Method for producing a meat analog product containing a protein deamidase

Abstract

The present invention relates to a method for producing a meat analog product which comprises texturing a non-animal protein, such as a vegetable protein, and adding a deamidase before or during the texturing process.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to a method for producing a plant-based meat analog that involves texturing a vegetable protein material. [Background technology]

[0003] A growing world population requires protein-rich, sustainably produced foods. Legumes, such as soybeans and beans, are attractive crops for the production of protein-rich foods. However, other non-animal protein sources, for example from plants, algae, or insects, are also widely used in food production.

[0004] Texturized products, such as extruded products, are widely used in the food industry. Extrusion is primarily used to impart a particular texture and unique mouthfeel to food products.

[0005] Osen et al. (2014), Journal of Food Engineering 127:67-74, studied high-moisture extrusion cooking of pea protein isolates. They found that the functional properties of three different pea proteins were affected by high cooking temperatures above the protein denaturation temperature, which played a minor role during fiber formation.

[0006] Xu et al. (2020), Trends in Food Science & Technology 99, 167-180, provide an overview of developments in reactive extrusion (REX) and enzyme-based processing of food-related biopolymers. They divide REX enzymatic processes into two main technology categories: (1) REX-EH processes, which are sequential systems involving extrusion pretreatment followed by enzymatic hydrolysis, and (2) eREX processes, which involve the introduction of exogenous enzymes before or during REX. Xu et al. conclude that eREX is still an immature area, with the exception of starch saccharification.

[0007] Chen et al. (2011), Food Hydrocolloids 25:887-897, studied the effect of combined extrusion pretreatment followed by controlled enzymatic hydrolysis on the emulsifying properties of soy protein isolate.

[0008] Czarnecki et al. (1993), Journal of Food Science 58:395-398, incubated a high-protein fraction of mung bean with papain and cellulase for 24 or 72 hours, followed by freeze-drying, grinding, sieving and extrusion to obtain a novel snack-type product derived from legumes.

[0009] Zhou et al. (2017), J Food Process Preserv. 41:e13301, studied the effect of extrusion and papain co-extrusion on pea proteins and on antioxidant peptides produced by further papain hydrolysis of the extrudates.

[0010] WO 2017 / 117398 (Abbott) discloses a method for preparing a protein hydrolysate comprising adding an intact protein source and a protease component to an extruder.

[0011] WO 2018 / 125920 (Abbott) discloses a method for preparing a nutritional powder comprising adding an intact protein source, a protease component, a fat component and water to an extruder.

[0012] Extrusion of vegetable proteins can give the proteins a fibrous, meat-like structure.

[0013] Meat analog products are meat substitutes made from, for example, vegetable proteins designed to mimic the visual appearance, texture, and taste of meat products. Meat analog products can be made, for example, by extrusion of soy, wheat, or pea proteins. Extrusion is a thermomechanical texturing process that unfolds proteins. In high-moisture (HM) extrusion processes, a cooling die allows the proteins to rearrange and form new intermolecular covalent and non-covalent bonds. The cooling die at the end of the extruder creates a dense, layered, fibrous, meat-like structure. In low-moisture (LM) extrusion processes, the product expands as it leaves the die, forming a solid structure with a fibrous, insoluble network that can absorb up to three times its weight in liquid.

[0014] Texturized vegetable proteins, such as high moisture or low moisture extruded vegetable proteins, can be formed into a variety of shapes (chunks, flakes, nuggets, granules and strips) and sizes.

[0015] Extrusion has been used for decades in the production of meat analogs to obtain meat-like structures from plant proteins. However, obtaining extrudates of plant proteins, such as legume proteins, with acceptable textural and functional properties for use in meat analogs remains challenging. This means there is room for improvement to better mimic meat-like structures without the need for the addition of non-natural ingredients. International Publication WO 2020 / 038541 (Raisio Nutrition) discloses a method for producing plant-based meat substitutes by passing a mixture containing a plant protein material, an oat material, and optionally a cross-linking enzyme and / or a protein deamidating enzyme through an extruder at a temperature between 25 and 55°C. Meat substitutes containing transglutaminase were produced using both low-temperature and high-temperature extrusion, and the low-temperature samples rated better than the corresponding high-temperature samples for each sensory attribute tested, as well as for overall quality evaluation.

[0016] Chinese Patent Application Publication No. 109619208A (UNIV HEILONGJIANG BAYI AGRICULTURAL) discloses a method for preparing an imitation meat food product with enhanced flavor by extrusion puffing of ingredients containing bean seed flour and soy protein. Ultrasonic enzymolysis using Alcalase and Flavorzyme can be applied for a period of 10-20 minutes before extrusion puffing, preferably after the Maillard reaction, to provide an intense flavor and persistent aroma.

[0017] Nisov et al. (2022), Food Research International 156 (2022) 111089, investigated the effect of pH and temperature on the fibrous structure formation of vegetable proteins during high-moisture extrusion processing. They concluded that the structure formation of the extrudates can be positively affected by increasing the pH of the feedstock, which facilitates the structuring of vegetable proteins into attractive meat analogue products.

[0018] WO 2022 / 218863 discloses that the application of proteases increases solubility, resulting in improved tissue functionality of the final product. Summary of the Invention [Problem to be solved by the invention]

[0019] It is an object of the present invention to provide a textured vegetable protein material with better functional properties that make it suitable for use in meat analog products such as burger patties, ground meat, sausage or chicken nugget analogs. [Means for solving the problem]

[0020] The inventors have surprisingly found that adding a deamidase to a plant protein material prior to or during a texturing process, such as an extrusion process, provides a texturized plant protein material that provides improved properties, such as higher firmness and / or higher water-holding capacity, when used in plant-based meat products, such as burger patties. The effect of adding a deamidase during the extrusion process is to provide a plant-based burger patty or similar with a higher firmness, resulting in a more meat-like texture. The inventors have further found, in accordance with the literature, that a higher water-holding capacity in the enzyme-treated extrudate results in a higher perceived umami flavor intensity of the plant-based meat product.

[0021] Accordingly, the present invention provides a method for producing a plant-based meat analog, comprising: a) preparing a mixture of a plant protein-containing material having a protein content of 15 w / w% to 95 w / w% of the dry weight of the plant material and water having a moisture content of 5 w / w% to 99 w / w% of the weight of the mixture; b) treating the mixture with a protein deamidase enzyme; c) passing the mixture through an extruder at a temperature greater than 60°C; d) optionally shredding or chopping the extruded protein material; e) optionally drying the product of c) or d); f) optionally mixing the vegetable protein material with other ingredients to obtain a meat analog product.

[0022] definition Deamidase: The term "deamidase" refers to protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase) activity as described in EC 3.5.1.44, which catalyzes the hydrolysis of gamma-amides of glutamine substituted at the carboxyl position or at 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 glutamic acid residues and are also referred to as protein glutamine deamidases. The deamidase contains the Cys-His-Asp catalytic triad (Cys-156, His-197, and Asp-217 as shown, e.g., 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 belongs to InterPro entry IPR041325.

[0023] Deamidase activity: Deamidase (protein glutaminase) activity can be determined using the following assay.

[0024] Glutaminase reacts with glutamine substrate (Z-GLN-GLY,C 15 H 19 N3O 6) deamidates glutamate, producing ammonia in the process, which in combination with α-ketoglutarate is used as a substrate for glutamate dehydrogenase to produce glutamate.

[0025] This latter enzymatic reaction requires NADH as a coenzyme. NADH depletion can be followed by kinetic absorbance measurement at 340 nm and is directly proportional to glutaminase activity. The reaction temperature is 37°C, pH 7.0, and the reaction time is 216 seconds.

[0026] Isolated: The term "isolated" refers to 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, other proteins, nucleic acids, cells, etc. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide.

[0027] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its mature form after N-terminal processing (e.g., removal of a signal peptide). In one aspect, the mature polypeptide is amino acids 1-294 of SEQ ID NO: 1, which contains the propeptide sequence of aminino acids 1-109 of SEQ ID NO: 1. In one embodiment, the deamidase after cleavage of the propeptide is amino acids 1-185 of SEQ ID NO: 2.

[0028] In another aspect, the mature polypeptide is amino acids 1-297 of SEQ ID NO: 3, which contains the propeptide sequence of aminino acids 1-112 of SEQ ID NO: 3. In one embodiment, the deamidase following cleavage of the propeptide is amino acids 1-185 of SEQ ID NO: 4.

[0029] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0030] Purified: The term "purified" refers to a nucleic acid or polypeptide that is substantially free of 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, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, typically 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 or molar basis). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule after application of a purification or concentration 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 higher relative or absolute concentration compared to the starting composition.

[0031] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0032] For the purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output 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 on the command line. The Needle output labeled "longest identity" is calculated as follows: (Identical residues × 100) / (length of alignment − total number of gaps in alignment) DETAILED DESCRIPTION OF THE INVENTION

[0033] The inventors have surprisingly found that adding a deamidase to a vegetable protein material prior to or during a texturing process, such as an extrusion process, provides a texturized vegetable protein material with improved properties, such as increased cut strength and water holding capacity.

[0034] Thus, in a first aspect, the present invention relates to a method for producing a plant-based meat analogue, comprising the steps of: a) preparing a mixture of a vegetable protein material having a protein content of between 15 w / w% and 95 w / w% of the dry weight of the vegetable material and water having a moisture content of between 5 w / w% and 99 w / w% of the weight of the mixture; b) treating the mixture with a protein deamidase enzyme; c) passing the mixture through an extruder at a temperature greater than 60°C; d) optionally shredding or chopping the extruded protein material; e) optionally drying the product of c) or d); f) optionally mixing the vegetable protein material with other ingredients to obtain a meat analog product.

[0035] The vegetable protein material may be obtained from, for example, pulses such as beans, peas, lentils, chickpeas, or oil grains such as soybeans, peanuts, canola, cereals such as rice, corn, seeds such as hemp, sunflower, flax, sesame, chia, canola, and any combination thereof.

[0036] In a preferred embodiment, the vegetable protein is soy protein or pea protein.

[0037] The vegetable protein may be a protein material such as a vegetable protein material or a legume protein material, preferably a vegetable protein or legume protein having a dry matter ratio of protein of 15 to 95% (w / w). Preferably, the vegetable protein material is a soybean protein material, a pea protein material, a chickpea protein material, a mungbean protein material, a lentil protein material or a fava bean protein material, more preferably a soybean protein or a pea protein.

[0038] Extrusion Extrusion cooking has been used to produce meat analogues since the 1960s, particularly using soy protein as a raw material, and until a few years ago it was primarily used as a meat extender in the traditional meat industry to reduce the cost of ground meat. However, especially over the last decade, much more attention has been paid to finding extrudates applicable as complete replacements for meat-based patties and sausages (see, e.g., Chapter 6 "Plant-Based Meat Analogues" by K. Kyriakopoulou et al. in "Sustainable Meat Products and Processing," 2018 ed. Charis Galanakis, ISBN electronic 9780128156889).

[0039] Extrusion is a thermomechanical process that plasticizes moist, expandable starchy and proteinaceous food materials and forces them through a die using a combination of pressure, heat, and mechanical shear. A typical extruder setup consists of a feed system, screw, barrel, die, and cutter. Additionally, a pretreatment system can optionally be incorporated prior to extrusion. The extruder barrel can be divided into five to nine sections, often referred to as temperature zones, which can be heated separately. Materials are added to the first section of the extruder using volumetrically or gravimetrically controlled feeders. A pump supplies water, optionally containing enzymes, or preconditioned materials with adjusted moisture content to the second section of the extruder. The screw configuration used can consist of forward and reverse conveying elements. In the feed zone, materials are mixed and homogenized before being conveyed to the compression zone, where there is a decrease in screw depth and pitch, resulting in an increase in shear rate, temperature, and pressure. Mechanical energy dissipated through screw rotation increases the processing temperature inside the extruder. Between 70°C and 180°C, proteins denature, creating a viscoelastic mass that can be aligned in the cooling die. In essence, this change in process conditions converts the solid material into a fluid melt. Maximum temperature and pressure are reached before exiting the extruder, immediately reducing the viscosity of the extruded material. In the case of high-moisture extrusion meat analogs, the cooling die is long to prevent excessive material expansion, which can create alignment and destroy the newly formed structure.

[0040] Successful preparation of meat analog products requires control of extrusion parameters such as screw speed, moisture content of the feedstock, barrel temperature, extruder characteristics, and chemical and physical composition of the feedstock. Those skilled in the art know how to adjust the extrusion parameters to optimize the process.

[0041] For meat analogue manufacturing, two product categories have developed based on the amount of water added during extrusion: high moisture and low moisture extruded.

[0042] In low-moisture extrusion, low-moisture flour or concentrate is converted into textured vegetable protein (TVP), also called dry extrudate; the ingredients are hydrated during extrusion, e.g., to 5-15% moisture, resulting in extrudates with a lower final moisture content. These are rehydrated and mixed with other ingredients before cooking, e.g., cooking in oil, for the final meat analog recipe. Products from low-moisture extrudates exhibit a sponge-like structure, expanding and rapidly absorbing water. They have typically been used as meat extenders, but more recently have also been used partially or completely as meat analogs, such as in sausages and beef patties.

[0043] In high-moisture extrusion, the ingredients are hydrated during extrusion, for example, to 45-70% moisture, resulting in an extrudate with a higher final moisture content. Most often, co-rotating twin-screw extruders are used for this application, and the product is either used directly for further processing or frozen after extrusion to extend shelf life and, in some cases, strengthen the structure. The high-moisture product can be used either as is, or shredded to imitate goulash, such as chicken or meat pieces, or chopped into various sizes for inclusion in patties or sausages.

[0044] According to Egbert and Borders, 2006, "Achieving success with meat analogs," Food Technology, Chicago, 60(1):28-34, meat analog products may contain water (50-80%), textured vegetable protein (10-25%), untextured protein (4-20%), flavor (3-10%), fat (0-15%), binder (1-5%), and color (0-0.5%). The combination of ingredients results in a meat analog that is acceptable in terms of organoleptic attributes. A high moisture content not only reduces product cost, but also provides the desired umami intensity and acts as a plasticizer during processing, aiding in emulsification.

[0045] In a preferred embodiment of the process of the present invention, the vegetable protein is passed through an extruder, preferably a twin screw extruder such as a co-rotating or counter-rotating twin screw, more preferably a co-rotating twin screw extruder.

[0046] The protein is preferably passed through the extruder at a temperature of 65 to 200°C, for example, 90 to 200°C, preferably 100 to 180°C, and more preferably 120 to 175°C.

[0047] In a preferred embodiment, the extruder has two or more temperature zones, for example, 2 to 10 zones, and preferably 5 to 9 zones. Therefore, the starting temperature in the first zone may be lower than the above-mentioned preferred temperature range. For example, the starting temperature may be in the range of 20 to 60°C.

[0048] According to the present invention, the deamidase can be added before or during step c).

[0049] In one embodiment, the deamidase is added before step c) and the water content of the mixture is in the range of 5 w / w% to 50 w / w%, 10 w / w% to 40 w / w%, for example 20 w / w% to 35 w / w% by weight.

[0050] In another embodiment, the deamidase is added during extrusion step c) and the water content of the mixture in the extruded product is in the range of 45 wt. % to 70 wt. % by weight, for example 50 wt. % to 65 wt. %. In particular, according to this embodiment, water is added during extrusion in an amount selected from 1.2 to 3.0 g water / g protein of the plant material.

[0051] In another embodiment, the deamidase is added such that the water content of the mixture in the extruded product after step c) is in the range of 1% to 45% w / w by weight, for example 2% to 25% w / w by weight, in particular 5% to 15% by weight. In particular, according to this embodiment, water is added during extrusion in an amount selected from 0.05 to 1.0 g water / g protein of the plant material.

[0052] The protein content of the plant material is in the range of 15 w / w% to 95 w / w%, more preferably 25 w / w% to 92 w / w%, for example 45 w / w% to 75 w / w%, on a dry weight basis.

[0053] In embodiments in which the deamidase treatment is carried out before the extrusion step, the reaction temperature and incubation time may vary depending on the deamidase activity of the added enzyme, but the incubation time is preferably in the range of 1 to 120 minutes, 1 to 60 minutes, e.g., 1 to 15 minutes, and the incubation temperature is in the range of 20 to 95°C, e.g., 30 to 70°C.

[0054] Shear Cell Technology Recognizing that extrusion, while effective, is not a well-defined process, a technology based on well-defined shear flow deformation was introduced a decade ago to produce textiles. Shearing devices, so-called shear cells, inspired by rheometer design, were developed to apply intense shear in cone-in-cone or couette geometries. The final structure achieved with this technology varies depending on the ingredients and processing conditions. Fibers can be obtained with several vegetable protein blends, such as soy protein concentrate or soy protein isolate (SPI) blended with wheat gluten (WG), pectin, and / or starch. Fibers can also be obtained with calcium caseinate. This 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 also typically applied when using the shear cell technology.

[0055] Similar to the extrusion process, deamidases can be applied in other, perhaps milder processes for making meat analogs, such as shear cell technology, which improves the performance of the extrudate and the final compounded product as well.

[0056] Deamidase In the methods of the present invention, a protein deamidase is added to a non-animal protein, such as a vegetable protein, before or during a texturing process, such as an extrusion process or a shear cell technology process.

[0057] In the present invention, protein deamidase refers to an enzyme that acts directly on the amide groups in the side chains of amino acids that constitute proteins, causing deamidation without cleaving the peptide bonds of proteins and cross-linked proteins, thereby releasing ammonia. A specific example of protein deamidase is protein glutaminase (EC 3.5.1.44), which acts directly on the amide groups in the side chains of glutamine residues contained in proteins, releasing ammonia and thereby converting the glutamine residues to glutamic acid residues. Deamidases also include protein asparaginase, which acts directly on the amide groups in the side chains of asparagine residues contained in proteins, releasing ammonia and thereby converting the asparagine residues to aspartic acid residues. In the present invention, either protein glutaminase or protein asparaginase can be used as the protein deamidase, or both can be used in combination. A preferred example of the protein deamidase used in the present invention is protein glutaminase.

[0058] The protein deamidase used in the methods 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 with reference to a given source, shall mean that the polypeptide encoded by the polynucleotide is produced by the source or a strain into which a polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0059] The type or origin of the protein deamidase used in the present invention is not particularly limited. Examples of protein deamidases include protein deamidases derived from the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroide.

[0060] European Patent No. 1839491 discloses the cloning of a protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum. Deamidases, such as protein glutaminases from the genus Chryseobacterium, are commercially available. Preferred examples include protein deamidases from the genus Chryseobacterium, and more preferred examples include protein deamidases from Chryseobacterium proteolyticum. Protein glutaminases from Chryseobacterium proteolyticum are commercially available, for example, as protein-glutaminase.

[0061] "Amano" 500 manufactured by Amano Enzyme Inc. and its commercially available products can be used.

[0062] For example, the protein deamidase can be obtained from the culture broth of the above microorganisms.

[0063] Deamidase activity: Deamidase (protein glutaminase) activity can be determined using the following assay.

[0064] Glutaminase reacts with glutamine substrate (Z-GLN-GLY,C 15 H 19 N3O6) deamidates, producing ammonia in the process, which in combination with α-ketoglutarate is used as a substrate for glutamate dehydrogenase to produce glutamate.

[0065] This latter enzymatic reaction requires NADH as a coenzyme. NADH depletion can be followed by kinetic absorbance measurement at 340 nm and is directly proportional to glutaminase activity. The reaction temperature is 37°C, pH 7.0, and the reaction time is 216 seconds.

[0066] According to a preferred embodiment, the deamidase applied in the process of the present invention is derived from or obtainable from a Chryseobacterium species, for example Chryseobacterium proteolyticus.

[0067] More specifically, the deamidase, in one embodiment, (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 the 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 to 30 mutations, for example, substitutions, deletions and / or insertions at one or more positions, for example, 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 mutations, particularly substitutions; (e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; (f) a fragment of the polypeptide of (a), (b), (c), or (d); The polypeptide has deamidase activity.

[0068] In another particular embodiment, the deamidase is (a) a polypeptide having at least 75% sequence identity to SEQ ID NO:3; (b) a polypeptide having at least 75% sequence identity to SEQ ID NO:4; (c) a polypeptide having at least 75% sequence identity to the 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 to 30 mutations, for example, substitutions, deletions and / or insertions at one or more positions, for example, 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 mutations, particularly substitutions; (e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; (f) a fragment of the polypeptide of (a), (b), (c), or (d); The polypeptide has deamidase activity.

[0069] 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.

[0070] 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.

[0071] 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 the mature polypeptide of SEQ ID NO:1.

[0072] 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.

[0073] 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.

[0074] 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 the mature polypeptide of SEQ ID NO:3.

[0075] In the context of the present invention, the term "variant" refers to a polypeptide having endopeptidase activity that contains an alteration, i.e., a substitution, insertion, and / or deletion, at one or more (e.g., several) positions. A substitution refers to the replacement of an amino acid at a position with a different amino acid, a deletion refers to the removal of an amino acid at a position, and an insertion refers to the addition of one or more (e.g., several) amino acids, e.g., 1 to 5 amino acids, adjacent to and immediately following the amino acid at a position.

[0076] Amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding domain.

[0077] Examples of conservative substitutions are within the group 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 low molecular weight amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not change the specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0078] Alternatively, the amino acid changes may be of such a nature that they alter the physicochemical properties of the polypeptide, e.g., they may affect the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0079] Important amino acids in a polypeptide can be identified using 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 a molecule to identify amino acid residues critical to the molecule's activity, and the resulting mutant molecules are tested for endopeptidase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., 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 identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0080] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by associated screening procedures such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. 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).

[0081] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0082] In a preferred embodiment of the present invention, the deamidase is added to the vegetable protein just before or during the extrusion step c), which is to be understood as meaning that the deamidase is not added to the vegetable protein just before or during the extrusion step c) by pre-incubation.

[0083] In a particularly preferred embodiment, the vegetable 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 vegetable protein and the aqueous solution of the deamidase are added separately to the feed zone of the extruder.

[0084] The deamidase may also be added to the aqueous solution or suspension of vegetable protein just before feeding into the extruder, i.e., without a pre-incubation step.

[0085] In other embodiments of the invention, the deamidase is added to and incubated with at least a portion of the vegetable protein prior to step c). In such embodiments, mixing with other ingredients may occur before or during incubation of at least a portion of the vegetable protein with the deamidase, but before step c). and / or mixing with other ingredients may occur after step c).

[0086] In the method of the present invention, the vegetable protein is mixed with other ingredients to obtain a meat analog product. The mixing with other ingredients can occur before step c) or it can occur after step c).

[0087] The one or more ingredients may be selected from, for example, soy protein isolate, disorganized vegetable protein such as wheat gluten, fiber such as pectin, starch such as corn starch, pea starch and / or potato starch, salt, color, aroma, flavor, spices, and / or oil or fat such as coconut oil, sunflower oil and / or rapeseed oil.

[0088] For example, disorganized vegetable proteins such as soy protein isolates can be added as a binding agent, for example as an ingredient in a burger patty.

[0089] The meat analog product produced by the method of the present invention can be, for example, a ground meat analog product, a burger patty, a sausage, a meatball analog product, a chicken nugget analog product, a goulash meat analog product, or a schnitzel analog product. In a preferred embodiment, the meat analog product produced by the method of the present invention is a burger patty.

[0090] Improved properties of the extruded products and meat analogs produced by the process of the present invention. The methods of the present invention surprisingly result in extruded and meat analog products with improved properties. In one embodiment, these improved properties include increased cut strength of the plant-based meat analog product after the extrusion process. In one embodiment, the plant-based extrudate has increased cut strength, with the relative increase in cut strength of the extrudate being at least 25%, at least 40%, at least 50%, e.g., at least 75%, compared to extrudate without added deamidase.

[0091] In another embodiment, the water holding capacity of the plant-based meat analog product is increased compared to plant-based material that has not been treated with a deamidase.

[0092] In one embodiment, the extrudate according to the invention has a water holding capacity of at least 4-6 g water / g extrudate.

[0093] In another embodiment, the patty made from the deamidase-treated extruded plant material has a relative increase in water holding capacity of at least 5%, at least 10%, at least 15%, such as at least 20%.

[0094] Another improved property of the meat analog products of the present invention is the increased chewiness and firmness of plant-based burger patties comprising the extrudates described in this invention.

[0095] In particular, in one embodiment, the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in firmness of at least 1%, at least 2%, at least 5%, at least 10%, e.g., at least 15%, compared to a patty made from a control extrudate without the deamidase.

[0096] In particular, in one embodiment, the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in chewiness of at least 5%, at least 10%, at least 15%, e.g., at least 20%, compared to a patty made from a control extrudate without the deamidase.

[0097] The present invention is further disclosed in the following numbered embodiments.

[0098] Embodiment 1. A method for producing a plant-based meat analog, comprising: a) preparing a mixture of a plant protein-containing material having a protein content of 15 w / w% to 95 w / w% of the dry weight of the plant material and water having a moisture content of 5 w / w% to 99 w / w% of the weight of the mixture; b) treating the mixture with a protein deamidase; c) passing the mixture through an extruder at a temperature greater than 60°C; d) optionally shredding or chopping the extruded protein material; e) optionally drying the product of c) or d); f) optionally mixing the vegetable protein material with other ingredients to obtain a meat analog product.

[0099] Embodiment 2. The method of embodiment 1, wherein the protein deamidase is a protein glutaminase.

[0100] Embodiment 3. The method of any of embodiments 1-2, wherein the vegetable protein material is derived from pulses, such as beans, peas, lentils, chickpeas, or oil grains, such as soybeans, peanuts, canola, cereals, such as rice, corn, seeds, such as hemp, sunflower, flax, sesame, chia, canola, and any combination thereof.

[0101] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the deamidase is added before or during step c).

[0102] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the deamidase is added before step c) and the water content of the mixture is in the range of 5 w / w% to 50 w / w%, 10 w / w% to 40 w / w%, for example 20 w / w% to 35 w / w% by weight.

[0103] Embodiment 6. The method of any one of embodiments 1 to 4, wherein a deamidase is added during step c) and the moisture content of the mixture in the extruded product after step c) is in the range of 45 w / w% to 70 w / w% by weight, for example 50 w / w% to 65 w / w%.

[0104] Embodiment 7. The method of embodiment 6, wherein water is added during extrusion in an amount selected from 1.2 to 3.0 g water / g protein in the plant material.

[0105] Embodiment 8. The method according to any one of embodiments 1 to 4, wherein a deamidase is added during step c) and the moisture content of the mixture in the extruded product after step c) is 1 w / w% to 45 w / w%, for example 2 w / w% to 25 w / w%, in particular 5 to 15%.

[0106] Embodiment 9. The method of embodiment 8, wherein water is added during extrusion in an amount selected from 0.05 to 1.0 g water / g protein in the plant material.

[0107] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the protein content of the plant material is in the range of 25 w / w% to 92 w / w%, for example in the range of 45 w / w% to 75 w / w%, by dry weight.

[0108] Embodiment 11. The method of any one of embodiments 1 to 10, wherein step c) is carried out at a temperature in the range of 65 to 200°C, 100 to 180°C, for example, 120 to 175°C.

[0109] Embodiment 12. The method according to any one of embodiments 1 to 11, wherein step b) is carried out before step c), and the incubation time is 1 to 120 minutes, 1 to 60 minutes, for example 1 to 15 minutes.

[0110] Embodiment 13. The method of embodiment 12, wherein the temperature is in the range of 20 to 95°C, for example, 30 to 70°C.

[0111] Embodiment 14. The method of any of embodiments 1-13, wherein the plant-based meat analog product after the extrusion step has increased cut strength, wherein the relative increase in cut strength of the extrudate compared to a control without deamidase is at least 25%, at least 40%, at least 50%, such as at least 75%.

[0112] Embodiment 15. The method of any one of embodiments 1-14, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in firmness of at least 1%, at least 2%, at least 5%, at least 10%, e.g., at least 15%, compared to a patty made from a control extrudate without the deamidase.

[0113] Embodiment 16. The method of any one of embodiments 1-15, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in chewiness of at least 5%, at least 10%, at least 15%, such as at least 20%, compared to a patty made from a control extrudate without the deamidase.

[0114] Embodiment 17. The method of any one of embodiments 1-16, wherein the plant-based meat analog product has an increased water-holding capacity compared to a plant-based material that has not been treated with a deamidase.

[0115] Embodiment 18. The method of embodiment 17, wherein the water holding capacity is at least 4-6 g of water / g of extrudate.

[0116] Embodiment 19. The method of embodiment 17, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in water holding capacity of at least 5%, at least 10%, at least 15%, such as at least 20%.

[0117] Embodiment 20. The deamidase is (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 the 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 to 30 mutations, for example, substitutions, deletions and / or insertions at one or more positions, for example, 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 mutations, particularly substitutions; (e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; (f) a fragment of the polypeptide of (a), (b), (c), or (d); 20. The method of any one of embodiments 1 to 19, wherein the polypeptide has deamidase activity.

[0118] Embodiment 21. The deamidase is (a) a polypeptide having at least 75% sequence identity to SEQ ID NO:3; (b) a polypeptide having at least 75% sequence identity to SEQ ID NO:4; (c) a polypeptide having at least 75% sequence identity to the 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 to 30 mutations, for example, substitutions, deletions and / or insertions at one or more positions, for example, 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 mutations, particularly substitutions; (e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; (f) a fragment of the polypeptide of (a), (b), (c), or (d); 21. The method of any one of embodiments 1 to 20, wherein the polypeptide has deamidase activity.

[0119] 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.

[0120] 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.

[0121] 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 the mature polypeptide of SEQ ID NO:1.

[0122] 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.

[0123] 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.

[0124] 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 the mature polypeptide of SEQ ID NO:3. [Example]

[0125] overview Deamidase enzymes were tested in the extrusion process to improve the firmness of the extrudate and, consequently, the chewability of the final plant-based meat product. Deamidase was added both in the pretreatment step and during extrusion. Both laboratory and pilot-scale extrusion equipment were included in the study.

[0126] Despite the harsh conditions (high temperature and shear) during the extrusion process, the addition of deamidase directly in the extrusion barrel in high moisture (HM) fully toasted defatted soy flour appeared to increase fiber formation in the extrudates, which is expected to correlate with increased bite strength / hardness in the final plant-based product. To optimize the enzyme effect, a pretreatment step was introduced prior to HM extrusion of the substrate. Deamidase pretreatment of soy protein concentrate (SPC) followed by HM extrusion improved the meat-like texture and resulted in significantly higher extrudate cut strength. Increased extrudate cut strength or hardness is generally desirable and can facilitate the upgrading of extrudates produced using less processed, less refined, and lower-cost raw materials.

[0127] The extrudates were formulated into patties similar to commercially available ones. Both the extrudates and patties were analyzed by chemical and physical analysis. The applied methods are well known from the existing food and meat industries. Texture analysis of patties made using deamidase-treated SPC extrudates showed that the addition of deamidase increased the hardness and chewiness of raw and cooked patties. Thus, the addition of deamidase results in plant-based meat with increased hardness and chewiness similar to conventional meat products. Furthermore, patties made using deamidase-treated extrudates significantly reduced cooking loss at minimal dosages.

[0128] Water holding capacity (WHC) analysis suggests that the addition of deamidase improved the WHC in the extrudates compared to the untreated samples, which is expected to correlate with increased umami intensity in the final product. Similarly, low-field NMR analysis indicates that deamidase treatment results in modified water binding, with the higher mobility of water in the deamidase-treated extrudates also indicating increased umami intensity.

[0129] Spectroscopic analysis (FT-IR) further showed modified secondary protein structure in the deamidase-pretreated substrate, reflecting better solubilization resulting in improved covalent and non-covalent bond formation in the final extrudate.

[0130] material KK Chryseobacterium sp.-62563 was isolated from a soil sample collected in September 2013 in Sibhult, Sweden.

[0131] Base material Substrate 1: Well-toasted defatted soybean flour (SBF), "NutriSoy®" (ADM, Decatur, IL, USA) 50.9 g / 100 g dry matter.

[0132] Substrate 2: Soy protein concentrate (SPC), "Arcon® F" (ADM, Decatur, IL, USA). 68.56 g / 100 g dry matter.

[0133] enzyme The deamidases (EC 3.5.1.44) used in the examples are as follows: Protein glutaminase from Chryseobacterium sp.-62563, having a mature polypeptide 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 was glutamyl endopeptidase from Bacillus licheniformis. The active deamidase obtained after maturation was the polypeptide shown as SEQ ID NO: 2.

[0134] Example 1 High moisture extrusion of fully roasted defatted soy flour (substrate 1) with direct feeding of deamidase in a laboratory-scale extruder (extrudates 1-5) Extrusion tests for the deamidase were performed using a laboratory-scale extruder (Process 11, Thermo Fisher Scientific, Karlsruhe, Germany). The extruder had intermeshing co-rotating twin screws. The screw diameter and extruder length to diameter ratio were 11 mm and 40:1, respectively. The raw materials were metered into the extruder by a gravimetric twin-screw feeder (MT-S, MiniTwin, Brabender Technologie, Duisburg, Germany). The extruder had seven internal heating zones and one external heating zone.

[0135] Extrudates were made from Substrate 1: fully roasted defatted soy flour (SBF), "NutriSoy" (ADM, Decatur, IL, USA) with a protein content of 50.9 g / 100 g DM (dry matter). The extrusion settings are shown in Table 1.

[0136] The raw material was fed into the extruder in the first zone. Deamidase was mixed with water at five doses (0 (blank), 16, 82, 164, and 328 PGLU / g protein in the raw material) and fed into the extruder in zones 2 / 8 using a peristaltic pump (Cole-Palmer Masterflex L / S, Illinois, USA) equipped with a silicone hose with an internal diameter of 3.2 mm (Tygon S3-3603, Saint-Gobain SA, Courbevoie, France). The mixture was conveyed through the extruder using a screw speed of 160 rpm. High-moisture (HM) extrudates were produced using a cooling die cooled with 30°C chilled water at a circulating water flow rate of 15 L / min. The extrudates were produced in the form of slabs measuring 4 x 20 mm and cut into slabs ranging from 100 to 150 mm. The extrudates were stored at 5°C. Further details regarding the testing as well as the results can be found in Example 4.

[0137] [Table 1]

[0138] Example 2 High moisture extrusion of deamidase pretreated fully roasted defatted soy flour (substrate 1) in a laboratory scale extruder (extrudates 6-9) Extrusion tests for the deamidase were performed using a laboratory-scale extruder (Process 11, Thermo Fisher Scientific, Karlsruhe, Germany). The extruder had intermeshing co-rotating twin screws. The screw diameter and extruder length to diameter ratio were 11 mm and 40:1, respectively. The raw materials were fed into the extruder by a gravimetric twin-screw feeder (MT-S, MiniTwin, Brabender Technologie, Duisburg, Germany). The extruder had seven internal heating zones and one external heating zone. High-moisture (HM) extrudates were produced using a cooling die cooled with 30°C cooling water at a circulating water flow rate of 15 L / min.

[0139] Extrudates were produced from deamidase-pretreated substrate 1: fully roasted defatted soy flour (SBF), “NutriSoy” (ADM, Decatur, IL, USA) with a protein content of 50.9 g / 100 g DM using the extrusion settings shown in Table 2 .

[0140] Pretreatment of substrates was performed in a Thermomix TM6 (Vorwerk, Wuppertal, Germany). In the mixing chamber, 280 g of SBF was mixed with MilliQ water (blank) or enzyme solution (16, 82, or 164 PGLU / g protein in the raw material) to obtain a total moisture content of 30 w / w%. The substrates were mixed with the water / enzyme solution by slowly pouring them into the thermomixer at a rotation speed of 1 / 10. The pretreatment temperature was set to 60°C, the time was set 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 -28°C freezer overnight. The frozen pretreated substrates were freeze-dried at 0.22 hPa (Heto PowerDry PL9000, Thermofisher Scientific Inc., Waltham, Massachusetts, USA) for 25 hours. The pretreated substrate was granulated into a fine powder through a 1 mm sieve (IKA®-Werke GmbH & Co, Staufen, Germany) using a laboratory MF10 hammer mill equipped with an MF10.1 cutting-grinding head rotating at 4000 rpm. The pretreated samples were stored at room temperature in a sealed container before extrusion. Further details regarding the testing as well as the results can be found in Examples 4 and 6.

[0141] [Table 2]

[0142] Example 3 High moisture extrusion of deamidase pretreated soy protein concentrate (substrate 2) using a pilot-scale extruder (extrudates 10-13) Extrusion tests for the deamidase were carried out using a pilot-scale twin-screw extruder (Coperion, ZsK 26 MPs, Stuttgart, Germany). The extruder was an intermeshing co-rotating twin-screw extruder equipped with a gravimetric twin-screw feeder (Model: KT20, Coperion K-Tron, Stuttgart, Germany). The extruder screw diameter was 27 mm, and the length / diameter ratio was 40:1. The extruder barrel consisted of five heating zones. High moisture (HM) extrudates were produced using a cooling die with cooling water at a temperature of 80°C.

[0143] Extrudates were prepared from Substrate 2: Arcon® F soy protein concentrate (SPC), manufactured by ADM, Decatur, IL, USA, with a protein content of 68.56 g / 100 g. Deamidase was diluted with water and preconditioned with soy protein concentrate (0, 82, 164, and 328 PGLU / g protein in the feed). For preconditioning, the enzyme-water mixture was sprayed onto the soy protein concentrate using a spray bottle and homogenized using a kneader for 5 minutes. The enzyme solution was added to adjust the dry matter content of the mixture to 70%. The mixture was then bagged and sealed. For the enzyme treatment step, the bag was heated in a convection oven at 50°C for 20 minutes. The mixture was transferred to a gravimetric feeder and fed to the first barrel zone of the extruder, where it was mixed with additional water to achieve a moisture content of 66 w / w%. The extrudate was 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.

[0144] Three different sets of tests were performed using the settings in Table 3. Further details regarding the tests as well as the results can be found in Examples 4, 5 and 8. The improvement in protein solubility of Substrate 2 used in this example can be seen in Example 7.

[0145] [Table 3]

[0146] Example 4 Visual Inspection of the Extrudates of Examples 1-3 and Texture Analysis (Cutting Strength) of the Extrudate (Example 3) Visual inspection The deamidase-treated extrudates resulted in a firmer texture, and visual inspection of the chopped extrudates showed that the deamidase-treated extrudates increased the formation of visible fibers, thereby resulting in a more meat-like texture.

[0147] [Table 4]

[0148] Measurement method Cutting strength texture analysis was performed using a TA.XT.Plus Texture Analyzer (Stable Micro Systems, Surrey, England) equipped with an HDP / BS probe (knife blade set) and an HDP / 90 Heavy Duty Platform, as described in Palanisamy et al., 2018 LWT Food Science and Technology Journal, Volume 87, pp. 546-552. The instrument was equipped with a 50 kg loading cell and calibrated using a 2 kg calibration weight.

[0149] Measurements were carried out at 20°C and samples were prepared in the following manner:

[0150] In Example 3, extrudates measuring 40 x 5 mm were cut into 4 cm slabs and placed under a cutting platform to cut in the cross-fiber direction.

[0151] The pre-test speed was set to 2 mm / s, the test speed to 5 mm / s, and the post-test speed to 10 mm / s. To ensure that the specimen was cut, the cutting distance was set to 15 mm, and the trigger value before the start of the measurement was set to 50 g.

[0152] Measurements were performed in replicates of 6 samples per treatment. The peak shear force (kg) was recorded as the "break strength" of the extrudate.

[0153] [Table 5]

[0154] Conclusion: Increased breaking strength was observed for the deamidase-treated extrudates compared to the control extrudates. The effect of pretreatment with deamidase and subsequent high moisture extrusion on Substrate 2 resulted in significant increases 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.

[0155] Example 5 Water retention capacity assessed by direct method and by LF-NMR of HM extruded SPC (Example 3) The water holding capacity (WHC) of plant-based extrudates is important for both cohesiveness and flavor intensity upon consumption. 1 H nuclear magnetic resonance (low field 1 The correlation between transverse relaxation T2 and WHC based on H nuclear magnetic resonance (LF-NMR) has been demonstrated 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).

[0156] WHC is defined as the ability to retain itself and added water during the application of force, pressure, centrifugation, or heat (Joseph F. Zayas, Functionalities of Proteins in Food, 1997, p77-79). The results are shown in Table 7.

[0157] The molecular mobility of water and biopolymers in foods can be studied using proton nuclear magnetic resonance (LF-NMR), which detects both the longitudinal or spin-lattice relaxation time (T1) and the transverse or spin-spin relaxation time (T2) of protons in a magnetic field.

[0158] LF-NMR analysis of the HM extrudates was performed at room temperature (22 °C) on an MQC-R pulsed NMR spectrometer (Oxford Instruments, Abingdon, United Kingdom) with a 23 MHz magnetic field. Transverse relaxation T was measured using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with 4096 echoes, 8 scans, and a 90-180 pulse interval (τ) value of 76.5 μs. Samples were analyzed in duplicate.

[0159] Relaxation time constant T 2n and the corresponding relative population size f n was determined by a discrete multiexponential fit, which involved deconvolution of the relaxation curve into n exponential components. This was accomplished using the software WinFit (Oxford Instruments, Abingdon, United Kingdom). The number of proton populations was determined by examining the residuals after fitting. The residuals revealed whether the curve was modeled by the correct number of components.

[0160] The results are shown in Table 6 (presented as the mean values ​​of duplicate analyses).

[0161] [Table 6]

[0162] [Table 7]

[0163] Conclusion: Three proton populations were identified using different T2 values, which were assigned to water populations of various mobilities. The fraction with low-mobility water was identified as T 21 and T 22 while the fraction representing the higher mobility water is denoted as T 23 This indicates:

[0164] Deamidase-treated samples showed higher relative amounts of T 23 The deamidase-treated samples exhibited a population (f3), while f1 and / or f2 were reduced. This indicates a higher relative amount of high-mobility / free water in the deamidase-treated samples compared to the control. The presence of more loosely bound water as an effect of deamidase treatment was confirmed by the increase in WHC (Table 7). This results in a higher umami intensity for the formulated patties. Umami intensity is a well-known consumer quality parameter for plant-based meat products.

[0165] It can be concluded that deamidase treatment increases WHC, but no conclusions could be drawn about the optimal dose of deamidase in terms of WHC.

[0166] Example 6 Changes in Secondary Protein Structure of Deamidase-Pretreated, Fully Toasted, Defatted Soybean Flour (Substrate 1) by FT-IR (Example 2) To investigate the secondary protein structure of HM extrudates, FT-IR analysis was performed (M. Carbonaro et al. (2012) Amino Acids. 43, pp. 911-921).

[0167] Absorbance measurements were performed using an MB3000 MID FT-IR spectrometer (ABB Ltd, Zuerich, Schweiz) equipped with a DTGS detector and an ATR (Attenuated Total Reflectance) setup with a single-reflection diamond crystal. All samples were run as six replicates taken from the deamidase-treated substrate 1. The samples were positioned on the crystal face and pressed towards the diamond crystal using a concave needle compressor. IR spectra were recorded using a 4 cm -1 Using a spectral resolution of 4000-500 cm -1 Each spectrum represents the average of 32 scans ratioed to the background (64 scans) collected on a blank crystal and saved as an absorbance spectrum (settings provided in Table 8).

[0168] [Table 8]

[0169] The spectra were analyzed using LatentiX (version 2.13). The amide I band (1700–1600 cm ) arising from the stretching vibration of C═O in the peptide bond was observed. -1 The spectral region containing the amide I band was examined. The vibrational energy of the carboxyl group varies depending on the different conformations of the protein, such as β-sheet and α-helical structures, β-turns and α-turns, and intra- or intermolecular aggregates. Calculating the second derivative of the spectrum allows the spectral content of the amide I band to be determined. In this example, the second derivative was calculated using Savitzky-Golay (window size: 13, polynomial order: 2, derivative: 2).

[0170] Principal component analysis (PCA) was performed to study relationships between and within different samples and variables, and scores and loadings were examined to detect trends, groupings, and outliers. PCA analysis was used as an exploratory analysis. Findings from the PCA score plot were converted to a semiquantitative scale given as a positive number (range: 1-3). Conclusions from the PCA are summarized in Table 9.

[0171] [Table 9]

[0172] Conclusion: Deamidase treatment of SBF resulted in higher levels of intermolecular protein complexes at all doses compared to the blank sample. A slight increase in α-helical and random coil structures was also observed in the deamidase-treated samples, while the amount of β-sheet was significantly reduced by deamidase treatment. Therefore, the protein structure of deamidase-treated SBF appears to 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 Tables 4 and 5.

[0173] Example 7 Protein Solubility of Substrate 2-SPC (Arcon® F) After Deamidase Treatment (Example 3) Substrate (Soy Protein Concentrate (SPC), "Arcon® F" (ADM, Decatur, IL, USA) 68.56% protein, PDI 6%) was prepared for the BCA assay by making a mixture with 5% protein substrate in DI water. The mixture was hydrated at room temperature for 30 minutes with stirring. Deamidase was added and incubated at 50°C for 1 hour in a thermomixer (ThermoFisher Scientific, Massachusetts, USA). The deamidase treatment was inactivated at 85°C / 10 minutes. The mixture was centrifuged at 14,000 rpm for 10 minutes, and the supernatant was used for protein solubility measurements.

[0174] Protein solubility analysis was performed using the Pierce™ Rapid Gold BCA Protein Assay Kit (ThermoFisher Scientific, Massachusetts, USA). The absorbance of the diluted supernatant was measured at 480 nm on a SpectraMax® Plus 384 absorbance microplate reader (Molecular Devices, CA, USA). Protein concentrations were calculated from the absorbance measurements of the supernatant using a standard curve with bovine serum albumin (BSA) (0.0, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL). The BCA assay was performed with a 5% protein solution, in which the concentration of solubilized protein was determined according to the BSA standard (n=4) (Table 10).

[0175] [Table 10]

[0176] Conclusion: Deamidase treatment of Substrate 2 - SPC (Arcon® F) clearly shows an increase in protein solubility that varies with deamidase dose.

[0177] Example 8 Formulation of meat analogue patties made with SPC HM extrudates (Extrudates 10-13) when the substrate was treated with deamidase prior to extrusion (Example 3) Analysis of the formulation and resistance to cutting (rank 1-5), texture profile analysis (TPA, hardness and chewiness), water holding capacity (WHC) and cooking loss of patties of high moisture (HM) extruded SPC treated with various dosages of deamidase.

[0178] The recipe for the burger patty can be found in Table 11.

[0179] [Table 11]

[0180] The HM SPC extrudates were chopped into minced meat in chunks of approximately 2–5 mm diameter in a food processor (Bosch Multitalent 3, Germany) with a rotating knife for approximately 10–30 s, and the resistance to cutting was ranked using visual inspection.

[0181] An emulsion was made by adding soy protein isolate (SPI), spice mix, potato starch, water, and beetroot color to rapeseed oil while mixing for 1 minute. Mixing was done manually for 1 minute. The shredded HM extrudate was then added to the emulsion and gently mixed for 30 seconds. Burger patties were formed using 50 grams of ground beef into burger patty shapes and allowed to rest at 5°C for 1 hour before cooking in oil or further analysis.

[0182] result The deamidase treated high moisture extrudates (82, 164 and 328 PGLU / g protein) were significantly more difficult to shred in a food processor than the blank extrudates (Table 12).

[0183] [Table 12]

[0184] Texture analysis of the patties was performed using a Texture Analyser (Ta.XT.Plus, Stable Micro Systems, England) equipped with a 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 at a test speed of 5 mm s-1 and a post-test speed of 5 mm s-1. The trigger force was set to 50 g, and the time between two cycles was set to 5 seconds. Hardness was calculated as the maximum peak force in one cycle, which can be used as a measure to describe the hardness of the product. Chewiness was calculated as maximum peak force * (area² / area¹) * distance² / distance¹. Chewiness can be used as a measure to describe the energy required to chew a solid food. The results of the texture analysis are presented in Tables 13 and 14.

[0185] [Table 13]

[0186] [Table 14]

[0187] The results show an increase in firmness and chewiness of patties based on deamidase-treated HM extrudates. The effect was particularly evident for the cooked patties and to a lesser extent for the raw patties. The effect appears to be dose-dependent. Increased firmness and chewiness of plant-based meat products similar to animal-based meat products are well known to lead to increased consumer preference.

[0188] Water holding capacity (WHC) was measured at 25°C using the following method:

[0189] Tubes (50 mL) were weighed (three measurements for each sample). 5.0 g of raw ground meat patty (formulation made from the ingredients in Table 11) was weighed into each tube. Deionized water was added in excess (8 mL). The samples were placed on a rotator (20 rpm) at room temperature for 15 minutes. The samples were centrifuged at 4600 rpm and 20°C for 10 minutes. The supernatant was carefully discarded using a cotton swab to remove any fat residue inside the tube. The tubes containing the sediment were weighed again and the WHC was calculated. The calculation was performed using the dry matter (DM) content of the samples. WHC can be defined as the water retained in the raw / uncooked ground meat patty.

[0190] DM was measured using the following method:

[0191] The aluminum trays were weighed (three times for each sample). 0.5 g of sample was weighed into each aluminum tray. The aluminum trays containing the samples were placed in an oven at 105°C for 16 hours. The aluminum trays containing the samples were weighed again and the DM was calculated.

[0192] The results from the WHC analysis are shown in Table 15.

[0193] [Table 15]

[0194] Conclusions: The WHC evaluated in raw ground meat patties appears to provide improved water-holding capacity at the lowest dose (82 PGLU / g protein), which represents the optimal dose of deamidase possible compared to the WHC.

[0195] Cooking loss was measured using the following method:

[0196] Raw ground burger patties were formed and weighed (approximately 50 g, calculated three times for each sample). The patties were then pan-fried in oil using an induction cooker (Steba IK 55, Germany) at the 3 / 10 level for 3 minutes and 30 seconds on each side. Cooking the patties in oil ensured a core temperature of at least 75°C. The cooked patties were re-weighed and cooking loss was calculated using the formula: (raw patty (g) / cooked patty (g)) / (raw patty (g) / 100).

[0197] [Table 16]

[0198] Conclusion: Cooking loss evaluated in raw ground beef patties resulted in a reduction in cooking loss at the lowest dose (82 PGLU / g protein), with the effect being less pronounced at intermediate doses. The results indicate an optimal possible dose of 82 PGLU / g protein for cooking loss reduction.

Claims

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

2. 2. The method of claim 1, wherein the protein deamidase is a protein glutaminase.

3. 3. The method according to any one of claims 1 to 2, wherein the vegetable protein material is derived from pulses such as beans, peas, lentils, chickpeas, or oil grains such as soybeans, peanuts, maize, seeds such as hemp, sunflower, flax, sesame, chia, canola, etc.

4. The method according to any one of claims 1 to 3, wherein the deamidase is added before or during step c).

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

6. 5. The method of any one of claims 1 to 4, wherein the deamidase is added during step c) and the moisture content of the mixture in the extruded product after step c) is in the range of 45 w / w% to 70 w / w%, for example 50 w / w% to 65 w / w% by weight.

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

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

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

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

11. A method according to any preceding claim, wherein step c) is carried out at a temperature in the range of from 65 to 200°C, from 100 to 180°C, for example from 120 to 175°C.

12. The method according to any one of claims 1 to 11, wherein step b) is carried out before step c), and the incubation time is between 1 and 120 minutes, between 1 and 60 minutes, for example between 1 and 15 minutes.

13. 13. The method of claim 12, wherein the temperature is in the range of 20 to 95°C, for example 30 to 70°C.

14. 14. The method of any of claims 1 to 13, wherein the plant-based meat analog product after the extrusion step has increased cut strength, the relative increase in cut strength of the extrudate being at least 25%, at least 40%, at least 50%, such as at least 75% compared to a control without deamidase.

15. 15. The method of any of claims 1 to 14, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in firmness of at least 1%, at least 2%, at least 5%, at least 10%, such as at least 15%, compared to a patty made from a control extrudate without the deamidase.

16. 16. The method of any of claims 1 to 15, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has a relative increase in chewiness of at least 5%, at least 10%, at least 15%, such as at least 20%, compared to a patty made from a control extrudate without the deamidase.

17. 17. The method of any of claims 1 to 16, wherein the plant-based meat analog product has an increased water holding capacity compared to the plant-based material that has not been treated with a deamidase, the water holding capacity of the extrudate being at least 4-6 g water / g extrudate, and / or the plant-based meat analog product, such as a patty made from the extrudate, has a relative increase in water holding capacity of at least 5%, at least 10%, at least 15%, such as at least 20%.

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