coarse-type sausage, even more preferably a hybrid burger patty or a hybrid coarse-type non-emulsified sausage, most preferably a hybrid burger patty.

[0264] The hybrid meat product of any of the eight preceding embodiments having a total protein content of 10-25% (w / w).

[0265] The hybrid meat product of any of the nine preceding embodiments, where the textured plantbased protein is extruded.

[0266] The hybrid meat product of any of the ten preceding embodiments, where the textured plantbased protein is low-moisture extruded plant protein, preferably low-moisture extruded plant protein having a moisture content of less than 30%.

[0267] The hybrid meat product of any of the 11 preceding embodiments, where the textured plantbased protein is protein from TVP, preferably from hydrated TVP.

[0268] The hybrid meat product of the preceding embodiment where TVP means texturized vegetable protein.

[0269] The hybrid meat product of any of the 13 preceding embodiments, where the textured plantbased protein is from a legume.

[0270] The hybrid meat product of any of the 14 preceding embodiments, where the textured plantbased protein is from soy, pea, faba bean, chickpea, lentil, and / or wheat, preferably from soy and / or pea.55. The hybrid meat product of any of the 15 preceding embodiments, where the animal-derived or cultivated meat protein is animal-derived, preferably from beef, pork, chicken, fish and / or seafood.

[0271] 56. The hybrid meat product of any of the 16 preceding embodiments, which comprises 0.1-100 TGHll(A) of transglutaminase per g of total protein, preferably 0.5-50, more preferably 1-25 TGHll(A) per g of total protein.

[0272] 57. The hybrid meat product of any of the 17 preceding embodiments, wherein the transglutaminase is a polypeptide having transglutaminase activity selected from the group consisting of:

[0273] (i) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of a mature polypeptide of any of SEQ ID NO: 1 or SEQ ID NO: 5, preferably a mature polypeptide of SEQ ID NO: 1, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2,

[0274] (ii) a polypeptide having a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably the polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold, preferably by Alphafold2 or newer, most preferably by Alphafold2,

[0275] (iii) a polypeptide having a sequence identity to a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably to a mature polypeptide of SEQ ID NO: 1, 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%,

[0276] (iv) a polypeptide having a sequence identity to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably to SEQ ID NO: 3, 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%,(v) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from a mature polypeptide of any of SEQ ID NO:1 or SEQ ID NO:5, preferably SEQ ID NO: 1, and (vi) a polypeptide differing by at most 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of any of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably SEQ ID NO: 3.

[0277] 58. The hybrid meat product of any of the 18 preceding embodiments, wherein the transglutaminase has a sequence which comprises or consists of a mature polypeptide sequence of any of SEQ I D NO: 1 or SEQ I D NO: 5, preferably SEQ I D NO: 1.

[0278] 59. The hybrid meat product of any of the 19 preceding embodiments, wherein the transglutaminase has a sequence which comprises or consists of the sequence of any of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, preferably SEQ ID NO: 3.

[0279] 60. The hybrid meat product of any of the 20 preceding embodiments, where the transglutaminase is a microbial transglutaminase, preferably a microbial transglutaminase derived from Streptoverticillium mobaraensis, Streptoverticillium caniferus or Streptoverticillium ladakanum, preferably from Streptoverticillium mobaraensis.

[0280] 61. The hybrid meat product of any of the 21 preceding embodiments, which is not a hybrid emulsified meat product.

[0281] 62. The hybrid meat product of any of the 22 preceding embodiments, which does not comprise methylcellulose.

[0282] EXAMPLES

[0283] Summary of examples

[0284] There are several challenges connected with developing high quality plant-based meat products, also including hybrid products containing protein sources from both meat and plant. One key challenge is the lack of good texture and chewing experience in these products.

[0285] In the present examples, we have developed recipes for producing hybrid burger patties and hybrid coarse-type sausages comprising a high amount of textured vegetable protein and investigated if use of MTGase can modify the textural properties of such products. Examples 1-3 show that the addition of MTGase in hybrid burger patties can improve the textural properties and lowerthe cooking loss. Examples 4-5 show that addition of MTGase to coarse-type hybrid sausages can increase the hardness.

[0286] Hardness in the context of using plant proteins for meat analogue products is perceived as a quality enhancing the chewing experience. Meat is often perceived as more resistant to the oral mastication while plant-based meat analogue products disrupt or disintegrate faster in the mouth. Meat can have a harder and chewier texture, and a generally accepted way to evaluate this is hardness analysis obtained by instrumental texture analysis. In this case hardness is to be understood as a positive and meaty like term. The present examples show that the use of MTGase can be a way to increase the hardness and thereby advance the chewing experience of hybrids. Furthermore, using MTGase and thereby enhancing the structural properties makes it possible to potentially avoid undesirable E-numbers such as hydrocolloids, methyl cellulose and / or phosphate.

[0287] MTGase activity assay:

[0288] T ransglutaminase 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). 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.

[0289] 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 is expressed as TGHU(A).

[0290] Enzyme:

[0291] Microbial transglutaminase (MTGase) from Streptoverticillium mobaraensis. The MTGase used is a mature transglutaminase.

[0292] Substrates used in Examples 1-3:Hybrid burger patties with plant and animal-based protein were prepared to evaluate the effect of microbial transglutaminase in these products. All samples were prepared with a final total protein content of 20%, thereof 25-75% animal protein and equally 75-25% plant protein.

[0293] The source of plant protein was a blend of two commercially available low moisture extrudates (TVP) from soy protein concentrate (SPC) (approximately 70% protein), namely PurePro Soy 70T Granule 300 and PurePro Soy 70T Flake 400. The blend consisted of 75% granules and 25% flakes. From here on referred to as SPC TVP or simply as TVP.

[0294] Additionally, soy protein isolate (SPI) (90% protein) Profam 974 was added to some of the samples (sample 9, 10, 13 and 14). The source of animal protein was commercially available organic minced beef (8-12% fat).

[0295] Example 1: Preparing hybrid burger patties with plant-based protein and animal-based protein

[0296] MTGase can be added in three different steps: 1) mixing, 2) hydration water and 3) in a binder. An overview of which step MTGase was added in the different samples can be found in Table 1 below.

[0297] The burger patties corresponding to sample 1-12 were prepared as follows:

[0298] 1. The TVP was rehydrated with tap water in the ration 1 :3 (TVP:water). Water and TVP was mixed for 15 minutes in a mixing bowl with a hook. After mixing, the mixture of TVP and water was transferred to a separate container to continue rehydration without mixing for an additional 15 minutes.

[0299] a. Sample 12: MTGase added with the hydration water in this step.

[0300] 2. Minced beef meat and the hydrated TVP was pushed through a meat grinder.

[0301] 3. The ground beef and TVP was mixed with a small amount of salt in a mixing bowl for 5 minutes.

[0302] a. Sample 2, 4, 6, 8 and 10: MTGase was added to the ground beef and TVP in this step before mixing.

[0303] b. Sample 9-10: SPI solution (22% SPI and 78% water) also added to the ground beef and TVP in this step before mixing.

[0304] 4. Finally, the patties were portioned into 50 g and shaped to a burger of 60 mm diameter and 15 mm height. The final patties were stored at 5°C overnight.The burger patties corresponding to sample 13-14 were prepared as follows:

[0305] 1. The TVP was rehydrated with tap water in the ration 1 :3 (TVP:water). Water and TVP was mixed for 15 minutes in a mixing bowl with a hook. After mixing, the mixture of TVP and water was transferred to a separate container to continue rehydration without mixing for an additional 15 minutes.

[0306] 2. Minced beef meat and the hydrated TVP was pushed through a meat grinder.

[0307] 3. In a separate mixing bowl, a binder was prepared consisting of 70% water, 15% oil, 15% SPI and (in sample 14) MTGase.

[0308] 4. The ground beef and TVP was mixed with the binder and a small amount of salt in a mixing bowl for 5 minutes.

[0309] 5. Finally, the patties were portioned into 50 g and shaped to a burger of 60 mm diameter and 15 mm height. The final patties were stored at 5°C overnight.

[0310] We have throughout the preparation of the hybrid patties secured minimum mechanical impact to maintain a high quality coarse product resembling meat.

[0311] In Table 1 below it is shown approximately how much of the total protein originates from animal protein, TVP and SPI in each sample.

[0312] Table 1:

[0313]

[0314]

[0315] Samples with MTGase added contained 5 TGHU(A) / g protein.

[0316] In Table 2 below, the relative amounts of each component for all samples are shown.

[0317] Table 2:

[0318]

[0319] aTVP:water mix in 1:3,b22% SPI and 78% water,c15% SPI, 15% canola oil and 70% water. The minced beef was 15-20% protein, the hydrated TVP 17-18% protein, the SPI solution added to samples 9-10 appr. 20% protein, and the SPI binder added to samples 13-14 was 13-14% protein.Example 2: Reduced cooking loss and shrinkage of hybrid patties with plant protein and animal-based protein when treated with microbial transglutaminase

[0320] The burger patties were cooked, and weighed before and after cooking, to examine the amount of water and fat lost during cooking.

[0321] The patties were cooked using following procedure:

[0322] • One tablespoon of canola oil was added to a frying pan and the pan was heated up for 5 minutes at medium heat

[0323] Patties were added to the warm pan, 3 patties pr. pan

[0324] • The patties were cooked for 5 minutes on each side

[0325] Cooking loss was calculated:

[0326] Weight of raw patty: W1

[0327] Weight of fried patty: W2

[0328] Cooking loss % was calculated using the equation below:

[0329] 100

[0330]

[0331] Table 3: Calculated cooking loss of hybrid products with and without MTGase based on weight before and after cooking. The cooking loss was decreased with addition of MTGase

[0332]

[0333]

[0334] After cooking it was observed that shrinkage was reduced in samples containing MTGase. From the calculated cooking loss it appears that MTGase can reduce the cooking loss in hybrid products. In the sample containing 75% plant based protein and 25% animal based protein (sam- pie 7 and 8) cooking loss was reduced from 19.4% to 15.4% with addition of MTGase in the mixing step, corresponding to a reduction of around 20% in cooking loss. Addition of MTGase can thereby prevent losing high amount of water and fat upon cooking. The effect is apparent both when MTGase is added in the mixing step and in the binder. Reduced cooking loss was not observed when the MTGase was added in hydration.

[0335] Example 3: Textural properties of hybrid patties altered with MTGase

[0336] Texture analysis of the cooked patties was performed on Stable Microsystems TST A1 TA. XT Plus Texture Analyzer with SMS P / 100 probe and the following settings:

[0337]

[0338] The outcome of the analysis was the parameter “Hardness” which is defined as the highest pos- itive peak force in grams measured during first compression.As can be seen in Table 4 below, the following key improvements on the hardness in hybrid burger patties were observed with addition of MTGase:

[0339] 1) A clear increase in hardness with recipe containing above 50% plant protein. In the samples with 50:50 animakplant protein the hardness was increased with almost 30% in the sample containing MTGase (sample 6) compared to the sample without (sample 5). And the effect is very clear in the samples with 25% animal based protein and 75% plant protein where the hardness was increased with around 80% in the sample with MTGase (sample 8) compared to the sample without enzyme (sample 7). No effect was observed in the samples with 75:25 animakplant (sample 3 and 4) when adding the MTGase..

[0340] 2) Adding MTGase in the TVP hydration step compared to the mixing step increases the effect of MTGase. Comparing the samples containing 50:50 meatplant where MTGase is added in the hydration step (sample 11 and 12 hardness increased with 140% with addition of MTGase) versus the final mixing (sample 5 and 6 hardness increased with 29% with addition of MTGase) surprisingly demonstrates that in which processing step the MTGase is added has a big effect on the final hybrid product.

[0341] 3) With the recipe containing 75% animal protein and 25% plant protein from SPI, we can by adding MTGase obtain hardness on par with regular 100% meat, (sample 1 and 10).

[0342] 4) In this example we saw that it was possible to increase hardness with MTGase of hybrid meat products containing only TVP as the plant protein source (sample 1-8 and 11-12). This was surprising because TVP is made of insoluble proteins that are less accessible to the enzyme compared to the proteins in protein powders like SPI.

[0343] Table 4:

[0344]

[0345]

[0346] Example 4: Method of preparation of coarse type sausages with meat and plant protein, using transglutaminase

[0347] The aim of this example was to study the effect of MTGase in high quality coarse type hybrid sausages with textured plant proteins contributing to a meat-like structure in the final product. The protein in TVP is very different regarding structure and solubility compared to proteins in powders so it was of great interest to investigate if the enzyme would have an effect on such products. Hybrid coarse type sausages containing TVP were prepared to evaluate the effect of MTGase on cooking loss and texture in these products. The sausages contained between 11.5 and 13.5% protein, of which 44 to 53% was plant protein, and 47 to 56% was animal based protein. All sausages contained around 15% fat.

[0348] Similar to a high-quality meat sausage process, we have throughout the preparation of the hybrid products secured minimum mechanical impact to maintain the high-quality coarse product. The source of plant protein was a commercially available pea based TVP NUTRALYS® T70S with around 70% protein and a soy protein concentrate, Arcon S IP with around 66% protein. The source of animal protein was lean pork shank (18.5%, 12.5% fat) and fatty pork shank (12.6% protein, 44.2% fat)

[0349] Throughout the process steps 1 to 5, the temperature was kept bellow 10° C.

[0350] 1. Textured pea protein concentrate (TVP) was hydrated in water

[0351] 2. Meat and fat cuts were ground in a meat grinder

[0352] 3. In a bowl chopper:

[0353] a. Half of the water / ice was added together with the ground meat, salt, preservatives, stabilizing salts, spices, flavours, aromas, and coloring at 10-20 RPM knife cutting speed.b. The other half of the water / ice was added together with the soy protein concentrate (SPC), the starch, the hydrated TVP and the microbial transglutaminase (MTGase) at 80-130 RPM chopping speed.

[0354] c. Finally, the ground fat was added and mixed gently to preserve the structure of the sausages.

[0355] 4. The dough was filled into 22-24 mm natural sausage casing using a sausage filler, to a final weigh of 65 g per sausage.

[0356] 5. Cased sausages were cooked and cooled according to the cooking program bellow. 1) Heating at a chamber temperature of 50°C for 26 minutes.

[0357] 2) Drying at a chamber temperature of 52°C for 15 minutes.

[0358] 3) Smoking at a chamber temperature of 50°C for 13 minutes.

[0359] 4) Boiling at a chamber temperature of 85°C for 20 minutes.

[0360] 5) Boiling at a chamber temperature of 80°C for 10 minutes.

[0361] 6) Venting at a chamber temperature of 80°C for 2 minutes.

[0362] 7) Spraying for 8 minutes, after which the sausages are moved to the cooler chamber. The produced sausages had between 11.5 to 13.5% protein, of which 44 to 53% was plant protein, and 47 to 56% was animal based protein. Besides, the sausages had 40 to 43% dry matter after cooking. Formulations N1 and N3 contain the same ingredients except for the enzyme MTGase that was only present in N3. Formulation N2 had the lowest total protein content and highest animal to plant protein ratio, besides containing the enzyme MTGase. At the same time N2 had a much higher TVP to SPC ratio to investigate the MTGase effect on coarse type sausages with higher levels of TVP.

[0363] Table 5:

[0364]

[0365]

[0366] Example 5: Modification of textural properties of raw and boiled coarse type sausages prepared with meat and plant protein, using MTGase

[0367] Coarse type hybrid sausages comprising meat and plant protein prepared with MTGase (formulation N2 and N3) had decreased adhesiveness and higher hardness, springiness, and chewiness as compared to formulation N1 (no enzyme), as measured on a Texture Analyzer. Summarized description of the recipes is presented in Table 6 below:

[0368] Table 6:

[0369] Summarized description% of total protein % of total protein % of total protein Enzyme Rec- % Total

[0370] originating from originating from originating from

[0371] ipe protein

[0372] plant source animal source TVP

[0373] N1 13.5% 53% 47% 23%

[0374] N2 11.5% 44% 56% 33% MTGase N3 13.5% 53% 47% 23% MTGase

[0375] Sausages from Example 4 including the cooking and cooling step (1-7), are here referred to as raw. A set of samples were boiled in tap water at 90° C for 5 min, now referred to as boiled. Raw and boiled sausages were cut into 1.5 cm slices, 4 cm off the edges. A circular cookie cutter (0= 1.5 cm) was used to cut out the centre of the sausage slice. The samples were analysed using a TA. XT Plus Texture Analyzer (Stable Micro Systems, Ltd.) with a SMS P / 75 probe, in a heavy duty platform, according to the following settings:

[0376]

[0377] Samples of raw and boiled sausages from the three formulations (N1, N2 and N3) were meas- ured in six independent replicates (n = 36).

[0378] Definition of the measured texture parameters as measured using a TA. XT Plus Texture Analyzer:

[0379]

[0380] Hardness The highest peak force measured during first compression.

[0381] The area under curve after peak force is reached divided by area under Resilience

[0382] curve before peak force is reached.

[0383] A ratio or percentage of a product’s recovery to its original height measured as the second compression’s time from start to the subsequent highest peak Springiness

[0384] force divided by the first compression’s time from start to the subsequent highest peak force

[0385] Table 7: The texture parameters for the hybrid sausages, N1, N2 and N3 obtained from the texture analyser. The values are given as a mean of the six independent replicates

[0386]

[0387] A linear model was applied to analyse the effects of the formulation (N1, N2, N3; n = 3), the treatment (raw, boiled; n = 2) and its interaction on the texture parameters of the sausages. Theresults were reported as least squares means and standard error. Tukey-test at an a = 0.05 was used to perform pairwise comparisons and detect significant differences between the reported least square means.

[0388] Table 8:

[0389]

[0390] *Significantly different at a = 0.05

[0391] The data from Table 8 are also shown in Figures 1-6.

[0392] Based on the measured texture parameters and the statistical analysis it is clear that the MTGase has a significant impact on the texture in hybrid sausages.Hardness and chewiness: the addition of MTGase significantly increased (a = 0.05) both the hardness and the chewiness of raw and boiled sausages produced with 53% of protein from vegetable origin (N3) in comparison with the reference (N1; 53% of protein from plant protein origin).

[0393] The addition of MTGase increased both the hardness and the chewiness of raw and boiled hybrid sausages with 11.5% total protein (N2; 44% from vegetable origin) to a level like that of the reference with 13.5% total protein (N1; 53% from vegetable protein origin, no enzyme).

[0394] Cohesiveness and Resilience: the addition of MTGase significantly increased (a = 0.05) both the cohesiveness and the resilience of both raw and boiled sausages produced with 44 (N2) to 53% (N3) of protein from plant origin in comparison with the reference (N1; 53% of protein from plant protein origin, no enzyme).

[0395] Adhesiveness: the addition of MTGase significantly decreased (a = 0.05) the adhesiveness of both raw and boiled sausages produced with 44 (N2) to 53% (N3) of protein from plant origin in comparison with the reference (N1; 53% of protein from plant protein origin, no enzyme).

[0396] Springiness: the addition of MTGase significantly increased (a = 0.05) the springiness of both raw and boiled sausages produced with 44 (N2) and 53% (N3) of protein from plant origin in comparison with the reference (N1; 53% of protein from plant protein origin, no enzyme).

[0397] A summary of the results and conclusions is presented in the table below:

[0398] Table 9:

[0399] Texture parameter N2 vs. N1 N3 vs. N1

[0400] MTGase increased both the

[0401] hardness and chewiness of MTGase significantly increased both raw and boiled sausages both the hardness and chewiness Hardness and

[0402] to a level like that of N1, which of both raw and boiled sausages Chewiness

[0403] contains 15% more total prowith similar total protein and protein, of which 16.5% more protein from plant origin

[0404] tein from plant origin

[0405] MTGase significantly in- MTGase significantly increased creased both the cohesiveness the cohesiveness, the resilience, Cohesiveness, Reand the resilience of both raw and the springiness of both raw silience and Springiand boiled sausages to a level and boiled sausages with similar ness

[0406] above that of N1, which contotal protein and protein from tains 15% more total protein, of plant originwhich 16.5% more protein from

[0407] plant origin

[0408] MTGase significantly decreased the adhesiveness of MTGase significantly decreased both raw and boiled sausages the adhesiveness of both raw and Adhesiveness to a level below that of N1 , boiled sausages with similar total which contains 15% more total protein and protein from plant protein, of which 16.5% more origin

[0409] protein from plant origin

Claims

CLAIMS1. A method for producing a hybrid meat product, which method comprises:a) providing a hydrated textured plant-based protein source having a water content of 50- 90% (w / w) by optionally hydrating textured plant-based protein in water;b) providing finely chopped or minced animal-derived or cultivated meat;c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging;wherein transglutaminase is added before, during or after any of steps a), b), c), and / or d).

2. The method of claim 1 , wherein the transglutaminase is added before or during the optional hydration in step a), to the binder of optional step c) during or after its preparation, and / or in step d) before or during the mixing.

3. A method for producing a hybrid meat product, which method comprises:a) providing a hydrated textured plant-based protein source having a water content of 50- 90% (w / w);b) providing finely chopped or minced animal-derived or cultivated meat;c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), transglutaminase, and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

4. A method for producing a hybrid meat product, which method comprises:a) hydrating a textured plant-based protein source to obtain a hydrated textured plant-based protein source having a water content of 50-90% (w / w), wherein a transglutaminase is added before or during the hydration;b) providing finely chopped or minced animal-derived or cultivated meat;c) optionally providing a binder comprising water, oil and a non-textured plant-based protein source;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), optionally the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein;e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

5. The method of any of the preceding claims, wherein the hybrid meat product comprises 20- 80% (w / w) textured plant-based protein out of total protein and 20-80% (w / w) animal-derived or cultivated meat protein out of total protein.

6. The method of any of the preceding claims, where in step d), a mixture comprising 40-80% (w / w), preferably 45-80%, textured plant-based protein out of total protein and 20-60% (w / w), preferably 20-55%, animal-derived or cultivated meat protein out of total protein is arrived at, and wherein the hybrid meat product comprises 40-80% (w / w), preferably 45-80%, textured plant-based protein out of total protein and 20-60% (w / w), preferably 20-55%, animal-derived or cultivated meat protein out of total protein.

7. A method for producing a hybrid meat product, which method comprises:a) providing a hydrated textured plant-based protein source having a water content of 50- 90% (w / w);b) providing finely chopped or minced animal-derived or cultivated meat;c) providing a binder comprising water, oil, a non-textured plant-based protein source and transglutaminase;d) mixing the hydrated textured plant-based protein source of step a), the animal-derived or cultivated meat of step b), the binder of step c), and optionally other ingredients, to arrive at a mixture comprising 20-75% (w / w) textured plant-based protein out of total protein, 5-40% (w / w) non-textured plant-based protein out of total protein, and 20-75% (w / w) animal-derived or cultivated meat protein out of total protein;e) optionally processing the mixture of step d), wherein the processing may include shaping, stuffing into casing, heat-treating, cooling, cooking, frying, boiling, smoking, and / or packaging.

8. The method of any of the preceding claims, where the textured plant-based protein source is obtained using extrusion or shear cell technology, preferably using extrusion.

9. The method of any of the preceding claims, where the textured plant-based protein source is a low-moisture extrudate, preferably a low-moisture extrudate having a moisture content of less than 30%.

10. The method of any of the preceding claims, where the textured plant-based protein is from a legume.

11. The method of any of claims 1-9, where the textured plant-based protein is from soy, pea, faba bean, chickpea, lentil, and / or wheat, preferably from soy and / or pea.

12. The method of any of the preceding claims, where the animal-derived or cultivated meat protein is animal-derived, preferably from beef, pork, chicken, turkey, fish and / or seafood.

13. The method of any of the preceding claims, where the concentration of transglutaminase is 0.1-100 TGHll(A) of transglutaminase per g of total protein, preferably 0.5-50, more preferably 1-25 TGHll(A) per g of total protein.

14. The method of any of the preceding claims, wherein the hybrid meat product is a hybrid burger patty, a hybrid sausage, a hybrid nugget, a hybrid schnitzel, a hybrid meatball, a hybrid deli cut, a hybrid fish product, or a hybrid minced meat, preferably wherein the hybrid meat product is a hybrid burger patty or a hybrid sausage, more preferably wherein the hybrid meat product is a hybrid burger patty or a hybrid coarse-type sausage, even more preferably wherein the hybrid meat product is a hybrid burger patty or a hybrid coarse-type nonemulsified sausage, most preferably wherein the hybrid meat product is a hybrid burger patty.

15. The method of any of the preceding claims, wherein the hybrid meat product has a total protein content of 10-25% (w / w).

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