WO2024121191A1 - Method for producing a meat analogue product involving protein-deamidase - Google Patents

Abstract

The present invention relates to a method for producing a meat analogue product which comprises texturizing a non-animal protein, e.g., a plant protein, and adding a deamidase before or during the texturization process.

Description

METHOD FOR PRODUCING A MEAT ANALOGUE PRODUCT INVOLVING PROTEIN-DEAMIDASE

Reference to sequence listing

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

FIELD OF THE INVENTION

The present invention relates to a method for producing a plant-based meat analogue which comprises texturizing a plant protein material.

BACKGROUND OF THE INVENTION

The growing world population demands sustainably produced protein-rich foods. E.g., legumes, such as soy and pulses, are attractive crops for production of protein-rich foods. But other nonanimal protein sources are also being used in food production, e.g., from plants broadly, from algae, or from insects.

Texturized products such as extruded products are used extensively in the food industry. Extrusion is used primarily to give the food a specific texture and distinctive mouthfeel.

Osen et al. (2014), Journal of Food Engineering 127: 67-74, have studied high moisture extrusion cooking of pea protein isolates. They find that the functional properties of 3 different pea protein isolates play a minor role during fiber formation due to the elevated cooking temperature, which exceeds the proteins’ denaturation temperature.

Xu et al. (2020), Trends in Food Science & Technology 99, 167-180, provide an overview of the development of processing of food-related biopolymers using reactive extrusion (REX) and enzymes. They divide REX-enzyme processes into two major technological categories: (1) REX- EH processes, which are series systems including an extrusion pre-treatment followed by enzymatic hydrolysis; and (2) eREX processes, which introduce exogenous enzymes before or during REX. They conclude that eREX is still an immature area except for starch saccharification.

Chen et al. (2011), Food Hydrocolloids 25: 887-897, have studied the effects of combined extrusion pre-treatment followed by controlled enzymatic hydrolysis on the emulsifying properties of soy protein isolates.

Czarnecki et al. (1993), Journal of Food Science 58: 395-398, incubated pinto bean high protein fraction with papain and cellulase for 24 or 72 hours followed by freeze-drying, grinding, sieving and extrusion, to obtain new snack type products from beans. Zhou et al. (2017), J Food Process Preserv. 41 : e13301 , have studied the effect of extrusion and of papain co-extrusion on pea protein and on antioxidant peptides created by further papain hydrolysis of the extrudates.

WO2017/117398 (Abbott) discloses a method of preparing a protein hydrolysate comprising adding an intact protein source and a protease component to an extruder.

WO2018/125920 (Abbott) discloses a method of preparing a nutritional powder comprising adding an intact protein source, a protease component, a fat component, and water to an extruder.

Extrusion of plant protein can give the protein a fibrous, meat-like structure.

Meat analogue products are meat replacers made from, e.g., plant protein that are designed to mimic the visual appearance, texture and taste of meat products. Meat analogue products may be made by extrusion of, e.g., soy, wheat or pea protein. Extrusion is a thermo mechanical texturization process that causes the protein to unfold. In a high-moisture (HM) extrusion process, a cooling die allows the proteins to re-arrange and form new intermolecular covalent and non-co- valent bonds. The cooling die at the end of the extruder creates dense, layered and fibrous meat like structures. In a low-moisture (LM) extrusion process, the products expand when they exit the die and form a solid structure with a fibrous, insoluble, porous network that can soak up as much as three times its weight in liquids.

Texturized vegetable protein such as high or low moisture extruded vegetable protein, may be formed into various shapes (chunks, flakes, nuggets, grains, and strips) and sizes.

Extrusion has been employed for many years in the production of meat analogues for obtaining meat-like structures from plant protein. It is difficult, though, to obtain extrudates of plant protein, such as legume protein, with acceptable textural and functional properties for them to be used in meat analogues in other words there is room for improvement to better mimic the meat-like structure without having to add non-natural ingredients. W02020/038541 (Raisio Nutrition) discloses a method for manufacturing a plant-based meat substitute by passing a mixture containing plant protein material and oat material and optionally a crosslinking enzyme and/or a protein deamidating enzyme at a temperature of 25-55°C through an extruder. Meat substitutes were produced where transglutaminase was included using low temperature and high temperature extrusion and for each sensory property tested, and also for the overall quality rating, the low temperature samples gained better ratings than the corresponding high temperature samples.

CN109619208A (UNIV HEILONGJIANG BAYI AGRICULTURAL) discloses a method for preparing a flavour-enhanced imitation meat food by extrusion puffing of raw materials comprising bean seed flour and soy protein. Ultrasonic enzymolysis using Alcalase and Flavourzyme may be applied for a duration of 10-20 minutes, preferably followed by a Maillard reaction, before the extrusion puffing to provide a strong flavour and a persistent aroma. Nisov et al (2022), Food Research International 156 (2022) 111089 have investigated effect of pH and temperature on fibrous structure formation of plant proteins during high-moisture extrusion processing. They conclude that the structure formation of the extrudate can be positively influenced by increasing the pH of the raw material, which facilitates the plant protein structuring into appealing meat analogue product.

WO2022/218863 discloses that application of proteases increased solubility and resulted in improved texture-functionalities of the final product.

It is an object of the present invention to provide texturized plant protein material having better functional properties making them suitable for use in meat analogue products such as burger patties, mince, sausages or chicken nugget analogues.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that by adding a deamidase to plant protein material before or during a texturization process, such as an extrusion process, a texturized plant protein material is provided which, when used in a plant-based meat product, such as a burger patty, gives improved properties such as higher firmness and/or higher water holding capacity. The effect of adding deamidase during the extrusion process results in higher firmness of e.g., the plant-based burger patty and consequently a more meat-like texture. The inventors further found a higher water holding capacity in the enzyme treated extrudates which according to the literature results in a higher perceived juiciness of the plant-based meat products.

The present invention therefore provides a method of producing a plant-based meat analogue comprising the steps: a) preparing a mixture of plant protein containing material, having a protein content by dry weight of the plant material of 15% w/w to 95% w/w, and water, with a water content by weight of the mixture of 5% w/w to 99% w/w; b) treating the mixture with a protein-deamidase enzyme; and c) passing the mixture at a temperature above 60°C, through an extruder; d) optionally mincing or shredding the extruded protein material; e) optionally drying the product of c) or d); and f) optionally mixing the plant protein material with other ingredients to obtain the meat analogue product. DEFINITIONS

Deamidase: The term “deamidase” means a protein-glutamine glutaminase (also known as glu- taminylpeptide glutaminase) activity, as described in EC 3.5.1.44, which catalyzes the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or both the alpha-amino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L- glutaminylglycine. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues and are also referred to as protein glutamine deamidase. Deamidases comprise a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al. “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belong to the InterPro entry IPR041325.

Deamidase activity: Deamidase (protein glutaminase) activity can be determined using thew below assay.

The glutaminase deamidates the glutamine substrate (Z-GLN-GLY, C15H19N3O6) and generates ammonia in the process. The ammonia is used as substrate for the glutamate dehydrogenase in combination with a-ketoglutarate to produce glutamate.

This latter enzymatic reaction requires NADH as a coenzyme. The depletion of NADH can be followed by kinetic absorbance measurement at 340 nm and is directly proportional to the glutaminase activity. The reaction temperature is 37°C, pH 7.0, reaction time 216 sec.

Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component with which it is naturally associated as found in nature, including but not limited to, for example, other proteins, nucleic acids, cells, etc. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N terminal processing (e.g., removal of signal peptide). In one aspect, the mature polypeptide is amino acids 1 to 294 of SEQ ID NO: 1 including a propetide sequence of aminino acids 1 to 109 of SEQ ID NO: 1. In one embodiment the deamidase after cleavage of the propetide is amino acids 1 to 185 of SEQ ID NO: 2.

In another aspect, the mature polypeptide is amino acids 1 to 297 of SEQ ID NO: 3 including a propetide sequence of aminino acids 1 to 112 of SEQ ID NO: 3. In one embodiment the deamidase after cleavage of the propetide is amino acids 1 to 185 of SEQ ID NO: 4.

Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process. Purified: The term “purified” means a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

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

For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labelled “longest identity” is calculated as follows:

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

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have surprisingly found that by adding a deamidase to plant protein material before or during a texturization process, such as an extrusion process, a texturized plant protein material is provided which, has improved properties such as increased cutting strength and increase water holding capacity.

In a first aspect the present invention therefore relates to a method of producing a plant-based meat analogue comprising the following steps: a) preparing a mixture of plant protein material, having a protein content by dry weight of the plant material of 15% w/w to 95% w/w, and water, with a water content by weight of the mixture of 5% w/w to 99% w/w; b) treating the mixture with a protein-deamidase enzyme; and c) passing the mixture at a temperature above 60°C, through an extruder; d) optionally mincing or shredding the extruded protein material; e) optionally drying the product of c or d); and f) optionally mixing the plant protein material with other ingredients to obtain the meat analogue product.

The plant protein raw material may be obtained from pulses, e.g., beans, peas, lentils, chickpea, or from oil crops, e.g., soybean, peanuts, canola; from cereals, e.g., rice, corn; from seeds, e.g., hemp, sunflower, flax, sesame, chia, canola, and any combination thereof.

In a preferred embodiment, the plant protein is soy protein or pea protein.

The plant protein may be a protein material, such as a plant protein material or a legume protein material, preferably a plant protein or legume protein material having a protein to dry matter ratio of 15-95% (w/w). Preferably, the protein material is a soy protein material, a pea protein material, a chickpea protein material, a mung bean protein material, a lentil protein material, or a fava bean protein material, more preferably a soy protein material or a pea protein material.

Extrusion

Since the 1960s extrusion cooking has been employed to produce meat analogues using especially soy protein as raw material and until a few years ago primarily used as meat extenders to reduce cost of minced meat in the traditional meat industry. However especially over the last 10 years much more focus has been on finding applicable extrudates for a full substitution of meatbased patties and sausages (see, e.g., chapter 6 “Plant-Based Meat Analogues” by K. Kyria- kopoulou et al. in “Sustainable Meat Products and Processing”, 2018 ed. Charis Galanakis, ISBN electronic 9780128156889).

Extrusion is a thermomechanical process by which moistened, expandable, starchy and proteinaceous food materials are plasticized and pushed through a die by a combination of pressure, heat, and mechanical shear. A typical extruder set-up consists of a feeding system, a screw, a barrel, a die, and a cutting machine. Furthermore, a preconditioning system can optionally be introduced before the extrusion. The extruder barrel may be divided into 5-9 sections, often referred to as temperature zones, which can be heated separately. Material is added to the first section of the extruder using a volumetrically or gravimetrically controlled feeder. A pump is used to feed water, possibly including enzymes, or preconditioned material with adjusted moisture content to the second section of the extruder. The screw configuration used can consist of forward and reverse transport elements. In the feeding zone, the material is mixed, homogenized, and then transported to the compression zone. In that zone, a reduction in screw depth and pitch exists, which results in an increase in shear rate, temperature, and pressure. The mechanical energy dissipated through the rotation of the screws increase the processing temperature inside the extruder. Between 70 and 180°C, the proteins denature resulting in a viscoelastic mass that can be aligned in the cooling-die. To summarize, this change in the process conditions convert the solid material into a fluid melt. Before exiting the extruder, a maximum temperature and pressure is reached leading to an immediate reduction of the viscosity of the extruded material. In case of meat-analogue production by high-moisture extrusion, the cooling-die is long to create alignment and to prevent severe material expansion, which could destroy the newly formed structure.

The successful preparation of meat-analogue products requires the control of the extrusion parameters, such as screw speed, moisture content of the feed, barrel temperature, extruder properties and chemical and physical composition of the feed. The skilled person will know how to adjust the extrusion parameters to optimize the process.

For meat-analogue production, two product categories have been developed, depending on the amount of water added during extrusion: high and low-moisture extrusion.

In low-moisture extrusion, flours or concentrates with low-moisture content are transformed into textured vegetable proteins (TVP) also called dry extrudates, ingredients are hydrated with e.g. 5-15% moisture during extrusion, resulting in extrudates with a lower final moisture content. For the final meat-analogue recipe they are rehydrated and mixed with other ingredients before being cooked, e.g., fried. Products from low-moisture extrusion, present a sponge-like structure and expand and absorb water rapidly. They have typically been used as meat extenders but are these days also used partly or fully as meat analogues such as sausages and beef patties.

In high-moisture extrusion, ingredients are hydrated with e.g., 45-70% moisture during extrusion, resulting in extrudates with higher final moisture content. Mostly, a co-rotating twin-screw extruder is used for this application and products are used directly for further processing or are frozen after extrusion to increase the shelf-life and possibly enhance the structure. The high moisture products can either be used as they are or shredded to mimic chicken or goulash like meat pieces or minced to different sizes to go into patties or sausages.

According to Egbert and Borders, 2006, Achieving success with meat analogs, Food Technology, Chicago, 60(1): 28-34, a meat analogue product may contain water (50-80%), textured vegetable protein (10-25%), nontextured protein (4-20%), flavorings (3-10%), fat (0-15%), binging agents (1-5%), and coloring agents (0-0.5%). The combination of ingredients yields meat analogues that are accepted in terms of sensory attributes. The high-water content not only reduces the costs of the product, but provides the desired juiciness, acts as a plasticizer during processing and helps on emulsification.

In preferred embodiments of the method of the invention, the plant protein is passed through an extruder, preferably a twin-screw extruder, such as a co-rotating or counter-rotating twin-screw extruder, more preferably a co-rotating twin-screw extruder.

The protein is preferably passed through the extruder at a temperature of 65-200°C, such as 90- 200 °C, preferably 100-180 °C, more preferably 120-175 °C.

In preferred embodiments, the extruder has more than one temperature zone, such as 2-10, preferably 5-9, temperature zones. The starting temperature in the first zone may therefore be lower than the preferred temperature ranges above. E.g., the starting temperature may be in the range from 20-60°C.

According to the invention, the deamidase may be added before or during step c).

In one embodiment, the deamidase is added before step c), and the water content by weight of the mixture is 5% w/w to 50% w/w, 10% w/w to 40% w/w, such as in the range from 20% w/w to 35% w/w.

In another embodiment, the deamidase is added during extrusion step c), and the water content by weight of the mixture in the extruded product after step c) is 45% w/w to 70% w/w, such as in the range from 50% w/w to 65% w/w. Particularly, according to this embodiment, water is added during extrusion in amounts selected from 1.2-3.0 g water/g protein in the plant material.

In another embodiment the deamidase is added during step c), and the water content by weight of the mixture in the extruded product after step c) is 1 % w/w to 45% w/w, such as 2% w/w to 25% w/w, particularly 5-15%. Particularly, according to this embodiment, water is added during extrusion in amounts selected from 0.05-1.0 g water/g protein in the plant material.

The protein content by dry weight of the plant material is in the range from 15% w/w to 95% w/w, more preferred in the range from 25% w/w to 92% w/w, such as 45% w/w to 75% w/w.

In the embodiment where deamidase treatment is performed before the extrusion step, the reaction temperature and incubation time may depend on the deamidase activity of the applied enzyme, however, the incubation time is preferably in the range from 1 to 120, 1 to 60 min, such as 1 to 15 min, and incubation temperature is in the range from 20-95 °C, such as 30-70 °C.

Shear cell technology Based on the recognition that extrusion is an effective, but not a well-defined process, a technology based on well-defined shear flow-deformation was introduced a decade ago to produce fibrous products. Shearing devices inspired on the design of rheometers so called shear cells, were developed in which intensive shear can be applied in a cone-in-cone or in a couette geometry. The final structure obtained with this technique depends on the ingredients and on the processing conditions. Fibrous products are obtained with several plant protein blends, such as soy protein concentrate or soy protein isolate (SPI) blended with, e.g., wheat gluten (WG), pectin and/or starch. Fibrous products can also be obtained by use of calcium caseinate. The technology has proven successful, at least up to pilot scale (BL Deckers et al., in Trends in Food Science & Technology 81 (2018) 25-36). High temperatures (above 100 °C) are usually also applied when using shear cell technology.

As in extrusion processes, deamidases can be applied in other perhaps more gentle processing for making meat analogues such as shear cell technology and hereby result in the same improved performance of the extrudate and the final formulated product.

Deamidase

In the methods of the present invention, a protein deamidase is added to non-animal protein such as plant protein, before or during a texturization process, such as an extrusion process or a shear cell technology process.

In the present invention, a protein deamidase refers to an enzyme having an effect of directly acting on an amide group of a side chain of an amino acid that constitutes a protein to cause deamidation and release ammonia without cleaving a peptide bond of the protein and crosslinking proteins. Specific examples of the protein deamidase include a protein glutaminase (EC 3.5.1.44) that directly acts on an amide group of a side chain of a glutamine residue contained in a protein to release ammonia and thus converts the glutamine residue into a glutamate residue. Deamidase may also include a protein asparaginase that directly acts on an amide group of a side chain of an asparagine residue contained in a protein to release ammonia and thus converts the asparagine residue into an aspartate residue. In the present invention, as a protein deamidase, any one of the protein glutaminase and the protein asparaginase can be used, or both can be used in combination. One preferred example of the protein deamidase used in the present invention is, for example, a protein glutaminase.

A protein deamidase to be used in a method of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained 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 polypeptide obtained from a given source is secreted extracellularly.

The types or origins of the protein deamidase used in the present invention are not particularly limited. Examples of the protein deamidase includes protein deamidases derived from Chryseobacterium genus, Flavobacterium genus, Empedobacter genus, Sphingobacterium genus, Aure- obacterium genus, or Myroides genus.

EP1839491 discloses cloning of a protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum. and deamidases are also commercially available, e.g., protein glutaminases derived from Chryseobacterium genus. Preferred examples include protein deamidases derived from Chryseobacterium genus, and more preferred examples include protein deamidases derived from Chryseobacterium proteolyticum. Protein glutaminases derived from Chryseobacterium proteolyticum are commercially available as, for example, Protein-glutaminase.

"Amano" 500 manufactured by Amano Enzyme Inc., and this commercially available products can be used.

For example, protein deamidases can be obtained from a culture broth of the above-described microorganisms.

Deamidase activity: Deamidase (protein glutaminase) activity can be determined using thew below assay.

The glutaminase deamidates the glutamine substrate (Z-GLN-GLY, C15H19N3O6 ) and generates ammonia in the process. The ammonia is used as substrate for the glutamate dehydrogenase in combination with a-ketoglutarate to produce glutamate.

This latter enzymatic reaction requires NADH as a coenzyme. The depletion of NADH can be followed by kinetic absorbance measurement at 340 nm and is directly proportional to the glutaminase activity. The reaction temperature is 37°C, pH 7.0, reaction time 216 sec.

According to a preferred embodiment the deamidase applied in the process of the invention is derived from or obtained from a Chryseobacterium species, e.g., Chryseobacterium proteolyticus.

More specifically the deamidase may in one embodiment be selected from: (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 ;

(b) a polypeptide having at least 75% sequence identity to SEQ ID NO: 2;

(c) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 1;

(d) a polypeptide derived from SEQ ID NO: 1, a mature polypeptide of SEQ ID NO: 1, or SEQ ID NO: 2 having 1-30 alterations, e.g., substitutions, deletions and/r insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids; and

(f) a fragment of the polypeptide of (a), (b), (c), or (d); wherein the polypeptide has deamidase activity.

In another specific embodiment the deamidase is selected from:

(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;

(b) a polypeptide having at least 75% sequence identity to of SEQ ID NO: 4;

(c) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 3;

(d) a polypeptide derived from SEQ ID NO: 3, a mature polypeptide of SEQ ID NO: 3, or SEQ ID NO: 4 having 1-30 alterations, e.g., substitutions, deletions and/r insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids; and

(f) a fragment of the polypeptide of (a), (b), (c), or (d); wherein the polypeptide has deamidase activity.

In one embodiment the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 1.

In one embodiment the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 2. In one embodiment the deamidase is selected from a polypeptide having 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% sequence identity to a mature polypeptide of SEQ ID NO: 1.

In one embodiment the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 3.

In one embodiment the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 4.

In one embodiment the deamidase is selected from a polypeptide having 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% sequence identity to a mature polypeptide of SEQ ID NO: 3.

In the context of the present invention, the term “variant” means a polypeptide having endopeptidase 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 one or more (e.g., several) amino acids, e.g., 1-5 amino acids, adjacent to and immediately following the amino acid occupying a position.

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.

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, AlaA/al, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA/al, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may affect 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 endopeptidase 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).

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.

In a preferred embodiment of the invention, the deamidase is added to the plant protein immediately before or during extrusion step c). It is to be understood that addition of the deamidase to the plant protein immediately before or during step c) means there is no pre-incubation.

In a particularly preferred embodiment, the plant protein is texturized by passing it through an extruder, and the deamidase is fed directly to the extruder, preferably an aqueous solution of the deamidase is fed directly to the extruder. Preferably, the plant protein and an aqueous solution of the deamidase are added separately to the feeding zone of the extruder.

The deamidase may also be added to an aqueous solution or suspension of the plant protein immediately before feeding it to the extruder, i.e. without a preincubation step.

In other embodiments of the invention, the deamidase is added to and incubated with at least part of the plant protein before step c). In such embodiments, the mixing with other ingredients may be performed before or during incubation of at least part of the plant protein with the deamidase but before step c). And/or the mixing with other ingredients may be performed after step c).

In the methods of the invention, the plant protein is mixed with other ingredients to obtain a meat analogue product. The mixing with other ingredients may be performed before step c), it may be performed after step c).

The one or more ingredients may be selected from non-texturized plant protein, such as soy protein isolate, wheat gluten, fiber such as pectin, starch such as corn starch, pea starch and/or potato starch, salt, colour, aroma, flavor, spices, and/or oil or fat such as coconut fat, sunflower oil and/or rapeseed oil.

Non-texturized plant protein, such as e.g., soy protein isolate, may be added as an ingredient in, e.g., burger patties as a binder.

The meat-analogue product produced by a method of the invention may be, e.g., a minced-meat analogue product, a burger patty, a sausage, a meat-ball analogue product, a chicken nugget analogue product, a goulash meat analogue product or a schnitzel analogue product. In a preferred embodiment, the meat-analogue product produced by a method of the invention is a burger patty.

Improved properties of the extrusion product and the meat analogue produced by the method of the invention.

The method of the invention has surprisingly resulted in extrusion product and meat analogue products having improved properties. Such improved property is in one embodiment that the plant-based meat analogue product after the extrusion step, has increased cutting strength. In one embodiment the plant-based extrudate has increased cutting strength, and wherein the relative increase in cutting strength of the extrudate is at least 25%, at least 40%, at least 50%, such as at least 75% compared to an extrudate where no deamidase has been added.

In another embodiment the plant-based meat analogue product has increased water holding capacity compared to the plant-based material not treated with a deamidase.

In one embodiment the extrudates according to the invention has a water holding capacity of at least 4-6 g water/g extrudate. In another embodiment patties made from deamidase treated extruded plant material have a relative increase in water holding capacity of at least 5%, at least 10%, at least 15%, such as at least 20%.

Another improved property of the meat analogue product of the invention is increased chewiness and hardness of plant-based burger patties including the extrudates according to the invention.

Particularly in one embodiment, the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative hardness increase of at least 1%, at least 2%, at least 5%, at least 10%, such as at least 15% compared to patties made from a no deamidase control extrudate.

Particularly in one embodiment, the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative increase in chewiness of at least 5%, at least 10%, at least 15%, such as at least 20% compared to patties made from a no deamidase control extrudate.

The invention is further disclosed in the following numbered embodiments.

Embodiment 1. A method for producing a plant-based meat analogue comprising the steps: a) preparing a mixture of plant protein containing material, having a protein content by dry weight of the plant material of 15% w/w to 95% w/w, and water, with a water content by weight of the mixture of 5% w/w to 99% w/w; b) treating the mixture with a protein-deamidase enzyme; and c) passing the mixture at a temperature above 60°C, through an extruder; d) optionally mincing or shredding the extruded protein material; e) optionally drying the product of c) or d); and f) optionally mixing the plant protein material with other ingredients to obtain the meat analogue product.

Embodiment 2. The method according to embodiment 1 , wherein the protein-deamidase is protein-glutaminase.

Embodiment 3. The method according to any of embodiments 1-2, wherein the plant protein material is derived from pulses, e.g., beans, peas, lentils, chickpea, or from oil crops, e.g., soybean, peanuts, canola; from cereals, e.g., rice, corn; from seeds, e.g., hemp, sunflower, flax, sesame, chia, canola, and any combination thereof.

Embodiment 4. The method according to any of the preceding embodiments, wherein the deamidase is added before or during step c).

Embodiment 5. The method of any of embodiments 1-4, wherein the deamidase is added before step c), and the water content by weight of the mixture is 5% w/w to 50% w/w, 10% w/w to 40% w/w, such as in the range from 20% w/w to 35% w/w.

Embodiment 6. The method of any of embodiments 1-4, wherein the deamidase is added during step c), and the water content by weight of the mixture in the extruded product after step c) is 45% w/w to 70% w/w, such as in the range from 50% w/w to 65% w/w.

Embodiment 7. The method of embodiment 6, wherein water is added during extrusion in amounts selected from 1.2-3.0 g water/g protein in the plant material.

Embodiment 8. The method of any of embodiments 1-4, wherein the deamidase is added during step c), and the water content by weight of the mixture in the extruded product after step c) is 1% w/w to 45% w/w, such as 2% w/w to 25% w/w, particularly 5-15%.

Embodiment 9. The method of embodiment 8, wherein water is added during extrusion in amounts selected from 0.05-1.0 g water/g protein in the plant material.

Embodiment 10. The method according to any of the preceding embodiments, wherein the protein content by dry weight of the plant material is in the range from 25% w/w to 92% w/w, such as 45% w/w to 75% w/w.

Embodiment 11. The method according to any of the preceding embodiments, wherein step c) is performed at a temperature in the range of 65-200 °C, 100-180 °C, such as 120-175 °C. Embodiment 12. The method of any of the preceding embodiments, wherein the step b) is performed before step c) and, wherein the incubation time is from 1 to 120 min, 1 to 60 min, such as 1 to 15 min.

Embodiment 13. The method of embodiment 12, wherein the temperature is in the range from 20-95 °C, such as 30-70 °C.

Embodiment 14. The method of any of the preceding embodiments, wherein the plant-based meat analogue product after the extrusion step has increased cutting strength, and wherein the relative increase in cutting strength of the extrudate is at least 25%, at least 40%, at least 50%, such as at least 75% compared to a no deamidase control.

Embodiment 15. The method of any of the preceding embodiments, wherein the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative hardness increase of at least 1%, at least 2%, at least 5%, at least 10%, such as at least 15% compared to patties made from a no deamidase control extrudate.

Embodiment 16. The method of any of the preceding embodiments, wherein the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative increase in chewiness of at least 5%, at least 10%, at least 15%, such as at least 20% compared to patties made from a no deamidase control extrudate.

Embodiment 17. The method of any of the preceding embodiments, wherein the plant-based meat analogue product has increased water holding capacity compared to the plant-based material not treated with a deamidase.

Embodiment 18. The method of embodiment 17, wherein the water holding capacity is at least 4- 6 g water/g extrudate.

Embodiment 19. The method of embodiment 17, wherein the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative increase in water holding capacity of at least 5%, at least 10%, at least 15%, such as at least 20%. Embodiment 20. The method of any of the preceding embodiments, wherein the deamidase is selected from:

(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 ;

(b) a polypeptide having at least 75% sequence identity to SEQ ID NO: 2;

(c) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 1;

(d) a polypeptide derived from SEQ ID NO: 1, a mature polypeptide of SEQ ID NO: 1 , or SEQ ID NO: 2 having 1-30 alterations, e.g., substitutions, deletions and/r insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids; and

(f) a fragment of the polypeptide of (a), (b), (c), or (d); wherein the polypeptide has deamidase activity.

Embodiment 21. The method of any of the preceding embodiments, wherein the deamidase is selected from:

(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;

(b) a polypeptide having at least 75% sequence identity to of SEQ ID NO: 4;

(c) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 3;

(d) a polypeptide derived from SEQ ID NO: 3, a mature polypeptide of SEQ ID NO: 3, or SEQ ID NO: 4 having 1-30 alterations, e.g., substitutions, deletions and/r insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids; and

(f) a fragment of the polypeptide of (a), (b), (c), or (d); wherein the polypeptide has deamidase activity.

Embodiment 22. The method of embodiment 20, wherein the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 1. Embodiment 23. The method of embodiment 20, wherein the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 2.

Embodiment 24. The method of embodiment 20, wherein the deamidase is selected from a polypeptide having 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% sequence identity to a mature polypeptide of SEQ ID NO: 1.

Embodiment 25. The method of embodiment 21, wherein the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 3.

Embodiment 26. The method of embodiment 21, wherein the deamidase is selected from a polypeptide having 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% sequence identity to SEQ ID NO: 4.

Embodiment 27. The method of embodiment 21, wherein the deamidase is selected from a polypeptide having 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% sequence identity to a mature polypeptide of SEQ ID NO: 3.

EXAMPLES

Summary

In order to improve hardness of extrudates and consequently the bite in the final plant-based meat products, deamidase enzyme has been tested in the extrusion process. Deamidase was added in both a pretreatment step and directly during extrusion. Both lab and pilot-scale extrusion facilities have been included in the testing.

Despite the harsh conditions (high temperature and shear) during the extrusion process, direct addition of deamidase in the extrusion barrel during high moisture (HM) extrusion of toasted, defatted soybean flour appeared to give the extrudates an increased fiber formation. This is expected to be correlated to increased bite strength/firmness of the final plant-based products.

To optimize the effect of the enzyme, a pretreatment step prior to the HM extrusion of the substrate was introduced. The deamidase pretreatment of a soy protein concentrate (SPC) and subsequent HM extrusion resulted in an improved meat-like texture giving a significant higher cutting strength of the extrudates. Increased cutting strength or firmness of the extrudates is generally desirable and can help to upgrade extrudates produced using less processed, less refined and lower cost raw materials.

The extrudates have been formulated into patties similar to what is commercially available. Both the extrudates and the patties have been analyzed by chemical and physical analyses. The methods applied are well known from the existing food and meat industry. Texture analysis of the patties made using deamidase treated SPC extrudates showed that deamidase addition increased the hardness and chewiness of the raw and fried patties. Hence, the deamidase addition would result in plant-based meats with enhanced firmness and bite, resembling regular meat products. Further, the patties produced using deamidase treated extrudates resulted in a significant reduced cooking loss at the lowest dosage.

Water holding capacity (WHC) analysis suggests that the addition of deamidase improves the WHC in the extrudates in comparison to an untreated sample. This is expected to correlate to increased juiciness in the final product. Similarly, low field NMR analysis showed that deamidase treatment results in modifications of the water binding, with a higher degree of more mobile water in the deamidase treated extrudates also indicating an increase in juiciness.

Spectroscopic analysis (FT-IR) further showed a modified secondary protein structure of the deamidase pretreated substrate, reflecting a better solubilization which leads to improved formation of covalent and non-covalent bonds in the final extrudate. Materials

Strains.

The Chryseobacterium sp-62563 strain was isolated from a soil sample collected in Sibhult, Sweden in September 2013.

Substrates

Substrate 1 : Toasted, defatted soybean flour (SBF), “NutriSoy®” (ADM, Decatur, IL, USA) 50.9 g/100 g dry matter.

Substrate 2: Soy protein concentrate (SPC), “Arcon® F” (ADM, Decatur, IL, USA). 68.56 g/100 g dry matter.

Enzymes

The deamidase (EC 3.5.1.44) used in the examples is the following:

Protein glutaminase derived from Chryseobacterium sp-62563 having the mature polypeptide sequence shown as SEQ ID NO: 2. Cleavage of the propeptide was achieved by treating the deamidase of SEQ ID NO: 1 with a site-specific endopeptidase. The site-specific endopeptidase used is a glutamyl endopeptidase from Bacillus licheniformis. The resulting active deamidase after maturation was the polypeptide shown in SEQ ID NO: 2.

Example 1

High moisture extrusion of toasted, defatted soybean flour (Substrate 1) with direct feed of deamidase on lab-scale extruder (Extrudate 1-5)

Extrusion trials for testing of deamidase were carried out using a laboratory scale extruder (Process 11 , Thermo Fisher Scientific, Karlsruhe, Germany). The extruder was an intermeshing, corotating twin-screw extruder. The screw diameter and the extruder length to diameter were 11 mm and 40:1 , respectively. The raw material was metered into the extruder by a gravimetric twin- screw feeder (MT-S, MiniTwin, Brabender Technologie, Duisburg, Germany). The extruder had 7 internal and 1 external heating zones.

Extrudates were produced from Substrate 1 : Toasted, defatted soybean flour (SBF), “NutriSoy” (ADM, Decatur, IL, USA.) with a protein content of 50.9 g/100 g DM (dry matter). The extrusion settings are given in Table 1.

The raw material was fed into the extruder in the first zone. Deamidase was mixed into the water, in five dosages (0 (blank), 16, 82, 164, 328 PGLU/g protein in raw material) which was fed into the extruder in zone 2/8 using a peristaltic pump (Cole-Palmer Masterflex L/S, Illinois, USA) equipped with a silicone hose (Tygon S3-3603, Saint-Gobain S.A., Courbevoie, France) with an inner diameter of 3,2 mm. The mixture was conveyed through the extruder using a screw speed of 160 rpm. High moisture (HM) extrudates were made using a cooling die cooled by 30 °C cooling water with a recirculation water flow rate of 15 L/min. The extrudates were produced in a slab shape with dimensions of 4x20 mm and cut into slabs ranging from 100-150 mm. The extrudates were stored at 5 °C. Further details on the trials as well as the outcome can be seen in Example 4.

Table 1: Extrusion settings.

Example 2

High moisture extrusion of deamidase pretreated toasted, defatted soybean flour (Substrate 1) on lab-scale extruder (Extrudate 6-9)

Extrusion trials for testing of deamidase were carried out using a laboratory scale extruder (Process 11 , Thermo Fisher Scientific, Karlsruhe, Germany). The extruder was an intermeshing, corotating twin-screw extruder. The screw diameter and the extruder length to diameter were 11 mm and 40: 1 , respectively. The raw material was fed into the extruder by a gravimetric twin-screw feeder (MT-S, MiniTwin, Brabender Technologie, Duisburg, Germany). The extruder had 7 internal and 1 external heating zones. High moisture (HM) extrudates were made using a cooling die cooled by 30 °C cooling water with a recirculation water flow rate of 15 L/min.

Extrudates were produced from deamidase pretreated Substrate 1 : Toasted, defatted soybean flour (SBF), “NutriSoy” (ADM, Decatur, IL, USA.) with a protein content of 50,9 g/100 g DM using the extrusion settings given in Table 2. The pretreatment of the substrate was made in a Thermomix TM6 (Vorwerk, Wuppertal Germany). In the mixing chamber, 280 g of SBF was mixed with Mil HQ water (blank) or the enzyme solution (16, 82, 164 PGLU/g protein in raw material) resulting in a total moisture content of 30% w/w. The substrate was mixed with water/enzyme solution by slowly pouring it into the thermomixer at rotation speed 1/10, the pretreatment temperature was set to 60 °C, the time to 30 min and the rotation speed was increased to 3/10. The pretreated substrates were spread on a metal tray and immediately placed in a freezer -28 °C overnight. The frozen pretreated substrates were freeze dried for 25 h at 0,22 hPa (Heto PowerDry PL9000, Thermofisher Scientific Inc, Waltham, Massachusetts USA). The pretreated substrates were granulated to a flour using a laboratory MF10 hammer mill equipped with a MF10.1 cutting-grinding head rotating at 4000 rpm and passing through a 1mm sieve (IKA®-Werke GmbH & Co, Staufen, Germany). The pretreated samples were stored at room temperature in a closed container before extrusion. Further details on the trials as well as the outcome can be seen in Example 4 and 6.

Table 2: Extrusion settings.

Example 3

High moisture extrusion of deamidase pretreated soy protein concentrate (Substrate 2) using a pilot-scale extruder (Extrudate 10-13)

Extrusion trials for testing of deamidase were carried out using a pilot-scale twin-screw extruder (Coperion, ZsK26 MP s, Stuttgart, Germany). The extruderwas an intermeshing, co-rotating twin- screw extruder with a gravimetric twin-screw feeder (Model: KT20, Coperion K-Tron, Stuttgart, Germany). The screw diameter of the extruder was 27 mm with a length/diameter ratio of 40:1 . The extruder barrel consisted of 5 heating zones. High moisture (HM) extrudates were made using a cooling die using cooling water with a temperature of 80 °C. Extrudates were produced from Substrate 2: Soy protein concentrate (SPC), Arcon® F with a protein content of 68,56 g/100 g DM from ADM, Decatur IL USA. The deamidase was diluted in water and preconditioned with soy protein concentrate (0, 82, 164, 328 PGLU/g protein in raw material). For preconditioning, the enzyme-water mixture was sprayed onto the soy protein concentrate using a spray bottle and homogeneously mixed using a kneading machine for 5 min. By adding the enzyme solution, the dry matter in the mixture was adjusted to 70 %. The mixture was then packed into bags and sealed. The bags were heated for 20 minutes at 50 °C in a convector oven as the enzyme treatment step. The mixture was transferred to the gravimetric feeder and was fed into the extruder in the first barrel zone where it was mixed with additional water resulting in a moisture content of 66% w/w. The extrudates were cut into 15-20 cm slabs and allowed to cool at room temperature. The extrudates were vacuum sealed and stored at -20 °C before further analysis.

Three different sets of trials were run using the settings of Table 3. Further details on the trials as well as the outcome can be seen in Examples 4, 5 and 8. The improvement of protein solubility of Substrate 2 used in this example can be seen in Example 7.

Table 3: Extrusion settings.

Example 4

Visual inspection of extrudates from Example 1-3 and texture analysis (cutting strength) of extrudates (Example 3)

Visual inspection

The deamidase treated extrudates resulted in a more tough texture and the visual inspection of the shredded extrudate showed that the deamidase treated extrudates had increased formation of visible fibres and thus a more meat-like texture.

Table 4: Arbitrary ranking of formation of visible fibres in extrudates (Example 1-3).

Method of measurement

The cutting strength texture analysis was performed using a TA.XT.PIus Texture Analyzer (Stable Micro Systems, Surrey, England) equipped with a HDP/BS probe (Blade set with knife) and a HDP/90 Heavy Duty Platform similar to method described by Palanisamy et al 2018 LWT Food Science and Technology Journal, Volume 87, 546-552. A 50 kg loading cell was equipped, and the equipment was calibrated using a 2 kg calibration weight.

The measurements were conducted at 20 °C and the samples were prepared the following way:

In Example 3, the extrudates with the dimension of 40x5mm were cut into 4 cm slabs, placed under on cutting platform and cut over the transverse direction of the fibers.

The pre-test speed was set to 2 mm/s, a test speed of 5 mm/s and a post-test speed of 10 mm/s. The cutting distance was set to 15 mm to ensure a cut through the sample and the trigger value before starting the measurement was set to 50 g.

The measurements were conducted in replicates for 6 samples per treatment. The peak shear force (kg) was recorded as the “cutting strength” of the extrudates.

Table 5: Cutting strength of HM extrudates produced in Example 3.

Conclusion: In comparison with the control extrudates, an increased cutting strength for the deamidase treated extrudates were observed. The effect of pre-treatment with deamidase on Substrate 2 and the subsequent high moisture extrusion resulted in a significant increase from 3.20 kg to 5.93, 6.04 and 6.44 kg for extrudates treated with 82, 164 and 328 PGLU/g protein, respectively.

Example 5

Water holding capacity evaluated by a direct method and by LF-NMR of the HM extruded SPC (Example 3)

The water holding capacity (WHC) of plant-based extrudates is of importance for both cohesiveness and juiciness when eaten. The correlation between transverse relaxation T2 based on low field ¹H nuclear magnetic resonance (LF-NMR) and WHC have been proven in various papers (e.g., HC Bertram et al. in Meat Science 57 (2001) 125-132 and Massimo Lucarini et al in Foods (2020) 9, 480).

The WHC is defined as ability to hold its own and added water during the application of forces, pressing, centrifugation, or heating (Joseph F. Zayas, Functionalities of Proteins in food, 1997, p 77-79). Results are shown in Table 7.

The molecular mobility of water and biopolymers in food products can be studied with proton nuclear magnetic resonance LF-NMR detecting both longitudinal or spin-lattice relaxation times (T1) and transverse or spin-spin relaxation times (T2) of protons in a magnetic field.

The LF-NMR analysis of HM extrudates were performed at room temperature (22 °C) on an MQC- R pulsed NMR spectrometer (Oxford Instruments, Abingdon, United Kingdom) with a magnetic field of 23 MHz. Transverse relaxation T2 was measured using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with 4096 echoes, 8 scans and a 90-180 pulse spacing (T) value of 76.5 ps. The samples were analysed in duplicates.

Relaxation time constants T2n and corresponding relative population size f_n were determined by discrete multi-exponential fitting including deconvolution of the relaxation curve into n exponential components. This was achieved using the software WinFit (Oxford Instruments, Abingdon, United Kingdom). The number of proton populations was determined by inspecting the residual error after fitting. The residual error revealed whether the curve was modelled by the correct number of components.

Results are given in Table 6 (presented as mean values of duplicate analysis).

Table 6: Relaxation Time constants T21, T22 and T23 and relative population size f1, f2 and f3 determined by LF-NMR of HM extrudates.

Table 7 WHC expressed as g water/g dry matter in extrudates (Example 3).

Conclusion: Three proton populations were identified with different T2 values, which were assigned to water populations of various mobilities. The fractions having the lower mobility water is designated T21 and T22, while the fraction representing the higher mobility water is designated T23.

Deamidase treated samples exhibited a higher relative amount of T23 population (fs) while fi and/or f2 de-creased. This indicates higher relative amounts of high mobility/free water in deamidase treated samples in comparison with the control. The presence of more loosely bound water as an effect of the deamidase treatment has been confirmed by increased WHC (Table 7). This will in turn lead to higher juiciness of the formulated patties. Juiciness is a well-known consumer quality parameter for plant-based meat products.

It can be concluded that the deamidase treatment will increase WHC, however it was not possible to conclude on an optimal dosage of deamidase in regard to WHC.

Example 6

Changes of secondary protein structure of deamidase pretreated, toasted, defatted soybean flour (Substrate 1) by FT-IR (Example 2)

FT-IR analysis was done in order to investigate the secondary protein structure of the HM extrudates (M. Carbonaro et al. (2012) Amino Acids. Vol. 43, pp. 911-921).

The absorbance measurements were performed using a MB3000 MID FT-IR Spectrometer (ABB Ltd, Zurich, Schweiz) with a DTGS detector and equipped with an ATR (Attenuated Total Reflectance) device with a single reflection diamond crystal. All samples were run as 6 replicates taken from the deamidase treated Substrate 1. The sample was positioned on the crystal surface and squeezed towards the diamond crystal by use of a concave needle compressor. IR spectra were recorded in the range from 4000-500 cm^-1 using a spectral resolution of 4 cm^-1. Each spectrum represents the average of 32 scans ratioed against the background (64 scans) collected with the empty crystal and stored as absorbance spectra (Settings are provided in Table 8).

Table 8 - FT-IR settings for producing the spectra .

The spectra were analysed using LatentiX (v. 2.13). The spectral region containing the amide I band (1700-1600 cm^-1) arising from the stretching vibrations of C=O in the peptide bonds was examined. The vibration energies of the carboxyl group depend on the different conformations of the protein, such as p-sheet and a-helix structures, - and a-turns, and inter- or intra-molecular aggregates. Calculating the second derivatives of the spectra makes it possible to assign the spectral components of the amide I band. In this example the second derivative was calculated using Savitzky-Golay (window size: 13, polynomial order: 2, derivatives: 2).

Principal component analysis (PCA) was performed, and scores and loadings examined in order to study the relationships between or within the different samples and variables and to detect trends, groupings and outliers. The PCA analysis is used as an explorative analysis. The findings from the PCA score plot have been translated into a semi quantitative measure given as a number of pluses (range: 1-3). The conclusions drawn from the PCA are summarised in Table 9.

Table 9: Secondary structure of proteins in pretreated SBF powders (Example 2) determined by FT-IR (range: 1-3).

Conclusion: The deamidase treatment of the SBF resulted in a higher level of intermolecular protein complexes for all dosages in comparison with the blank sample. A slight increase in a- helixes and random coil structure was also seen for deamidase treated samples, while the amount of p-sheets decrease significantly with deamidase treatment. Hence, it seems that the protein structure of the deamidase treated SBF will be more flexible/less ordered when the substrate enters the extruder. This is expected to result in a higher level of fibrous structure in the extrudate. This is confirmed by the results in Table 4 and 5.

Example 7

Protein solubility of Substrate 2 - SPC (Arcon® F) after deamidase treatment (Example 3)

The substrate (Soy protein concentrate (SPC), “Arcon® F” (ADM, Decatur, IL, USA). 68,56% protein, PDI 6%) was prepared for BCA assay by making a mixture with 5% protein substrate in Dl-water. The mixture was hydrated for 30 min at room temperature while being stirred. Deamidase was added and incubated in a Thermomixer (ThermoFisher Scientific, Massachusetts, USA) at 50 °C for 1 hour. The deamidase treatment was inactivated at 85 °C/10 min. The mixture was centrifuged at 14000 rpm for 10 min and the supernatant was used for protein solubility measurement.

The protein solubility analysis was made using a Pierce™ Rapid Gold BCA Protein Assay Kit (ThermoFisher Scientific, Massachusetts, USA). Absorbance of the diluted supernatants was measured at 480 nm on a SpectraMax® Plus 384 absorbance microplate reader (Molecular Devices, CA, USA). A standard curve using bovine serum albumin (BSA) (0.0, 0.2, 0.4, 0.6, 0.8, 1 mg/mL) was used to calculate the protein concentration from the absorbance measurement of the supernatants. The BCA assay was made on a 5% protein solution where the concentration of soluble protein was measured according to the BSA standard (n=4) (Table 10).

Table 10: Protein solubility of SPC (Arcon® F) treated with deamidase at 50 °C for 1 h and subsequent inactivation at 85 °C for 10 min.

Conclusion: The deamidase treatment of Substrate 2 - SPC (Arcon® F) clearly demonstrates increased protein solubility dependent on deamidase dosage.

Example 8

Formulation of meat analogue patties made with SPC HM extrudates (Extrudate 10-13) where the substrate was treated with deamidase before extrusion (Example 3)

Formulation of patties with varying dose of deamidase treated high moisture (HM) extruded SPC and analysis of resistance to cutting (rank 1-5), Texture Profile Analysis (TPA, hardness and chewiness), water holding capacity (WHC) and cooking loss.

The recipe for the burger patties can be seen in Table 11.

Table 11: Recipe fora plant-based burger patty.

HM SPC extrudates were shredded to a mince with an approximate diameter of 2-5mm chunks in a food processor (Bosch Multitalent 3, Germany) by a rotating knife -10-30 sec, visual inspection was used to rank the resistance to cutting.

An emulsion was made by adding soy protein isolate (SPI), spice mix, potato starch, water and beetroot color to the rapeseed oil while mixing for 1 min. The mixing was done by hand for 1 min. Hereafter the shredded HM extrudates were added to the emulsion and gently mixed for 30 sec. Burger patties were shaped in a burger patty form using 50 grams mince and allowed to rest for 1 h at 5 °C before frying or further analysis. Results

The high moisture extrudates treated with deamidase (82, 164 and 328 PGLU/g protein) were noticeable harder to shred in the food processer than the blank extrudate (Table 12).

Table 12: Resistance to cutting - amount of pulse/force to cut the HM extrudates in the food pro- cesser by a rotating knife. Visual inspection and arbitrary ranking ( 1-5).

Texture analysis of the patties was performed using a Texture Analyser (Ta. XT. Plus, Stable Micro Systems, England) fitted with cylinder probe, SMS p/125 mm. All burger patties were subjected to a two-cycle compression test (TPA) (Breene WM, Application of texture profile analysis to instrumental food texture evaluation. J Texture Stud 6:53-82 (1975)). Samples were compressed to 50% of their original height with a test speed of 5 mm s-1 and post-test speed of 5 mm s-1.

Trigger force was set to 50 g and time between the two cycles was set to 5 seconds. Hardness was calculated as max peak force of the first cycle. It can be used as a measure to describe the hardness of a product. Chewiness was calculated as: max peak force * (Area 2 I Area 1) * Distance 2 I Distance 1. Chewiness can be used as a measure describing the energy required to chew a solid food. The results of the texture analysis are presented in Table 13 and Table 14.

Table 13: Texture analysis (hardness: g, force) of HM patties with deamidase treated HM extrudates from SPC (n=3).

Table 14: Texture analysis (chewiness) of HM patties with deamidase treated HM extrudates from SPC (n=3).

The results indicate an increased hardness and chewiness of patties based on deamidase treated HM extrudates. The effect was especially clear for the fried patties and to a lesser extent for the raw patties. The effect appears to be dosage dependent. Increased hardness and chewiness of plant-based meat products similar to animal meat products is well-known to lead to increased consumer liking.

Water holding capacity (WHC) was measured at 25 °C by use of the following method:

Tubes (50 mL) were weighed (triple determinations for each sample). 5.0 g raw patty mince (formulation produced from ingredients from Table 11) was weighed into each tube. Deionized water was added in excess (8 mL). Samples were placed in a rotator (20 rpm) at room temperature for 15min. The samples were centrifuged at 4600 rpm for 10 min at 20 °C. The supernatant was carefully discarded, using a cotton swab to remove fatty residues on the inside of the tube. The tubes containing precipitate were weighed again and WHC was calculated. The calculations were done using dry matter (DM) content in samples. The WHC can be defined as water retained in raw/unfried patty mince.

DM was measured by use of following method:

Alu-trays were weighed (triple determinations for each sample). 0.5 g sample was weighed into each alu-tray. The alu-trays with sample were placed in an oven at 105 °C for min. 16 hours. Alu- trays with sample were reweighed a DM was calculated.

Resutls from the WHC analysis are given in Table 15.

Table 15: %WHC of raw burger mince with deamidase treated HM extrudates (n=3).

Conclusion: WHC evaluated on raw patty mince appears to result in an improved water holding capacity at the lowest dosage (82 PGLU/g protein) indicating a possible optimal dosage deamidase in relation to WHC.

Cooking loss were measured by use of the following method:

The raw burger patty mince was formed and weighed (approx. 50 g. and triple determinations for each sample). Hereafter, the patties were fried in a pan at level 3/10 for 3 min 30 secs, on each side using an induction stove (Steba IK 55, Germany). The frying of the patties ensured a center temperature of at least 75 °C. The fried patties were reweighed, and cooking loss was calculated using the formula: (Raw patty (g)/cooked patty(g))/(Raw patty (g)/100).

Table 16: Amount of cooking loss (%) when HM patties were fried at level 3/10 for 3 min and 30 sec on each side in preheated steel pan on an induction stove (n=3).

Conclusion: Cooking loss evaluated on raw patty mince resulted in a reduced cooking loss at the lowest dosage (82 PGLU/g protein), and a less pronounced effect at the medium dose. The results indicate a possible optimal dosage at 82 PGLU/g protein for reduced cooking loss.

Claims

1. A method for producing a plant-based meat analogue comprising the steps: a) preparing a mixture of plant protein containing material, having a protein content by dry weight of the plant material of 15% w/w to 95% w/w, and water, with a water content by weight of the mixture of 5% w/w to 99% w/w; b) treating the mixture with a protein-deamidase enzyme; and c) passing the mixture at a temperature above 60°C, through an extruder; d) optionally mincing or shredding the extruded protein material; e) optionally drying the product of c) or d); and f) optionally mixing the plant protein material with other ingredients to obtain the meat analogue product.

2. The method according to claim 1 , wherein the protein-deamidase is protein-glutaminase.

3. The method according to any of claims 1-2, wherein the plant protein material is derived from pulses, e.g., beans, peas, lentils, chickpea, or from oil crops, e.g., soybean, peanuts, corn; from seeds, e.g., hemp, sunflower, flax, sesame, chia, canola.

4. The method according to any of the preceding claims, wherein the deamidase is added before or during step c).

5. The method of any of claims 1-4, wherein the deamidase is added before step c), and the water content by weight of the mixture is 5% w/w to 50% w/w, 10% w/w to 40% w/w, such as in the range from 20% w/w to 35% w/w.

6. The method of any of claims 1-4, wherein the deamidase is added during step c), and the water content by weight of the mixture in the extruded product after step c) is 45% w/w to 70% w/w, such as in the range from 50% w/w to 65% w/w.

7. The method of claim 6, wherein water is added during extrusion in amounts selected from 1.2- 3.0 g water/g protein in the plant material.

8. The method of any of claims 1-4, wherein the deamidase is added during step c), and the water content by weight of the mixture in the extruded product after step c) is 1 % w/w to 45% w/w, such as 2% w/w to 25% w/w, particularly 5-15%.

9. The method of claim 8, wherein water is added during extrusion in amounts selected from 0.05-1.0 g water/g protein in the plant material.

10. The method according to any of the preceding claims, wherein the protein content by dry weight of the plant material is in the range from 25% w/w to 92% w/w, such as 45% w/w to 75% w/w.

11 . The method according to any of the preceding claims, wherein step c) is performed at a temperature in the range of 65-200 °C, 100-180 °C, such as 120-175 °C.

12. The method of any of the preceding claims, wherein step b) is performed before step c) and, wherein the incubation time is from 1 to 120 min, 1 to 60 min, such as 1 to 15 min.

13. The method of claim 12, wherein the temperature is in the range from 20-95 °C, such as 30- 70 °C.

14. The method of any of the preceding claims, wherein the plant-based meat analogue product after the extrusion step has increased cutting strength, and wherein the relative increase in cutting strength of the extrudate is at least 25%, at least 40%, at least 50%, such as at least 75% compared to a no deamidase control.

15. The method of any of the preceding claims, wherein the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative hardness increase of at least 1%, at least 2%, at least 5%, at least 10%, such as at least 15% compared to patties made from a no deamidase control extrudate.

16. The method of any of the preceding claims, wherein the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative increase in chewiness of at least 5%, at least 10%, at least 15%, such as at least 20% compared to patties made from a no deamidase control extrudate.

17. The method of any of the preceding claims, wherein the plant-based meat analogue product has increased water holding capacity compared to the plant-based material not treated with a deamidase, and wherein the water holding capacity of the extrudate is at least 4-6 g water/g extrudate, and/or wherein the plant-based meat analogue product, e.g., patties made from the extrudate, have a relative increase in water holding capacity of at least 5%, at least 10%, at least 15%, such as at least 20%.