Process for production of meat analogue products involving protein-deamidase

Abstract

The present invention relates to a method for producing a meat analogue product comprising texturizing a non-animal protein, such as a vegetable protein, and adding a deamidase prior to or during the texturizing process.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for producing plant-based meat analogs, the method comprising texturizing a plant protein material. Background Art

[0004] The growing world population requires sustainably produced protein-rich foods. For example, legumes (such as soybeans and beans) are attractive crops for producing protein-rich foods. However, other non-animal protein sources are also being used in food production, such as from various plants, algae, or insects.

[0005] Texturized products, such as extruded products, are widely used in the food industry. Extrusion is primarily used to impart a specific texture and unique mouthfeel to food.

[0006] Osen et al. (2014), Journal of Food Engineering 127:67–74, have studied high-moisture extrusion cooking of pea protein isolates. They found that the functional properties of three different pea protein isolates played a secondary role during fiber formation due to the elevated cooking temperature (above the denaturation temperature of the protein).

[0007] Xu et al. (2020), Trends in Food Science & Technology 99, 167–180, provide an overview of the development of reactive extrusion (REX) and enzymes for processing food-related biopolymers. They categorize REX-enzyme processes into two main technology categories: (1) REX–EH processes, which are serial systems that include extrusion pretreatment followed by enzymatic hydrolysis; and (2) eREX processes, which introduce exogenous enzymes before or during REX. They conclude that, with the exception of starch saccharification, eREX remains an immature field.

[0008] Chen et al. (2011), Food Hydrocolloids 25:887-897, have studied the effect of extrusion pretreatment combined with subsequent controlled enzymatic hydrolysis on the emulsification properties of soy protein isolate.

[0009] Czarnecki et al. (1993), Journal of Food Science 58:395-398, incubated a high protein fraction of pinto beans with papain and cellulase for 24 or 72 hours and then freeze-dried, ground, sieved and extruded to obtain a new snack-type product from the beans.

[0010] Zhou et al. (2017), J Food Process Preserv. 41:e13301, have studied the effects of extrusion and papain co-extrusion on pea proteins and on antioxidant peptides produced by further papain hydrolysis of the extrudate.

[0011] WO 2017 / 117398 (Abbott) discloses a method for preparing a protein hydrolysate comprising adding a complete protein source and a protease component to an extruder.

[0012] WO 2018 / 125920 (Abbott Laboratories) discloses a method for preparing a nutritional powder comprising adding a complete protein source, a protease component, a fat component, and water to an extruder.

[0013] Extrusion of vegetable proteins can give the protein a fibrous, meat-like structure.

[0014] Meat analog products are meat substitutes made from, for example, plant proteins, which are intended to simulate the visual appearance, texture and taste of meat products. Meat analog products can be made by extruding, for example, soy, wheat or pea proteins. Extrusion is a thermomechanical texturizing process that unfolds proteins. In the high moisture (HM) extrusion process, a cooled die allows the proteins to rearrange and form new intermolecular covalent and non-covalent bonds. The cooled die at the end of the extruder produces a dense, layered and fibrous meat-like structure. In the low moisture (LM) extrusion process, the product expands upon leaving the die and forms a solid structure with a fibrous, insoluble, porous network that can absorb up to three times its weight in liquid.

[0015] Textured vegetable proteins (such as high or low moisture extruded vegetable proteins) can be formed into a variety of shapes (chunks, flakes, nuggets, pellets, and bars) and sizes.

[0016] For many years, extrusion has been used in the production of meat analogs to obtain a meat-like structure from plant proteins. However, it is difficult to obtain extrudates of plant proteins (such as leguminous plant proteins) with acceptable texture and functional properties for use in meat analogs. In other words, there is room for improvement in better simulating a meat-like structure without having to add non-natural ingredients. WO 2020 / 038541 (Raisio Nutrition) discloses a method for making plant-based meat substitutes by passing a mixture containing a plant protein material and an oat material and, optionally, a cross-linking enzyme and / or a protein deamidase through an extruder at a temperature of 25°C-55°C. Meat substitutes with the addition of transglutaminase were produced using low and high temperature extrusion, and the low temperature samples received better ratings than the corresponding high temperature samples for each sensory property tested, as well as for the overall quality rating.

[0017] CN 109619208A (Heilongjiang Bayi Agricultural University) discloses a method for preparing a flavored meat-like food by extruding and puffing a raw material comprising bean seed powder and soy protein. Ultrasonic enzymatic hydrolysis using Alcalase and Flavorzyme can be applied for a duration of 10-20 minutes, preferably followed by a Maillard reaction, and then extrusion to provide a rich flavor and long-lasting aroma.

[0018] Nisov et al. (2022), Food Research International 156 (2022) 111089 studied the effects of pH and temperature on the fibrous structure formation of plant proteins during high-moisture extrusion processing. They concluded that the structure formation of the extrudate can be positively influenced by increasing the pH of the raw material, which helps to structure the plant proteins into attractive meat analog products.

[0019] WO 2022 / 218863 discloses that the use of proteases increases solubility and leads to improved texture functionality of the final product.

[0020] It is an object of the present invention to provide texturized vegetable protein materials with improved functional properties making them suitable for use in meat analog products such as hamburger patties, ground meat, sausage or chicken nugget analogs. Summary of the Invention

[0021] The inventors of the present invention have surprisingly found that by adding a deamidase to a plant protein material before or during a texturizing process (such as an extrusion process), a texturized plant protein material is provided that imparts improved properties, such as greater hardness and / or greater water holding capacity, when used in plant-based meat products (such as hamburger patties). The effect of adding the deamidase during the extrusion process results in, for example, a plant-based hamburger patty having a greater hardness and, therefore, a more meat-like texture. The inventors have further found that the enzyme-treated extrudate has a higher water holding capacity, which, according to the literature, results in a plant-based meat product having a higher perceived juiciness.

[0022] Therefore, the present invention provides a method for producing a plant-based meat analog, the method comprising the steps of:

[0023] a) preparing a mixture of a vegetable protein-containing material and water, the vegetable protein-containing material having a protein content of from 15% w / w to 95% w / w based on the dry weight of the vegetable material and a water content of from 5% w / w to 99% w / w based on the weight of the mixture;

[0024] b) treating the mixture with a protein-deamidase; and

[0025] c) passing the mixture through an extruder at a temperature above 60° C.;

[0026] d) optionally chopping or slicing the extruded protein material;

[0027] e) optionally drying the product of c) or d); and

[0028] f) optionally mixing the vegetable protein material with other ingredients to obtain the meat analog product.

[0029] definition

[0030] Deamidase: The term "deamidase" refers to protein-glutamylglutaminase (also known as glutamyl peptide 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 at both the α-amino and carboxyl positions, such as L-glutamylglycine and L-phenylalanyl-L-glutamylglycine. Thus, deamidase can deamidate glutamine residues in proteins to glutamic acid residues, and deamidase is also referred to as protein glutamine deamidase. Deamidase enzymes include 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 proforms converted into enzyme-substrate complex," Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691–38702) and are included in InterPro entry IPR041325.

[0031] Deamidase activity: Deamidase (protein glutaminase) activity can be determined using the following assay.

[0032] Glutaminase converts glutamine substrate (Z-GLN-GLY, C 15 H 19 N3O6) and generates ammonia in the process. The ammonia is used as a substrate for glutamate dehydrogenase in combination with α-ketoglutarate to produce glutamate.

[0033] The latter enzymatic reaction requires NADH as a coenzyme. NADH consumption can be tracked by measuring kinetic absorbance at 340 nm and is proportional to glutaminase activity. The reaction temperature is 37°C, pH 7.0, and the reaction time is 216 seconds.

[0034] 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.). Isolated polypeptides include, but are not limited to, culture fluid containing secreted polypeptides.

[0035] Mature polypeptide: The term "mature polypeptide" means 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 to 294 of SEQ ID NO: 1, comprising the propeptide sequence of amino acids 1 to 109 of SEQ ID NO: 1. In one embodiment, the deamidase after cleavage of the propeptide is amino acids 1 to 185 of SEQ ID NO: 2.

[0036] In another aspect, the mature polypeptide is amino acids 1 to 297 of SEQ ID NO: 3, comprising a propeptide sequence of amino acids 1 to 112 of SEQ ID NO: 3. In one embodiment, the deamidase after cleavage of the propeptide is amino acids 1 to 185 of SEQ ID NO: 4.

[0037] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0038] Purified: The term "purified" means 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 can form discrete bands in an electrophoretic gel, a chromatography eluate, and / or a culture 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., by weight on a molar basis). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other designated material or component in a composition at a relative or absolute concentration greater than that of the starting composition.

[0039] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0040] For purposes of the present invention, use Needleman-Wunsch algorithm (Needleman-Wunsch algorithm) (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453) to determine the sequence identity between two amino acid sequences as the output of " longest identity ", this algorithm is as EMBOSS software package (EMBOSS:TheEuropean Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [genetics trend] 16:276-277, preferred 6.6.0 version or more recent version) Needle program (Needle program) is implemented.The parameter used is gap open penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.In order to make Needle program report the longest identity, must specify non-simplified option in command line.The output of " longest identity " of Needle mark is calculated as follows:

[0041] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment) DETAILED DESCRIPTION

[0042] The inventors of the present invention have surprisingly found that by adding a deamidase to a plant protein material before or during a texturizing process (such as an extrusion process), a texturized plant protein material is provided that has improved properties, such as increased cutting strength and increased water holding capacity.

[0043] Thus, in a first aspect, the present invention relates to a method for producing a plant-based meat analogue, the method comprising the steps of:

[0044] a) preparing a mixture of a vegetable protein material and water, the vegetable protein material having a protein content of from 15% w / w to 95% w / w based on the dry weight of the vegetable material and a water content of from 5% w / w to 99% w / w based on the weight of the mixture;

[0045] b) treating the mixture with a protein-deamidase; and

[0046] c) passing the mixture through an extruder at a temperature above 60° C.;

[0047] d) optionally chopping or slicing the extruded protein material;

[0048] e) optionally drying the product of c or d); and

[0049] f) optionally mixing the vegetable protein material with other ingredients to obtain the meat analog product.

[0050] The plant protein raw materials may be obtained from legumes, such as beans, peas, lentils, chickpeas, or oilseed crops, such as soybeans, peanuts, rapeseed; cereal crops, such as rice, corn; seeds, such as sunflower, flax, sesame, chia, rapeseed, and any combination thereof.

[0051] In a preferred embodiment, the plant protein is soy protein or pea protein.

[0052] 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% to 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 faba bean protein material, more preferably a soy protein material or a pea protein material.

[0053] Extrusion

[0054] Since the 1960s, extrusion cooking has been used to produce meat analogs that use, in particular, soy protein as a raw material and, until a few years ago, were primarily used as a meat supplement to reduce the cost of minced meat in the conventional meat industry. However, particularly in the past 10 years, more attention has been focused on finding suitable extrudates to completely replace meat-based patties and sausages (see, for example, Chapter 6 "Plant-Based Meat Analogues" by K. Kyriakopoulou et al. in "Sustainable Meat Products and Processing" (2018, ed. Charis Galanakis, e-ed., ISBN 9780128156889).

[0055] Extrusion is a thermomechanical process by which moist, swellable, starchy, and proteinaceous food materials are plasticized and pushed through a die using a combination of pressure, heat, and mechanical shear. A typical extruder assembly consists of a feed system, a screw, a barrel, a die, and a cutter. Furthermore, a pretreatment system may optionally be introduced prior to extrusion. The extruder barrel can be divided into 5-9 sections (often referred to as temperature zones), which can be heated individually. The material is added to the first section of the extruder using a volume- or weight-controlled feeder. Water (which may include enzymes) or pretreated material with a regulated moisture content is fed to the second section of the extruder using a pump. The screw configuration used may consist of forward and reverse conveying elements. In the feed zone, the material is mixed and homogenized before being conveyed to the compression zone. In this zone, the screw depth and pitch decrease, resulting in increased shear rate, temperature, and pressure. The mechanical energy dissipated by the screw rotation increases the processing temperature within the extruder. Between 70°C and 180°C, proteins denature, creating a viscoelastic mass that can align in the cooling die. Overall, this change in process conditions transforms the solid material into a fluid melt. Before leaving the extruder, the highest temperatures and pressures are reached, resulting in an immediate decrease in the viscosity of the extruded material. In the case of meat analogs produced by high-moisture extrusion, the cooling die is very long to promote alignment and prevent significant material expansion, which could disrupt the newly formed structure.

[0056] The successful preparation of meat analog products requires control of extrusion parameters such as screw speed, moisture content of the feed, barrel temperature, extruder characteristics, and the chemical and physical composition of the feed. A skilled person will know how to adjust the extrusion parameters to optimize the process.

[0057] For meat analogue production, two product categories have been developed depending on the amount of water added during extrusion: high-moisture extrusion and low-moisture extrusion.

[0058] In low-moisture extrusion, flours or concentrates with low moisture content are converted into textured vegetable protein (TVP), also known as dry extrudates. These ingredients are hydrated during extrusion, for example, to 5%-15% moisture, resulting in an extrudate with a lower final moisture content. They are then rehydrated and mixed with other ingredients before cooking (e.g., frying) for the final meat analog formulation. Low-moisture extruded products exhibit a spongy structure and expand and absorb water quickly. They are typically used as meat supplements, but are now also used partially or fully as meat analogs, such as sausages and beef patties.

[0059] 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 potentially enhance structure. The high-moisture product can be used as is, cut into strips to simulate chicken or goulash-like cutlets, or chopped into different sizes to make patties or sausages.

[0060] According to Egbert and Borders, 2006, Achieving success with meat analogs, Food Technology, Chicago, 60(1):28–34, meat analog products can contain water (50%–80%), textured vegetable protein (10%–25%), untextured protein (4%–20%), flavoring (3%–10%), fat (0–15%), binders (1%–5%), and colorants (0–0.5%). This combination of ingredients results in meat analogs that are acceptable in terms of sensory attributes. High water content not only reduces the cost of the product but also provides the desired juiciness, acts as a plasticizer, and aids in emulsification during processing.

[0061] In a preferred embodiment of the method 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 extruder, more preferably a co-rotating twin-screw extruder.

[0062] 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.

[0063] In a preferred embodiment, the extruder has more than one temperature zone, such as 2-10, preferably 5-9 temperature zones. Therefore, the starting temperature in the first zone can be lower than the above preferred temperature range. For example, the starting temperature can be in the range of 20°C-60°C.

[0064] According to the present invention, the deamidase may be added before or during step c).

[0065] 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 of 20% w / w to 35% w / w.

[0066] In another embodiment, the deamidase is added during the extrusion step c), and the water content of the extruded product after step c) by weight of the mixture is 45% w / w to 70% w / w, such as in the range of 50% w / w to 65% w / w. In particular, according to this embodiment, water is added during extrusion in an amount selected from 1.2-3.0 g water / g protein in the plant material.

[0067] In another embodiment, the deamidase is added during step c), and the water content of the extruded product after step c) by weight of the mixture is 1% w / w to 45% w / w, such as 2% w / w to 25% w / w, in particular 5%-15%. In particular, according to this embodiment, water is added during extrusion in an amount selected from 0.05-1.0 g water / g protein in the plant material.

[0068] The protein content by dry weight of the plant material is in the range of 15% w / w to 95% w / w, more preferably in the range of 25% w / w to 92% w / w, such as 45% w / w to 75% w / w.

[0069] In embodiments where a deamidase treatment is performed prior to the extrusion step, the reaction temperature and incubation time may depend on the deamidase activity of the enzyme used, however, the incubation time is preferably in the range of 1 to 120 min, 1 to 60 min, such as 1 to 15 min, and the incubation temperature is in the range of 20°C-95°C, such as 30°C-70°C.

[0070] Shear cell technology

[0071] Based on the recognition that extrusion is an efficient but undefined process, a decade ago, technologies based on well-defined shear flow deformation were introduced to produce fibrous products. Inspired by rheometer design, shear devices, so-called shear cells, were developed, in which strong shear can be applied in a stacked cone or Couette geometry. The final structure achieved with this technology depends on the ingredients and processing conditions. Fibrous products have been obtained using blends of several plant proteins, such as soy protein concentrate or soy protein isolate (SPI) blended with, for example, wheat gluten (WG), pectin, and / or starch. Fibrous products can also be obtained using calcium caseinate. This technology has proven successful, at least at pilot scale (BL Deckers et al., Trends in Food Science & Technology 81 (2018) 25–36). When using shear cell technology, high temperatures (above 100°C) are also typically applied.

[0072] As well as in the extrusion process, the deamidase can be applied in other, potentially milder, processes for making meat analogs, such as shear cell technology, in such a way as to result in the same improved properties of the extrudate and the final formulated product.

[0073] Deamidase

[0074] In the methods of the present invention, a protein deamidase is added to a non-animal protein, such as a plant protein, before or during a texturization process, such as an extrusion process or a shear cell technology process.

[0075] In the present invention, protein deamidase refers to an enzyme with an amide group that directly acts on the amino acid side chain constituting the protein to cause deamidation and release ammonia without cutting the peptide bond of the protein and the cross-linked protein. The specific example of protein deamidase includes protein glutaminase (EC 3.5.1.44), which directly acts on the amide group of the glutamine residue side chain contained in the protein to release ammonia, and therefore glutamine residues are converted into glutamic acid residues. Deamidase can also include protein asparaginase, which directly acts on the amide group of the asparagine residue side chain contained in the protein to release ammonia, and therefore asparagine residues are converted into aspartic acid residues. In the present invention, as protein deamidase, any one of protein glutaminase and protein asparaginase can be used, or the two can be used in combination. A preferred embodiment of the protein deamidase used in the present invention is, for example, protein glutaminase.

[0076] The protein deamidase used in the method of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from the given source is secreted extracellularly.

[0077] The type or source of the protein deamidase used in the present invention is not particularly limited. Examples of protein deamidase include protein deamidase derived from Chryseobacterium genus, Flavobacterium genus, Empedobacter genus, Sphingobacterium genus, Aureobacterium genus or Myroides genus.

[0078] EP 1839491 discloses cloning protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum, and deamidase is also commercially available, for example derived from the protein glutaminase of Chryseobacterium.Preferred examples include the protein deamidase derived from Chryseobacterium, and more preferred examples include the protein deamidase derived from Chryseobacterium proteolyticum. Protein glutaminase derived from Chryseobacterium proteolyticum is commercially available as for example protein-glutaminase.

[0079] "Amano" 500 is manufactured by Amano Enzyme Inc., and this commercially available product can be used.

[0080] For example, protein deamidase can be obtained from the culture broth of the above-mentioned microorganisms.

[0081] Deamidase activity: Deamidase (protein glutaminase) activity can be determined using the following assay.

[0082] Glutaminase converts glutamine substrate (Z-GLN-GLY, C 15 H 19 N3O6) and generates ammonia in the process. The ammonia is used as a substrate for glutamate dehydrogenase in combination with α-ketoglutarate to produce glutamate.

[0083] The latter enzymatic reaction requires NADH as a coenzyme. NADH consumption can be tracked by measuring kinetic absorbance at 340 nm and is proportional to glutaminase activity. The reaction temperature is 37°C, pH 7.0, and the reaction time is 216 seconds.

[0084] According to a preferred embodiment, the deamidase used in the method of the present invention is derived from or obtained from a Chryseobacterium species, such as Chryseobacterium utilis.

[0085] More particularly, in one embodiment, the deamidase may be selected from:

[0086] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1;

[0087] (b) a polypeptide having at least 75% sequence identity to SEQ ID NO: 2;

[0088] (c) a polypeptide having at least 75% sequence identity to the mature polypeptide of SEQ ID NO: 1;

[0089] (d) has 1-30 alterations, e.g., substitutions, deletions and / or insertions, 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, particularly substitutions, at one or more positions derived from SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 1, or the polypeptide of SEQ ID NO: 2;

[0090] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and

[0091] (f) a fragment of the polypeptide of (a), (b), (c) or (d);

[0092] The polypeptide has deamidase activity.

[0093] In another particular embodiment, the deamidase is selected from the group consisting of:

[0094] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;

[0095] (b) a polypeptide having at least 75% sequence identity to SEQ ID NO: 4;

[0096] (c) a polypeptide having at least 75% sequence identity to the mature polypeptide of SEQ ID NO: 3;

[0097] (d) has 1-30 alterations, e.g., substitutions, deletions and / or insertions, 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, particularly substitutions, at one or more positions derived from SEQ ID NO: 3, the mature polypeptide of SEQ ID NO: 3, or the polypeptide of SEQ ID NO: 4;

[0098] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and

[0099] (f) a fragment of the polypeptide of (a), (b), (c) or (d);

[0100] The polypeptide has deamidase activity.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] In the context of the present invention, the term "variant" means a polypeptide having endopeptidase activity that comprises an alteration (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions. A substitution means replacing the amino acid occupying a position with a different amino acid; a deletion means removing 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.

[0108] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions of typically 1-30 amino acids; small amino-terminal 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 the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0109] Examples of conservative substitutions are within the group consisting 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 generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are 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.

[0110] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are altered. For example, these amino acid changes can affect the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0111] Essential amino acids in polypeptides 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 resulting mutant molecules are tested for endopeptidase activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutating putative contact site amino acids. 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.

[0112] 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 relevant 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).

[0113] 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 in a polypeptide.

[0114] In a preferred embodiment of the present invention, the deamidase is added to the vegetable protein just before or during the extrusion step c). It should be understood that adding the deamidase to the vegetable protein just before or during step c) means no pre-incubation.

[0115] In a particularly preferred embodiment, the vegetable protein is texturized by passing it through an extruder and the deamidase is fed directly into the extruder, preferably an aqueous solution of the deamidase is fed directly into the extruder. Preferably, the vegetable protein and the aqueous solution of the deamidase are added separately to the feed zone of the extruder.

[0116] The deamidase may also be added to the aqueous solution or suspension of the vegetable protein before feeding it to the extruder immediately, ie without a pre-incubation step.

[0117] In other embodiments of the present invention, the deamidase is added to at least a portion of the vegetable protein and incubated therewith prior to step c). In such embodiments, mixing with the other ingredients can occur before or during the incubation of at least a portion of the vegetable protein with the deamidase but prior to step c). And / or mixing with the other ingredients can occur after step c).

[0118] 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 be carried out before step c), or it can be carried out after step c).

[0119] The one or more ingredients can be selected from untextured vegetable proteins (such as soy protein isolate, wheat gluten), fibers (such as pectin), starches (such as corn starch, pea starch and / or potato starch), salt, coloring agents, aromas, flavorings, fragrances, and / or oils or fats (such as coconut fat, sunflower oil and / or rapeseed oil).

[0120] Untexturized vegetable proteins, such as soy protein isolate, for example, can be added as an ingredient to, for example, hamburger patties as a binder.

[0121] The meat analog product produced by the method of the present invention can be, for example, a ground meat analog product, a hamburger patty, a sausage, a meatball analog product, a chicken nugget analog product, a gula 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 hamburger patty.

[0122] Improved properties of extruded products and meat analogs produced by the process of the present invention.

[0123] The methods of the present invention unexpectedly produce extruded products and meat analog products with improved properties. In one embodiment, such improved property is that the plant-based meat analog product after the extrusion step has increased cut strength. In one embodiment, the plant-based extrudate has increased cut strength, and wherein the relative increase in cut strength of the plant-based extrudate compared to an extrudate without the addition of a deamidase is at least 25%, at least 40%, at least 50%, such as at least 75%.

[0124] In another embodiment, the plant-based meat analog product has increased water holding capacity compared to a plant-based material that has not been treated with a deamidase.

[0125] In one embodiment, the extrudates according to the present invention have a water holding capacity of at least 4-6 g water / g extrudate.

[0126] In another embodiment, the patties made from the 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%.

[0127] Another improved property of the meat analog product of the present invention is the increased chewiness and firmness of the plant-based burger patty comprising the extrudate according to the present invention.

[0128] In particular, in one embodiment, the plant-based meat analog product (e.g., a patty made from the extrudate) has an increase in relative hardness 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 deamidase.

[0129] 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%, such as at least 20%, compared to a patty made from a control extrudate without deamidase.

[0130] The invention is further disclosed in the following numbered examples.

[0131] Example 1. A method for producing a plant-based meat analog, the method comprising the following steps:

[0132] a) preparing a mixture of a vegetable protein-containing material and water, the vegetable protein-containing material having a protein content of from 15% w / w to 95% w / w based on the dry weight of the vegetable material and a water content of from 5% w / w to 99% w / w based on the weight of the mixture;

[0133] b) treating the mixture with a protein-deamidase; and

[0134] c) passing the mixture through an extruder at a temperature above 60° C.;

[0135] d) optionally chopping or slicing the extruded protein material;

[0136] e) optionally drying the product of c) or d); and

[0137] f) optionally mixing the vegetable protein material with other ingredients to obtain the meat analog product.

[0138] Embodiment 2. The method according to embodiment 1, wherein the protein-deamidase is protein-glutaminase.

[0139] Embodiment 3. The method according to any one of embodiments 1-2, wherein the plant protein material is derived from legumes, such as beans, peas, lentils, chickpeas, or oil crops, such as soybeans, peanuts, rapeseed; cereal crops, such as rice, corn; seeds, such as sunflower, flax, sesame, chia, rapeseed, and any combination thereof.

[0140] Embodiment 4. The method according to any one of the preceding embodiments, wherein the deamidase is added before or during step c).

[0141] Embodiment 5. The method of any one 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 of 20% w / w to 35% w / w.

[0142] Embodiment 6. A method according to any one of embodiments 1-4, wherein the deamidase is added during step c) and the water content of the extruded product after step c) by weight of the mixture is 45% w / w to 70% w / w, such as in the range of 50% w / w to 65% w / w.

[0143] Embodiment 7. The method of embodiment 6, wherein water is added during extrusion in an amount selected from 1.2-3.0 g water / g protein in the plant material.

[0144] Embodiment 8. The method according to any one of embodiments 1-4, wherein the deamidase is added during step c) and the water content of the extruded product after step c) is from 1% w / w to 45% w / w, such as from 2% w / w to 25% w / w, in particular from 5% to 15%, by weight of the mixture.

[0145] Embodiment 9. The method of embodiment 8, wherein water is added during extrusion in an amount selected from 0.05-1.0 g water / g protein in the plant material.

[0146] Embodiment 10. The method according to any one of the preceding embodiments, wherein the protein content by dry weight of the plant material is in the range of 25% w / w to 92% w / w, such as 45% w / w to 75% w / w.

[0147] Embodiment 11. The method according to any one 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.

[0148] Embodiment 12. The method according to any one of the preceding embodiments, wherein step b) is performed before step c), and wherein the incubation time is 1 to 120 min, 1 to 60 min, such as 1 to 15 min.

[0149] Embodiment 13. The method according to embodiment 12, wherein the temperature is in the range of 20°C-95°C, such as 30°C-70°C.

[0150] Embodiment 14. A method according to any of the preceding embodiments, wherein the plant-based meat analog product after the extrusion step has increased cutting strength, and wherein the relative increase in cutting strength of the extrudate compared to a no deamidase control is at least 25%, at least 40%, at least 50%, such as at least 75%.

[0151] Embodiment 15. A method according to any of the preceding embodiments, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has an increase in relative hardness 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 deamidase.

[0152] Embodiment 16. A method according to any of the preceding embodiments, 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 deamidase.

[0153] Embodiment 17. The method of any preceding embodiment, wherein the plant-based meat analog product has increased water holding capacity compared to a plant-based material not treated with a deamidase.

[0154] Embodiment 18. The method of embodiment 17, wherein the water holding capacity is at least 4-6 g water / g extrudate.

[0155] Embodiment 19. The method of embodiment 17, wherein 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%.

[0156] Embodiment 20. The method according to any one of the preceding embodiments, wherein the deamidase is selected from:

[0157] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1;

[0158] (b) a polypeptide having at least 75% sequence identity to SEQ ID NO: 2;

[0159] (c) a polypeptide having at least 75% sequence identity to the mature polypeptide of SEQ ID NO: 1;

[0160] (d) has 1-30 alterations, e.g., substitutions, deletions and / or insertions, 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, particularly substitutions, at one or more positions derived from SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 1, or the polypeptide of SEQ ID NO: 2;

[0161] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and

[0162] (f) a fragment of the polypeptide of (a), (b), (c) or (d);

[0163] The polypeptide has deamidase activity.

[0164] Embodiment 21. The method according to any one of the preceding embodiments, wherein the deamidase is selected from:

[0165] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;

[0166] (b) a polypeptide having at least 75% sequence identity to SEQ ID NO: 4;

[0167] (c) a polypeptide having at least 75% sequence identity to the mature polypeptide of SEQ ID NO: 3;

[0168] (d) has 1-30 alterations, e.g., substitutions, deletions and / or insertions, 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, particularly substitutions, at one or more positions derived from SEQ ID NO: 3, the mature polypeptide of SEQ ID NO: 3, or the polypeptide of SEQ ID NO: 4;

[0169] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and

[0170] (f) a fragment of the polypeptide of (a), (b), (c) or (d);

[0171] The polypeptide has deamidase activity.

[0172] Embodiment 22. A method according to 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.

[0173] Embodiment 23. A method according to 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.

[0174] Embodiment 24. A method according to 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 with the mature polypeptide of SEQ ID NO: 1.

[0175] Embodiment 25. A method according to 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.

[0176] Embodiment 26. A method according to 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.

[0177] Embodiment 27. A method according to 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 with the mature polypeptide of SEQ ID NO: 3.

[0178] Examples

[0179] Overview

[0180] To improve the hardness of the extrudate and thus the bite of the final plant-based meat product, deamidase has been tested in the extrusion process. Deamidase was added both in the pretreatment step and directly during extrusion. Both laboratory and pilot-scale extrusion facilities were included in the tests.

[0181] Despite the harsh conditions (high temperature and shear) during the extrusion process, the addition of deamidase directly in the extruder barrel during high moisture (HM) extrusion of toasted defatted soy flour appears to impart increased fiber formation to the extrudate. This is expected to correlate with increased bite strength / hardness of the final plant-based product.

[0182] To optimize the action of the enzyme, a pretreatment step was introduced prior to HM extrusion of the substrate. Deamidase pretreatment of soy protein concentrate (SPC) and subsequent HM extrusion resulted in an improved meat-like texture, imparting significantly higher cutting strength to the extrudate. Increased cutting strength or hardness of the extrudate is generally desirable and can help upgrade extrudates produced using less processed, less refined, and lower-cost raw materials.

[0183] The extrudates have been formulated into patties similar to commercially available patties. Both the extrudates and patties have been analyzed by chemical and physical analysis. The methods applied are well known in the existing food and meat industries. Texture analysis of patties made from deamidase-treated SPC extrudates showed that deamidase addition increased the hardness and chewiness of both raw and fried patties. Therefore, deamidase addition will produce plant-based meats with enhanced hardness and bite, similar to conventional meat products. In addition, patties produced from extrudates treated with deamidase resulted in significantly reduced cooking losses at the lowest dosage.

[0184] Water holding capacity (WHC) analysis showed that the addition of deamidase improved the WHC of the extrudates compared to the untreated samples. This is expected to correlate with increased juiciness in the final product. Similarly, low-field NMR analysis showed that deamidase treatment resulted in altered water binding, with higher levels of more mobile water in the deamidase-treated extrudates also indicating increased juiciness.

[0185] Spectroscopic analysis (FT-IR) further revealed altered protein secondary structure in the deamidase pretreated substrate, reflecting better solubility, which resulted in improved formation of covalent and non-covalent bonds in the final extrudates.

[0186] Material

[0187] strains.

[0188] Chryseobacterium sp. strain 62563 was isolated from a soil sample collected in Sibhult, Sweden in September 2013.

[0189] substrate

[0190] Substrate 1: Toasted defatted soy flour (SBF), (ADM, Decatur, IL, USA) 50.9 g / 100 g dry matter.

[0191] Substrate 2: Soy Protein Concentrate (SPC), F" (ADM Company, Decatur, IL, USA). 68.56 g / 100 g dry matter.

[0192] enzymes

[0193] The deamidase (EC 3.5.1.44) used in the examples is as follows:

[0194] The protein glutaminase derived from Chryseobacterium sp. 62563 has the mature polypeptide sequence shown in SEQ ID NO:2. Cleavage of the propeptide is achieved by treating the deamidase of SEQ ID NO:1 with a site-specific endopeptidase. The site-specific endopeptidase used is glutamyl endopeptidase from Bacillus licheniformis. The active deamidase obtained after maturation is the polypeptide shown in SEQ ID NO:2.

[0195] Example 1

[0196] High Moisture Extrusion of Toasted Defatted Soy Flour (Substrate 1) with Direct-Fed Deamidase on a Lab-Scale Extruder (Extrudates 1-5)

[0197] Extrusion experiments to test the deamidase were performed using a laboratory-scale extruder (Process 11, Thermo Fisher Scientific, Karlsruhe, Germany). The extruder was an intermeshing, co-rotating twin-screw extruder. The screw diameter and extruder length-to-diameter ratio were 11 mm and 40:1, respectively. The raw materials were metered into the extruder via a gravimetric twin-screw feeder (MT-S, MiniTwin, Brabender Technologie, Duisburg, Germany). The extruder had 7 internal heating zones and 1 external heating zone.

[0198] Extrudates were produced from Substrate 1: toasted 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 given in Table 1.

[0199] The raw materials are fed into the first zone of the extruder. Deamidase is mixed into water with five doses (0 (blank), 16, 82, 164, 328 PGLU / g of protein in the raw materials) and fed into the 2 / 8 zone of the extruder using a peristaltic pump (Cole-Palmer Masterflex L / S, Illinois, the U.S.) equipped with a silicone hose (Tygon S3-3603, Saint-Gobain SA, Courbevoie, France) with an internal diameter of 3,2 mm. The mixture is conveyed through the extruder at a screw speed of 160 rpm. A cooling die cooled by 30 ℃ of cooling water with a recirculating water flow rate of 15 L / min is used to prepare a high moisture (HM) extrudate. The extrudate is produced in a slab shape of 4 x 20 mm in size and cut into slabs in the range of 100-150 mm. The extrudate is stored at 5 ℃. Further detailed information and results of the test can be found in Example 4.

[0200] Table 1: Extrusion settings.

[0201]

[0202]

[0203] Example 2

[0204] High Moisture Extrusion of Deamidase Pretreated Toasted Defatted Soy Flour (Substrate 1) on a Lab-Scale Extruder (Extrudates 6-9)

[0205] Extrusion experiments to test the deamidase were carried out using a laboratory-scale extruder (Process 11, Thermo Fisher Scientific, Karlsruhe, Germany). The extruder was an intermeshing, co-rotating twin-screw extruder. The screw diameter and extruder length-to-diameter ratio were 11 mm and 40:1, respectively. The raw materials were fed into the extruder via a gravity twin-screw feeder (MT-S, MiniTwin, Brabender Technologies, Duisburg, Germany). The extruder had 7 internal heating zones and 1 external heating zone. High moisture (HM) extrudates were prepared using a cooling die cooled by 30°C cooling water at a recirculating water flow rate of 15 L / min.

[0206] Extrudates were produced from deamidase pretreated substrate 1: toasted defatted soy 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.

[0207] The pretreatment of the substrate was carried out in a Thermomix TM6 (Vorwerk, Wuppertal, Germany). In the mixing chamber, 280 g of SBF was mixed with MilliQ water (blank) or enzyme solution (protein in 16, 82, 164 PGLU / g raw material) to produce a total moisture content of 30% w / w. The substrate was slowly poured into a thermomixer at a rotation speed of 1 / 10 to mix the substrate with the water / enzyme solution, 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 substrate was spread on a metal tray and immediately placed in a freezer at -28 ° C overnight. The frozen pretreated substrate was freeze-dried at 0,22 hPa for 25 h (Heto PowerDry PL9000, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The samples were collected using a laboratory MF10 hammer mill equipped with a MF10.1 cutting mill head (rotating at 4000 rpm) and passed through a 1 mm sieve ( GmbH, Staufen, Germany ( The pretreated substrate was granulated into a powder by Eppendorf GmbH & Co. KG, Staufen, Germany. Prior to extrusion, the pretreated sample was stored in a closed container at room temperature. Further details and results of the experiments can be found in Examples 4 and 6.

[0208] Table 2: Extrusion settings.

[0209]

[0210] Example 3

[0211] High Moisture Extrusion of Deamidase Pretreated Soy Protein Concentrate (Substrate 2) Using a Pilot Scale Extruder (Extrudates 10-13)

[0212] Extrusion experiments to test the deamidase were conducted using a pilot-scale twin-screw extruder (Coperion, ZsK 26MPs, Stuttgart, Germany). The extruder was an intermeshing, co-rotating twin-screw extruder with a gravity 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 five heating zones. A cooling die was used, and high-moisture (HM) extrudates were produced using cooling water at a temperature of 80°C.

[0213] Extrudates were produced from Substrate 2: Soy Protein Concentrate (SPC), F, protein content is 68,56g / 100g DM, from ADM, Decatur, Illinois, USA. Deamidase is diluted in water and pre-conditioned with soy protein concentrate (0, 82, 164, 328PGLU / g protein in raw material). For pre-conditioning, enzyme-water mixture is sprayed onto soy protein concentrate using a spray bottle and evenly mixed for 5min using a kneader. By adding enzyme solution, the dry matter in the mixture is adjusted to 70%. The mixture is then packaged into bags and sealed. The bag is heated in a convection oven at 50°C for 20 minutes as an enzyme treatment step. The mixture is transferred to a gravity feeder and fed into the first barrel zone of the extruder, where the mixture is mixed with additional water to produce a moisture content of 66%w / w. The extrudate is cut into 15-20cm thick slices and allowed to cool at room temperature. The extrudate is vacuum sealed and stored at -20°C, then further analyzed.

[0214] Three different experiments were run using the settings in Table 3. Additional details and results of the experiments can be found in Examples 4, 5, and 8. The improvement in protein solubility of Substrate 2 used in this example can be found in Example 7.

[0215] Table 3: Extrusion settings.

[0216]

[0217] Example 4

[0218] Visual inspection of extrudates from Examples 1-3 and textural analysis (cut strength) of the extrudate (Example 3)

[0219] Visual inspection

[0220] The deamidase treated extrudates produced a tougher texture, and visual inspection of the cut extrudates showed that the deamidase treated extrudates had increased visible fiber formation and therefore a more meat-like texture.

[0221] Table 4: Arbitrary ranking of visible fiber formation in the extrudates (Examples 1-3).

[0222]

[0223] Measurement method

[0224] Cutting strength texture analysis was performed using a TA.XT.Plus texture analyzer (Stable Micro Systems, Surrey, England) equipped with an HDP / BS probe (blade equipped with a knife) and an HDP / 90 weight-bearing platform, similar to the method described in Palanisamy et al. 2018 LWT Food Science and Technology Journal, Vol. 87, 546-552. A 50 kg loading cell was used and a 2 kg calibration weight was used to calibrate the instrument.

[0225] The measurements were performed at 20°C, and the samples were prepared as follows:

[0226] In Example 3, the extrudate having dimensions of 40 x 5 mm was cut into 4 cm thick pieces, placed under a cutting platform and cut in the transverse direction of the fibers.

[0227] 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. The cutting distance was set to 15 mm to ensure cutting through the sample, and the trigger value before starting measurement was set to 50 g.

[0228] The measurement was repeated with 6 samples / treatment.The peak shear force (kg) was recorded as the "cut strength" of the extrudate.

[0229] Table 5: Cut strength of HM extrudates produced in Example 3.

[0230]

[0231] Conclusion: An increase in shear strength was observed for the deamidase treated extrudates compared to the control extrudates. The effect of pre-treating Substrate 2 with deamidase followed by 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.

[0232] Example 5

[0233] Evaluation of the water holding capacity of HM-extruded SPC (Example 3) by direct method and by LF-NMR

[0234] The water holding capacity (WHC) of plant-based extrudates is important for both cohesiveness and juiciness during consumption. 1The correlation between the transverse relaxation T2 of H nuclear magnetic resonance (LF-NMR) and WHC has been demonstrated in various papers (e.g., HC Bertram et al., Meat Science 57 (2001) 125-132 and Massimo Lucarini et al., Foods (2020) 9, 480).

[0235] WHC is defined as the ability to retain its own water and added water during application of force, squeezing, centrifugation, or heating (Joseph F. Zayas, Functionalities of Proteins in food, 1997, pp. 77-79). The results are shown in Table 7.

[0236] The molecular mobility of water and biopolymers in food products can be studied by proton nuclear magnetic resonance (LF-NMR), which measures both the longitudinal or spin-lattice relaxation time (T1) and the transverse or spin-spin relaxation time (T2) of protons in a magnetic field.

[0237] LF-NMR analysis of HM extrudates was performed at room temperature (22° C.) on a MQC-R pulsed NMR spectrometer (Oxford Instruments, Abingdon, United Kingdom) with a magnetic field of 23 MHz. Transverse relaxation T2 was measured using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with 4096 echoes, 8 scans, and a pulse interval (τ) value of 90-180 μs. Samples were analyzed in duplicate.

[0238] The relaxation time constant T was determined by discrete multi-exponential fitting (including deconvolution of the relaxation curve into n exponential components). 2n and the corresponding relative population size f n This was done using the software WinFit (Oxford Instruments, Abingdon, UK). The number of proton populations was determined by examining the residuals after fitting. The residuals reveal whether the curve is modeled by the correct number of components.

[0239] The results are given in Table 6 (presented as the mean of replicate analyses).

[0240] Table 6: Relaxation time constants T21, T22 and T23 and relative population sizes fl, f2 and f3 determined by LF-NMR of HM extrudates.

[0241]

[0242] Table 7: WHC expressed as g water / g dry matter in the extrudate (Example 3).

[0243]

[0244] Conclusion: Three proton populations with different T2 values were identified, which were assigned to water populations with different mobilities. The fraction with lower mobility water was assigned T 21 and T 22 , while the fraction representing higher mobility water was designated as T 23 .

[0245] Deamidase-treated samples showed higher T 23 The relative amount of the population (f3) increased, while f1 and / or f2 decreased. This indicates that the relative amount of high mobility / free water was higher in the deamidase-treated samples compared to the control. The presence of more loosely bound water as a result of the deamidase treatment has been confirmed by increased WHC (Table 7). This, in turn, results in a more juicy prepared patty. Juiciness is a well-known consumer quality parameter for plant-based meat products.

[0246] It can be concluded that deamidase treatment will increase WHC, however it is not possible to draw conclusions regarding the optimal dose of deamidase for WHC.

[0247] Example 6

[0248] Determination of changes in secondary protein structure of deamidase-pretreated, toasted defatted soy flour (substrate 1) by FT-IR (Example 2)

[0249] FT-IR analysis was performed to investigate the secondary protein structure of the HM extrudates (M Carbonaro et al. (2012) AminoAcids. Vol. 43, pp. 911-921).

[0250] Absorbance measurements were performed using an MB3000 MID FT-IR spectrometer (ABB Ltd, Zurich, Switzerland) with a DTGS detector and equipped with an ATR (attenuated total reflectance) setup with a single reflection diamond crystal. All samples were run as 6 replicates of deamidase-treated substrate 1. The sample was positioned on the crystal surface and pressed against the diamond crystal using a concave needle compressor. A 4 cm -1 The spectral resolution is between 4000-500cm -1 IR spectra were recorded over a range of 400 nm. Each spectrum represents the average of 32 scans relative to a background (64 scans) collected with a blank crystal and stored as an absorbance spectrum (settings are provided in Table 8).

[0251] Table 8 - FT-IR settings used to generate the spectra.

[0252]

[0253] The spectra were analyzed using LatentiX (version 2.13). The amide I band (1700-1600 cm -1 ) spectral region. The vibrational energy of the carboxyl group depends on different conformations of the protein, such as β-sheet and α-helical structures, β- and α-turns, and intermolecular or intramolecular aggregates. Calculating the second derivative of the spectrum allows the spectral components of the amide I band to be assigned. In this example, the second derivative was calculated using the Savitzky-Golay method (window size: 13, polynomial order: 2, derivative: 2).

[0254] Carry out principal component analysis (PCA), and check score and loading, to study the relationship between different samples and variables or inside, and detect trend, grouping and outliers.Use PCA analysis as exploratory analysis.The result of PCA score graph has been converted into semi-quantitative measurement, provides (scope: 1-3) with multiple plus signs.Table 9 summarizes the conclusion drawn from PCA.

[0255] Table 9: Secondary protein structure of pre-treated SBF powder (Example 2) determined by FT-IR (range: 1-3).

[0256]

[0257] 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 for the deamidase-treated samples, while the amount of β-sheets decreased significantly with deamidase treatment. Therefore, the protein structure of deamidase-treated SBF appears to be more flexible / disordered when the substrate enters the extruder. This is expected to result in higher levels of fibrillar structure in the extrudate. This is confirmed by the results in Tables 4 and 5.

[0258] Example 7

[0259] After deamidase treatment, the substrate 2-SPC ( F) Protein solubility of (Example 3)

[0260] The substrate was prepared by preparing a mixture with 5% protein substrate in DI water (soy protein concentrate (SPC), " F" (ADM, Decatur, IL, USA), 68, 56% protein, PDI 6%) for BCA assay. The mixture was hydrated at room temperature for 30 min while 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 min. The mixture was centrifuged at 14,000 rpm for 10 min, and the supernatant was used for protein solubility measurement.

[0261] Using Pierce TM Protein solubility analysis was performed using Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). The absorbance of the diluted supernatant was measured at 480 nm on a Sigma Plus 384 absorbance microplate reader (Molecular Devices, CA, USA). Protein concentration was calculated from the absorbance measurements of the supernatant using a standard curve using bovine serum albumin (BSA) (0.0, 0.2, 0.4, 0.6, 0.8, 1 mg / mL). The 5% protein solution was subjected to a BCA assay, where the concentration of soluble protein was measured based on a BSA standard (n=4) (Table 10).

[0262] Table 10: SPC treated with deamidase at 50°C for 1 h and subsequently inactivated at 85°C for 10 min ( F) Protein solubility.

[0263]

[0264] Conclusion: Substrate 2-SPC ( Deamidase treatment of F) clearly demonstrates a deamidase dosage-dependent increase in protein solubility.

[0265] Example 8

[0266] Formulation of meat analog patties made with SPC HM extrudates (Extrudates 10-13) wherein the substrate was treated with a deamidase prior to extrusion (Example 3)

[0267] Patties were prepared with high moisture (HM) extruded SPC treated with different doses of deamidase and analyzed for cut resistance (ranked 1-5), total texture analysis (TPA, hardness and chewiness), water holding capacity (WHC) and cook loss.

[0268] The recipe for the hamburger patties can be found in Table 11.

[0269] Table 11: Formulation of plant-based burger patties.

[0270]

[0271]

[0272] The HM SPC extrudate was cut into strips with a rotating knife in a food processor (Bosch Multitalent 3, Germany) for about 10-30 seconds into minced meat pieces with a diameter of approximately 2-5 mm, and cut resistance was ranked using visual inspection.

[0273] An emulsion was prepared by adding soy protein isolate (SPI), a spice blend, potato starch, water, and beetroot colorant to rapeseed oil while mixing for 1 minute. Manual mixing was performed for 1 minute. Afterwards, the cut HM extrudate was added to the emulsion and gently mixed for 30 seconds. Hamburger patties were formed into hamburger patties using 50 grams of ground meat and allowed to rest at 5°C for 1 hour before frying or further analysis.

[0274] result

[0275] The high moisture extrudates treated with deamidase (82, 164 and 328 PGLU / g protein) were significantly more difficult to cut in a food processor than the blank extrudates (Table 12).

[0276] Table 12: Cutting resistance - Pulse amount / force to cut HM extrudates by rotating knife in food processor. Visual inspection and arbitrary ranking (1-5).

[0277]

[0278] The texture analysis of the patties was performed using a texture analyzer (Ta.XT.Plus, Stable Microsystems, England) equipped with a cylindrical probe SMS p / 125 mm. All hamburger 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)). The sample was compressed to 50% of its original height with 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 cycles was set to 5 seconds. The hardness was calculated as the maximum peak force of the first cycle. It 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 solid food. The results of the texture analysis are presented in Tables 13 and 14.

[0279] Table 13: Texture analysis (hardness: g, force) of HM patties with deamidase treated HM extrudates from SPC (n=3).

[0280]

[0281] Table 14: Texture analysis (chewiness) of HM patties with deamidase treated HM extrudates from SPC (n=3).

[0282]

[0283] The results indicate that the firmness and chewiness of patties based on deamidase-treated HM extrudates increased. The effect was particularly pronounced for fried patties, and to a lesser extent for raw patties. The effect appeared to be dose-dependent. It is well known that increased firmness and chewiness in plant-based meat products, similar to animal meat products, increases consumer preference.

[0284] The water holding capacity (WHC) was measured at 25°C by using the following method:

[0285] Weigh the tubes (50 mL) (three determinations were made for each sample). 5.0 g of raw patty mince (a formulation produced from the ingredients in Table 11) was weighed into each tube. Excess deionized water (8 mL) was added. The samples were placed in a rotator (20 rpm) for 15 min at room temperature. The samples were centrifuged at 4600 rpm for 10 min at 20 ° C. The supernatant was carefully discarded and the fat residue on the inside of the tube was removed using a cotton swab. The tube containing the sediment was weighed again and the WHC was calculated. The dry matter (DM) content in the sample was used for calculation. WHC can be defined as the water retained in the raw / unfried patty mince.

[0286] DM is measured by using the following methods:

[0287] Weigh the aluminum trays (three determinations for each sample). Weigh 0.5 g of sample into each aluminum tray. Place the aluminum trays with samples in an oven at 105°C for at least 16 hours. Reweigh the aluminum trays with samples and calculate DM.

[0288] The results from the WHC analysis are given in Table 15.

[0289] Table 15: % WHC of raw hamburger grounds with deamidase treated HM extrudates (n=3).

[0290]

[0291] Conclusion: The WHC evaluated on raw patty ground meat appeared to produce improved water holding capacity at the lowest dose (82 PGLU / g protein), indicating a possible optimal deamidase dose for WHC.

[0292] Cooking loss is measured by using the following method:

[0293] Raw hamburger mince was formed and weighed (approximately 50 g, and each sample was determined three times). Afterwards, the patties were fried in a pan at 3 / 10 level for 3 min 30 sec on each side using an induction cooker (Steba IK 55, Germany). The patties were fried to a core temperature of at least 75°C. The fried patties were reweighed and the cooking loss was calculated using the following formula: (raw patty (g) / cooked patty (g)) / (raw patty (g) / 100).

[0294] Table 16: Amount of cooking loss (%) when frying HM patties in a preheated steel pan on an induction hob at 3 / 10 level for 3 min 30 sec per side (n=3).

[0295]

[0296] Conclusion: Cooking loss evaluated on raw patty ground meat resulted in reduced cooking loss at the lowest dose (82 PGLU / g protein), with the effect being less pronounced at intermediate doses. The results indicate that 82 PGLU / g protein is the likely optimal dose for reducing cooking loss.

Claims

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

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

3. The method according to any one of claims 1-2, wherein the vegetable protein material is derived from legumes, such as beans, peas, lentils, chickpeas, or oil crops, such as soy, peanuts, corn; seeds, such as sunflower, flax, sesame, chia, rapeseed.

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

5. The method according to any one of claims 1 to 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 of 20% w / w to 35% w / w.

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

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

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

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

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

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

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

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

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

15. A method according to any preceding claim, wherein the plant-based meat analog product, e.g., a patty made from the extrudate, has an increase in relative 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 deamidase.

16. A method according to any one of the preceding claims, wherein the plant-based meat analog product, such as 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 deamidase.

17. A method according to any one of the preceding claims, wherein the plant-based meat analogue product has an increased water holding capacity compared to plant-based material which has not been 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, 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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