Controlled enzymatic browning reaction of non-meat protein materials containing pigments and laccases

Abstract

This invention relates to the use of laccase in the controlled enzymatic browning reaction of cultured meat products containing a red pigment that changes color upon oxidation. Preferably, the laccase is embedded in fat with a melting temperature of at least 25°C, such that the oxidation of the red pigment occurs only at elevated temperatures, such as during the cooking of the cultured meat product.

Description

[0001] References to sequence lists

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

[0003] This invention relates to a controlled enzymatic browning reaction in artificial meat products containing a red pigment that changes color upon oxidation. Background Technology

[0004] Due to the benefits of protein, including providing a feeling of fullness and enhancing muscle mass, consumer demand for protein-containing foods has increased.

[0005] As consumer interest grows towards healthier and more eco-friendly diets, vegetarianism (and especially vegetarian sources of protein) is gaining popularity. Meat alternatives are also becoming increasingly popular as many consumers wish to continue preparing common, traditional, or favorite meals without or with minimal meat consumption. Many known meat alternatives include plant-based proteins, such as those derived from soy.

[0006] Meat substitutes preferably mimic natural or processed meat products in any one or more aspects of appearance, odor, flavor, texture, nutritional characteristics, and response to heat.

[0007] Extruded products are widely used in the food industry. Extrusion is mainly used to give food specific textures and unique flavors.

[0008] The moisture content of dry and wet extrusion products is approximately 15% and 65%, respectively, and they exhibit varying degrees of fibrous, meat-like structure depending on processing conditions. Dry extrusion and wet extrusion are increasingly being used for plant proteins to provide sustainable alternatives to animal products.

[0009] An important target parameter for plant-based meat substitutes is the color of the extrudate.

[0010] However, coloring the non-meat protein materials used in artificial meat has proven difficult because meat changes color when cooked. Uncolored protein materials (such as soy or pea protein) are beige or yellowish-brown, unlike the red of uncooked meat, and lighter than the brown of cooked meat. Therefore, artificial meat containing this protein has a mottled appearance in both its uncooked and cooked states. This mottled appearance is less appealing and less appetizing because, whether uncooked or cooked, artificial meat doesn't look "natural" to consumers.

[0011] Meat's red color comes from the presence of myoglobin, which is absent in plant-based meat. To mimic the red color of raw meat, plant-based colorants are often added to artificial meat.

[0012] For example, US 4536406 (General Foods Corp.) provides a red artificial meat product made from soy protein isolate and red food coloring containing paprika and shellac.

[0013] The most common coloring agent nowadays is beetroot, which contains the red pigment betaine.

[0014] When conventional meat products are fried, browning occurs, likely primarily due to the Maillard reaction and the breakdown of the tetrapyrrole ring in the muscle protein myoglobin. In plant-based meat, browning is mainly attributed to caramelization of the outer surface. This means that, for example, the outer surface of a burger patty will be more prone to charring and blackening, and the center of a plant-based burger patty containing ingredients such as betaine will remain red after cooking, even though the center temperature reaches over 75°C.

[0015] A combination of food coloring dyes has been used to provide color to the protein material, so the color of the protein material is similar to the color of browned meat in its uncooked and cooked states.

[0016] US 2017 / 0303558 A1 (Fraunhofer) disclosed colored extruded plant protein products containing plant proteins and a second plant compound derived from oilseeds. This second plant compound contains polyphenols that change color upon oxidation, such as sinapic acid, chlorogenic acid, caffeic acid, or open-ring isolarcisinol.

[0017] US 3958019 (Armour and Co) provides a color treatment for soy protein materials that imparts the color of uncooked raw meat to the protein material and alters its color during cooking to resemble that of cooked meat. The color treatment is a mixture of betaine and canthaxanthin food coloring.

[0018] EP 1180332 A1 (Solae) discloses a composition for coloring non-meat protein materials, the composition comprising a heat-labile pigment (such as betaine) and a browning agent, preferably formed from reducing sugar, added in sufficient quantity to react with the amino groups of the protein material in a Maillard-type reaction, so that the protein material undergoes browning to the same degree as meat through the same type of browning reaction.

[0019] Further development is needed for methods of controlled browning of materials containing non-meat proteins to make them more meat-like both before and after cooking. Of particular interest are methods that apply as few E-numbers as possible (due to the widespread need for clean labels) or that do not involve the addition of, for example, sugar (which provides extra calories and contradicts the “no added sugar” trend).

[0020] WO 2016 / 097270 (DSM) discloses the use of laccases with a specific prosite motif for bleaching food products, particularly dairy products. It discloses the use of laccases for bleaching whey colored with annatto from cheddar cheese production. Natto is commonly used for coloring cheese, but annatto remaining in the whey after cheese production is undesirable.

[0021] JP 2009165450 discloses the use of polyphenol oxidase to change the color of red bean rice (sekihan) (rice cooked with red beans).

[0022] WO 07097433 (Meiji Corporation) relates to a deodorizing composition for chewing gum, the deodorizing composition comprising an oxidoreductase. The preparation of a capsule containing laccase suspended in hardened palm oil at 40°C is disclosed.

[0023] JP 2004321077 also relates to the use of laccase in chewing gum for eliminating bad breath. The laccase is encapsulated in a capsule, which further contains, for example, hydrogenated oil. Summary of the Invention

[0024] This invention provides a means to make artificial meat products resemble browned meat in both uncooked and cooked states.

[0025] The inventors of this invention have discovered that when a red pigment that changes color upon oxidation is added together with an enzyme having laccase activity to a non-meat protein material (e.g., plant-based artificial meat products, such as hamburger patties), the enzymatic oxidation of the red pigment contributes to meat-like browning, for example, in hamburger patties.

[0026] By using enzymes, preferably isolated enzymes rather than, for example, another plant extract, enzyme activity can be easily controlled. Furthermore, enzymes with appropriate properties can be selected, such as temperature characteristics that match cooking conditions.

[0027] Therefore, the present invention relates to a non-meat protein material comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0028] The present invention also relates to a method for producing materials containing non-meat proteins, the method comprising:

[0029] a) Provide materials containing non-meat proteins; and

[0030] b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to this material.

[0031] The present invention also relates to the use of an enzyme with laccase activity for a controlled enzymatic browning reaction of a non-meat protein material containing a red pigment that changes color upon oxidation.

[0032] The present invention also relates to a composition for a controlled enzymatic browning reaction of a non-meat protein material containing a red pigment that changes color upon oxidation, said composition containing an enzyme having laccase activity.

[0033] The present invention also relates to a composition for controlled enzymatic browning reaction of non-meat protein materials, the composition comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0034] Preferably, the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of WYHXHX(3)-QYXDGX(2)-G, such as a prosite sequence, where X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0035] In a preferred embodiment, to further control browning to occur primarily during cooking, an enzyme with laccase activity is encapsulated in fat with a melting temperature of at least 25°C, preferably 25°C-60°C. In this way, the enzyme will only be released during the final cooking step and come into contact with the red pigment in non-meat protein materials, rather than during storage.

[0036] The present invention also relates to compositions comprising an enzyme having laccase activity, wherein the enzyme having laccase activity is embedded in a fat having a melting temperature of at least 25°C. Detailed Implementation

[0037] According to the present invention, a red pigment that changes color upon oxidation and an enzyme with laccase activity are added to a non-meat protein material that can be used as, for example, artificial meat or a meat extender, to make it turn brown in the same way as browned meat, and thus have a similar color to browned meat when cooked. During cooking, browned meat undergoes a Maillard reaction, which involves the condensation of amino and reducing groups to form intermediates. These intermediates eventually polymerize to form brown pigments (protein melanin), which impart a brown color to the cooked meat.

[0038] This invention relates to non-meat protein materials (such as cultured meat products) containing a red pigment that changes color upon oxidation and an enzyme with laccase activity.

[0039] In a preferred embodiment, the present invention relates to a cultured meat product containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% w / w, plant protein in total protein, the cultured meat product containing a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0040] The present invention also relates to a method for producing materials containing non-meat proteins (such as cultured meat products), the method comprising:

[0041] a) Provide materials containing non-meat proteins; and

[0042] b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to this material.

[0043] In a preferred embodiment, the present invention relates to a method for producing cultured meat products, the method comprising:

[0044] a) Provide materials containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% w / w, of plant protein in total protein; and

[0045] b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to this material.

[0046] The present invention also relates to the use of enzymes with laccase activity for controlled enzymatic browning reactions of non-meat protein materials (such as cultured meat products) containing a red pigment that changes color upon oxidation.

[0047] In a preferred embodiment, the present invention relates to the use of an enzyme with laccase activity for a controlled enzymatic browning reaction in a cultured meat product containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% w / w, of plant protein in total protein, and the cultured meat product containing a red pigment that changes color upon oxidation.

[0048] The present invention also relates to a composition for a controlled enzymatic browning reaction of a non-meat protein material containing a red pigment that changes color upon oxidation, said composition containing an enzyme having laccase activity.

[0049] The present invention also relates to a composition for controlled enzymatic browning reaction of non-meat protein materials, the composition comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0050] A non-meat protein-containing material is a material that contains non-meat proteins. The material may also contain meat proteins. The non-meat protein-containing material may be a mixture of non-meat proteins and meat proteins. In some preferred embodiments, the non-meat protein-containing material contains at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, and most preferably 100% non-meat proteins in the total protein. In some preferred embodiments, the non-meat protein-containing material does not contain meat proteins. In some preferred embodiments, the non-meat protein-containing material does not contain animal-derived proteins.

[0051] Non-meat proteins can be plant proteins or non-meat proteins obtained from other non-meat sources (e.g., algae, eggs, and / or dairy products), such as whey protein and / or casein. Non-meat proteins can be a mixture of plant proteins and non-meat proteins obtained from other non-meat sources. The proteins in non-meat protein-containing materials can be a mixture of proteins obtained from one or more plants, or a mixture of proteins obtained from one or more plants and proteins obtained from one or more other sources (e.g., algae, eggs, and / or dairy products) (e.g., whey protein or casein). In some preferred embodiments, the non-meat protein-containing material contains at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, and most preferably 100% plant protein in the total protein content.

[0052] In some embodiments, the non-meat protein material is a cultured meat product, such as a hamburger patty, or a plant-based cultured meat product, such as a plant-based hamburger patty.

[0053] In some embodiments, the non-meat protein material is an artificial meat product selected from artificial ground meat products, hamburger patties, sausages, artificial meatballs, and artificial gullasch meat products, preferably hamburger patties.

[0054] In some embodiments, the non-meat protein material is a plant-based artificial meat product, which is selected from artificial meat mince products, hamburger patties, sausages, artificial meatballs and artificial gouramis, preferably hamburger patties.

[0055] In some embodiments, the non-meat protein material is a plant protein material containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, and most preferably 100% plant protein in the total protein.

[0056] In some embodiments, the non-meat protein material is a plant protein material, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from cereals (e.g., rice, corn, wheat, oats), false cereals (e.g., buckwheat, quinoa), tubers (e.g., potatoes), oilseeds (e.g., soybeans, sunflowers), legumes (e.g., peas, chickpeas, lentils, lupins), or leafy plants (e.g., grasses), preferably at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow or green peas.

[0057] In some embodiments, the non-meat protein material is a plant protein material containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, and most preferably 100% plant protein in the total protein, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% plant protein obtained from cereals (e.g., rice, corn, wheat, oats), false cereals (e.g., buckwheat, quinoa), tubers (e.g., potatoes), oilseeds (e.g., soybeans, sunflowers), legumes (e.g., peas, chickpeas, lentils, lupins) or leafy plants (e.g., grasses), preferably wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% plant protein obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow or green peas.

[0058] In some embodiments, the non-meat protein material has been extruded, preferably extruded and shredded. In other embodiments, the non-meat protein material may have been textured by any of many other known methods for texturizing protein materials to provide a meat-like texture to the non-meat protein material.

[0059] Typically, the non-meat protein material in the non-meat protein material has been extruded, shredded, and formulated with other ingredients such as potato starch or other starch materials, cellulose, and / or salt. The extrusion and / or shredding of the non-meat protein can be performed before or after formulation with other ingredients, preferably before. Alternatively, the extrusion and / or shredding of the non-meat protein can be performed after the addition of some other ingredients (but not all).

[0060] As used herein, the terms “protein-containing material” or “material containing plant protein” refer to both materials that are primarily composed of protein and materials that contain protein and other components.

[0061] The non-meat protein in the non-meat protein material can be a plant-based protein material or an animal-derived protein material. Animal-derived non-meat protein materials that can be used include, but are not limited to, collagen, casein or caseinate protein, whey protein, and / or albumin. Plant-based protein materials that can be used include, but are not limited to, gluten, soy protein, and / or pea protein. Most preferably, the non-meat protein in the non-meat protein material is a pea protein material, such as pea protein isolate, pea protein concentrate, pea flour, pea flakes, or mixtures thereof, wherein the pea protein material preferably contains at least about 50% pea protein.

[0062] Such protein materials can be commercially available or manufactured.

[0063] Non-meat protein materials (such as cultured meat products) may also include adjuvants, including but not limited to starch, gums, and fibers, and mixtures thereof. Adjuvants may be included to impart various functionalities to non-meat protein materials, thereby improving their meat-like characteristics. For example, starch may be included in non-meat protein materials to increase their viscosity and gel-forming ability upon hydration. Gum may be included to enhance flowability. Fiber may be included to enhance their structure upon hydration.

[0064] Non-meat protein materials can be hydrated with water at a ratio of approximately 1 to 4 parts by weight of water to approximately 1 part by weight of protein material. Red pigments and / or enzymes can be mixed into the water before addition.

[0065] Sufficient water should be added to promote the mixing of pigments and enzymes into the non-meat protein-containing material, and excessive water should be avoided to ensure that the non-meat protein-containing material absorbs most of the pigment. The non-meat protein-containing material should be thoroughly mixed to hydrate and structure it, so that the pigment is well dispersed in the non-meat protein-containing material to provide uniform coloring.

[0066] If desired, seasonings can be added to ingredients containing non-meat proteins to provide a meat-like flavor.

[0067] According to the present invention, a red pigment is included to impart a color similar to that of uncooked browned meat to non-meat protein materials, thus making the non-meat protein materials resemble meat when uncooked. The red pigment used according to the present invention changes color upon oxidation, and when an enzyme with laccase activity is further added to the non-meat protein materials, the red color gradually disappears during cooking, and the non-meat protein materials will also resemble meat when cooked.

[0068] Red pigments can be extracted from the fruits, leaves, flowers, and / or roots of plants. Plant pigments are colored chemical substances produced by plants that absorb visible radiation between 380-780 nm. Betalains are a class of red-purple (betalain) and yellow (betaflavin) pigments found in plants of the Caryophyllales order. Common primary sources of betalains are Amaranthaceae plants, red beets, bougainvillea, and Cactaceae plants.

[0069] The pigment betaine is derived from red beet (Beta vulgaris).

[0070] In some preferred embodiments, the red pigment is obtained from plants, preferably from beetroot or amaranth grains.

[0071] In some preferred embodiments, the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet roots or amaranth pigment obtained from amaranth grains.

[0072] Preferably, the hydrated non-meat protein material contains about 0.0005% to about 0.5% of a pigment by weight, such as 0.001% to about 0.1%, more preferably about 0.001% to about 0.05% of a pigment.

[0073] In some preferred embodiments, the red pigment is betaine in the form of beetroot juice or beetroot powder, preferably betaine in the form of beetroot powder.

[0074] Non-meat protein materials (such as cultured meat products) may contain about 0.5% to about 20% beetroot powder by weight, such as about 1% to about 10%.

[0075] The enzyme possessing laccase activity is preferably laccase, more preferably laccase classified as EC1.10.3.2. Laccase (phenylene glycol: oxygen oxidoreductase, EC 1.10.3.2) is a polyphenol oxidase containing a copper atom at its catalytic center, and may be called a polycopper oxidase. Laccase-mediated catalysis typically involves the reduction of oxygen to water, accompanied by the oxidation of the substrate, which can be, for example, polyphenols, methoxylated phenols, aromatic diamines, or any of a range of other compounds.

[0076] For the purposes of this invention, laccase activity can be determined using a spectrophotometer using syringaldehyde azide as a substrate, through which laccase oxidizes syringaldehyde azide to tetramethoxyazobis(methylenequinone). The catalytic rate is measured at 540 nm.

[0077] Protein sequences can be classified into subclasses based on conserved patterns determined by sequence alignment. One such systematic pattern is the prosite pattern or prosite sequence, which is a conserved amino acid segment within a protein sequence subclass (such as a subclass of laccases).

[0078] In some embodiments of the present invention, the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of WYHXHX(3)-QYXDGX(2)-G, such as a prosite sequence, wherein X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0079] In the prosite sequence, the values ​​(3) and (2) after X indicate the presence of three or two amino acid residues, or in other words, X(3) corresponds to XXX and X(2) corresponds to XX. The amino acid residues are represented by their IUPAC single-letter codes, where A = alanine, R = arginine, N = asparagine, D = aspartic acid, C = cysteine, Q = glutamine, E = glutamic acid, G = glycine, H = histidine, I = isoleucine, L = leucine, K = lysine, M = methionine, F = phenylalanine, P = proline, S = serine, T = threonine, W = tryptophan, Y = tyrosine, and V = valine.

[0080] In some embodiments of the present invention, the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of GTXWYHX(5)-QY, such as a prosite sequence, wherein X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0081] In some embodiments, the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of [IL]-XD-[YW]-[YFNM]-[YHG], such as a prosite sequence, wherein [IL] indicates that the amino acid can be I or L, [YW] indicates that the amino acid can be Y or W, [YFNM] indicates that the amino acid can be Y, F, N or M, [YHG] indicates that the amino acid can be Y, H or G, and X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0082] In some embodiments, the enzyme having laccase activity has at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the mature polypeptide of SEQ ID NO:1.

[0083] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 any one of SEQ ID NO:1-8. In some embodiments, the enzyme having laccase activity differs from the mature polypeptide of any one of SEQ ID NO:1-8 by at most 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0084] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 any one of SEQ ID NO:1-4. In some embodiments, the enzyme having laccase activity differs from the mature polypeptide of any one of SEQ ID NO:1-4 by at most 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0085] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Niedel program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the nobrief option must be specified in the command line. The Niedel-marked "longest identity" output is calculated as follows:

[0086] (identical residues x 100) / (alignment length - total number of vacancies in the alignment)

[0087] The term "mature polypeptide" refers to a polypeptide in its mature form after N-terminal processing (e.g., removal of the signal peptide). In some embodiments, the mature polypeptide of SEQ ID NO:1 has the amino acid sequence of SEQ ID NO:1. In some embodiments, the mature polypeptide of SEQ ID NO:2 has the amino acid sequence of SEQ ID NO:2. In some embodiments, the mature polypeptide of SEQ ID NO:3 has the amino acid sequence of SEQ ID NO:3. In some embodiments, the mature polypeptide of SEQ ID NO:4 has the amino acid sequence of SEQ ID NO:4.

[0088] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 a polypeptide having an amino acid sequence of any one of SEQ ID NO: 1-4. In some embodiments, the enzyme having laccase activity differs from a polypeptide having an amino acid sequence of any one of SEQ ID NO: 1-4 by at most 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0089] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 polypeptide having the amino acid sequence of SEQ ID NO:1. In some embodiments, the enzyme having laccase activity is obtained from the genus *Myceliophthora*, preferably from *Myceliophthora thermophila*.

[0090] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 polypeptide having the amino acid sequence of SEQ ID NO:2. In some embodiments, the enzyme having laccase activity is obtained from the genus Rhizoctonia, preferably from Rhizoctonia solani.

[0091] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 polypeptide having the amino acid sequence of SEQ ID NO:3. In some embodiments, the enzyme having laccase activity is obtained from the genus Coprinus, preferably from Coprinus cinereus.

[0092] In some embodiments, the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 polypeptide having the amino acid sequence of SEQ ID NO:4. In some embodiments, the enzyme having laccase activity is obtained from the genus Polyporus, preferably from Polyporus pinsitus.

[0093] Enzymes with laccase activity can be obtained from any genus of microorganisms, such as bacteria or fungi, or from, for example, plants or insects. For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In some embodiments, enzymes obtained from a given source are secreted extracellularly.

[0094] In some embodiments, the enzyme with laccase activity is recombinantly produced. In some embodiments, the enzyme with laccase activity is heterologous to the recombinant host cell. The host cell can be any microbial or plant cell that can be used to recombinantly produce an enzyme with laccase activity, such as a prokaryotic cell or fungal cell.

[0095] In a preferred embodiment, the enzyme having laccase activity is isolated. The term "isolated" can mean that the enzyme is isolated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.) found in nature and naturally associated with it. Isolated enzymes include, but are not limited to, culture media containing secreted enzymes. The term "isolated" can also mean that the enzyme is largely isolated from the cells expressing the enzyme, for example, after applying purification or enrichment techniques (such as centrifugation or filtration).

[0096] In a preferred embodiment, the enzyme having laccase activity is purified. The term "purified" can mean an enzyme that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified enzymes form discrete bands). The purity of the purified enzyme can be at least 50%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% (e.g., by weight). The term "purified" can also mean that the enzyme is present in the composition at a relative or absolute concentration higher than that of the starting composition (e.g., the fermentation broth from which the enzyme was obtained).

[0097] In a preferred embodiment, the concentration of the enzyme with laccase activity is 0.1-100 mg EP / kg of non-meat protein material, preferably 0.5-50 mg EP / kg, such as 1-20 mg EP / kg. EP is an enzyme protein.

[0098] The amounts of red pigment and enzyme can be varied to adjust the color of non-meat protein materials to the desired color.

[0099] A red pigment that changes color upon oxidation and an enzyme with laccase activity can be added to a non-meat protein-containing material in the form of a dry powder or an aqueous solution. In some embodiments, the dry pigment and the non-meat protein-containing material can be dispersed together in water to hydrate the non-meat protein-containing material.

[0100] In a preferred embodiment, the enzyme with laccase activity is embedded in fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0101] The fat preferably has a melting temperature above room temperature.

[0102] Typically, plant-based fats can undergo melting within a certain temperature range. Coconut oil, often referred to as coconut fat, can have a melting temperature of, for example, 27°C–31°C. However, this range may vary depending on the type of coconut oil.

[0103] Palm oil, depending on whether it is hydrogenated or non-hydrogenated, can have a chain-breaking temperature of, for example, 37°C to 60°C.

[0104] In the context of this invention, the terms coconut resin and coconut oil are used interchangeably, as are the terms palm resin and palm oil.

[0105] In some preferred embodiments, the fat is a plant-based fat, preferably coconut oil or palm oil.

[0106] Enzymes with laccase activity can be encapsulated in fat by decomposing the fat and mixing enzymes into the molten fat. The enzyme-containing solid fat is then blended into non-meat ingredients using a meat mixer.

[0107] Non-meat protein materials containing red dye and enzymes (preferably textured and / or flavored) can be used as artificial meat. In one aspect of the invention, the non-meat protein material containing red dye and enzymes can be formed into patties or filled into casings to form artificial meat patties or sausages. The artificial meat patties and sausages can be cooked at effective cooking temperatures (e.g., from about 50°C to about 260°C) and for a given time, for example, by frying or baking.

[0108] The present invention also relates to compositions comprising an enzyme having laccase activity, the enzyme having laccase activity being embedded in a fat having a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C. Preferably, the fat is a plant-derived fat, more preferably coconut oil or palm oil. The enzyme having laccase activity is preferably isolated and / or purified. The enzyme having laccase activity is preferably laccase, more preferably laccase classified as EC1.10.3.2.

[0109] Preferred embodiments

[0110] 1. An artificial meat product containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% w / w, of plant protein in total protein, the artificial meat product containing a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0111] 2. A method for producing cultured meat products, the method comprising:

[0112] a) Provide materials containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% w / w, of plant protein in total protein; and

[0113] b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to this material.

[0114] 3. Use of an enzyme with laccase activity for the controlled enzymatic browning reaction of a cultured meat product containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% w / w, of plant protein in total protein, and the cultured meat product containing a red pigment that changes color upon oxidation.

[0115] 4. The artificial meat product, method or use as described in any of the foregoing embodiments, wherein the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of WYHXHX(3)-QYXDGX(2)-G, such as a prosite sequence, wherein X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0116] 5. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0117] 6. The artificial meat product, method, or use as described in the preceding embodiment, wherein the fat is a plant-based fat, preferably coconut oil or palm oil.

[0118] 7. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the red pigment is obtained from a plant, preferably from beetroot or amaranth grain.

[0119] 8. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet root or amaranth pigment obtained from amaranth grain.

[0120] 9. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the red pigment is beetroot juice or beetroot powder, preferably betaine in the form of beetroot powder.

[0121] 10. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the artificial meat product is selected from artificial ground meat products, hamburger patties, sausages, artificial meatball products, and artificial goura meat products, preferably hamburger patties.

[0122] 11. The cultured meat product, method, or use as described in the preceding embodiment, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from cereals, such as rice, corn, wheat, oats; false cereals, such as buckwheat, quinoa; tubers, such as potatoes; oilseeds, such as soybeans, sunflowers; legumes, such as peas, chickpeas, lentils, lupins; or leafy plants, such as grasses; preferably at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow or green peas.

[0123] 12. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the plant protein has been extruded, preferably extruded and chopped.

[0124] 13. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the enzyme having laccase activity is isolated.

[0125] 14. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the enzyme having laccase activity is laccase, preferably laccase classified as EC1.10.3.2.

[0126] 15. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the concentration of the enzyme having laccase activity is 0.1-100 mg EP / kg of the artificial meat product, preferably 0.5-50 mg EP / kg, such as 1-20 mg EP / kg.

[0127] 16. The artificial meat product, method, or use as described in any of the foregoing embodiments, wherein the enzyme having laccase activity is purified.

[0128] 17. A non-meat protein material comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0129] 18. A method for producing a material containing non-meat protein, the method comprising:

[0130] a) Provide materials containing non-meat proteins; and

[0131] b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to this material.

[0132] 19. The use of an enzyme with laccase activity for the controlled enzymatic browning reaction of a non-meat protein material containing a red pigment that changes color upon oxidation.

[0133] 20. A composition for a controlled enzymatic browning reaction of a non-meat protein material containing a red pigment that changes color upon oxidation, said composition containing an enzyme having laccase activity.

[0134] 21. A composition for controlled enzymatic browning reaction of non-meat protein materials, said composition comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0135] 22. The composition, material, method, or use as described in any one of the preceding five embodiments, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0136] 23. The composition, material, method, or use as described in the preceding embodiment, wherein the fat is a plant-derived fat, preferably coconut oil or palm oil.

[0137] 24. The composition, material, method or use as described in any one of the preceding seven embodiments, wherein the red pigment is obtained from a plant, preferably from beetroot or amaranth grain.

[0138] 25. The composition, material, method, or use as described in any one of the preceding eight embodiments, wherein the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet root or amaranth pigment obtained from amaranth grain.

[0139] 26. The composition, material, method, or use as described in any of the preceding nine embodiments, wherein the red pigment is betaine juice or betaine powder, preferably betaine glycoside in the form of betaine powder.

[0140] 27. The composition, material, method, or use as described in any of the foregoing ten embodiments, wherein the non-meat protein material is a plant-based artificial meat product, such as a plant-based hamburger patty.

[0141] 28. The composition, material, method, or use as described in any one of the preceding eleven embodiments, wherein the non-meat protein material is a plant protein material containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% plant protein, in the total protein.

[0142] 29. The compositions, materials, methods, or uses described in the foregoing twelve embodiments, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from cereals, such as rice, corn, wheat, oats; false cereals, such as buckwheat, quinoa; tubers, such as potatoes; oilseeds, such as soybeans, sunflowers; legumes, such as peas, chickpeas, lentils, lupins; or leafy plants, such as grasses; preferably at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow peas or green peas.

[0143] 30. The composition, material, method, or use as described in any of the preceding thirteen embodiments, wherein the non-meat protein in the non-meat protein-containing material has been extruded, preferably extruded and chopped.

[0144] 31. The composition, material, method, or use as described in any of the foregoing fourteen embodiments, wherein the enzyme having laccase activity is isolated.

[0145] 32. A non-meat protein material comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0146] 33. The material as described in the preceding embodiment, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0147] 34. The material as described in the preceding embodiment, wherein the fat is a plant-derived fat, preferably coconut oil or palm oil.

[0148] 35. The material as described in any one of the three embodiments above, wherein the red pigment is obtained from a plant, preferably from beetroot or amaranth grain.

[0149] 36. The material as described in any one of the four embodiments above, wherein the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet root or amaranth pigment obtained from amaranth grain.

[0150] 37. The material as described in any one of the five embodiments above, wherein the red pigment is beetroot juice or beetroot powder, preferably betaine in the form of beetroot powder.

[0151] 38. The material as described in any one of the six preceding embodiments, wherein the material is a plant-based artificial meat product, such as a plant-based hamburger patty.

[0152] 39. The material as described in any one of the seven embodiments above, wherein the material is a plant-based artificial meat product selected from artificial meat mince products, hamburger patties, sausages, artificial meatball products and artificial goura meat products, preferably hamburger patties.

[0153] 40. The material as described in any one of the preceding eight embodiments, wherein the material is a plant protein-containing material containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% plant protein, in the total protein.

[0154] 41. The material as described in the preceding embodiment, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from cereals (e.g., rice, corn, wheat, oats), false cereals (e.g., buckwheat, quinoa), tubers (e.g., potatoes), oilseeds (e.g., soybeans, sunflowers), legumes (e.g., peas, chickpeas, lentils, lupins) or leafy plants (e.g., grasses), preferably wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow or green peas.

[0155] 42. The material as described in any one of the ten embodiments above, wherein the material has been extruded, preferably extruded and shredded.

[0156] 43. The material as described in any one of the eleven preceding embodiments, wherein the enzyme having laccase activity is isolated.

[0157] 44. The material as described in any one of the twelve preceding embodiments, wherein the enzyme having laccase activity is purified.

[0158] 45. The material as described in any one of the thirteen preceding embodiments, wherein the enzyme having laccase activity is laccase, preferably laccase classified as EC1.10.3.2.

[0159] 46. ​​The material as described in any one of the fourteen preceding embodiments, wherein the concentration of the enzyme having laccase activity is 0.1-100 mg EP / kg of non-meat protein material, preferably 0.5-50 mg EP / kg, such as 1-20 mg EP / kg.

[0160] 47. A method for producing a material containing non-meat protein, the method comprising:

[0161] a) Provide materials containing non-meat proteins; and

[0162] b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to this material.

[0163] 48. The method as described in the preceding embodiment, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0164] 49. The method as described in the preceding embodiment, wherein the fat is a plant-based fat, preferably coconut oil or palm oil.

[0165] 50. The method of any one of the three embodiments described above, wherein the red pigment is obtained from a plant, preferably from beetroot or amaranth grain.

[0166] 51. The method of any one of the four embodiments described above, wherein the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet root or amaranth pigment obtained from amaranth grain.

[0167] 52. The method of any one of the five embodiments described above, wherein the red pigment is beetroot juice or beetroot powder, preferably betaine in the form of beetroot powder.

[0168] 53. The method as described in any one of the six preceding embodiments, wherein the non-meat protein material is a plant-based artificial meat product, such as a plant-based hamburger patty.

[0169] 54. The method of any one of the preceding seven embodiments, wherein the non-meat protein material is a plant-based artificial meat product, the plant-based artificial meat product being selected from artificial meat mince products, hamburger patties, sausages, artificial meatball products and artificial goura meat products, preferably hamburger patties.

[0170] 55. The method of any one of the preceding eight embodiments, wherein the non-meat protein material is a plant protein material containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% plant protein, in the total protein.

[0171] 56. The method as described in the preceding embodiment, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from cereals (e.g., rice, corn, wheat, oats), false cereals (e.g., buckwheat, quinoa), tubers (e.g., potatoes), oilseeds (e.g., soybeans, sunflowers), legumes (e.g., peas, chickpeas, lentils, lupins), or leafy plants (e.g., grasses), preferably wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow or green peas.

[0172] 57. The method as described in any one of the foregoing ten embodiments, wherein the non-meat protein in the non-meat protein material has been extruded, preferably extruded and chopped.

[0173] 58. The method as described in any one of the preceding eleven embodiments, wherein the enzyme having laccase activity is isolated.

[0174] 59. The method as described in any one of the twelve preceding embodiments, wherein the enzyme having laccase activity is purified.

[0175] 60. The method as described in any one of the preceding thirteen embodiments, wherein the enzyme having laccase activity is a laccase, preferably a laccase classified as EC1.10.3.2.

[0176] 61. The method as described in any one of the foregoing fourteen embodiments, wherein the concentration of the enzyme having laccase activity is 0.1-100 mg EP / kg of non-meat protein material, preferably 0.5-50 mg EP / kg, such as 1-20 mg EP / kg.

[0177] 62. The use of an enzyme with laccase activity for the controlled enzymatic browning reaction of a non-meat protein material containing a red pigment that changes color upon oxidation.

[0178] 63. The use as described in the preceding embodiment, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0179] 64. The use as described in the preceding embodiment, wherein the fat is a plant-derived fat, preferably coconut oil or palm oil.

[0180] 65. The use as described in any one of the three embodiments above, wherein the red pigment is obtained from a plant, preferably from beetroot or amaranth grain.

[0181] 66. The use as described in any one of the four embodiments above, wherein the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet root or amaranth pigment obtained from amaranth grain.

[0182] 67. The use as described in any one of the five embodiments above, wherein the red pigment is betaine in the form of beetroot juice or beetroot powder, preferably betaine in the form of beetroot powder.

[0183] 68. The use as described in any one of the preceding six embodiments, wherein the non-meat protein material is a plant-based artificial meat product, such as a plant-based hamburger patty.

[0184] 69. The use as described in any one of the preceding seven embodiments, wherein the non-meat protein material is a plant-based artificial meat product selected from artificial meat mince products, hamburger patties, sausages, artificial meatballs and artificial gouramis, preferably hamburger patties.

[0185] 70. The use as described in any one of the preceding eight embodiments, wherein the non-meat protein material is a plant protein material containing at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% plant protein, in the total protein.

[0186] 71. The use as described in the preceding embodiment, wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from cereals (e.g., rice, corn, wheat, oats), false cereals (e.g., buckwheat, quinoa), tubers (e.g., potatoes), oilseeds (e.g., soybeans, sunflowers), legumes (e.g., peas, chickpeas, lentils, lupins) or leaves (e.g., grasses), preferably wherein at least 50% w / w, preferably at least 70%, at least 90%, at least 95%, or at least 98%, such as 100% of the plant protein is obtained from peas, soybeans, or wheat, more preferably from peas, and most preferably from yellow or green peas.

[0187] 72. The use as described in any one of the foregoing ten embodiments, wherein the non-meat protein in the non-meat protein material has been extruded, preferably extruded and chopped.

[0188] 73. The use as described in any one of the preceding eleven embodiments, wherein the enzyme having laccase activity is isolated.

[0189] 74. The use as described in any one of the twelve preceding embodiments, wherein the enzyme having laccase activity is purified.

[0190] 75. The use as described in any one of the thirteen preceding embodiments, wherein the enzyme having laccase activity is a laccase, preferably a laccase classified as EC1.10.3.2.

[0191] 76. The use as described in any one of the foregoing fourteen embodiments, wherein the concentration of the enzyme having laccase activity is 0.1-100 mg EP / kg of non-meat protein material, preferably 0.5-50 mg EP / kg, such as 1-20 mg EP / kg.

[0192] 77. A composition for controlled enzymatic browning reaction of non-meat protein materials, said composition comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity.

[0193] 78. The composition as described in the preceding embodiment, wherein the enzyme having laccase activity is embedded in a fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0194] 79. The composition as described in the preceding embodiment, wherein the fat is a plant-derived fat, preferably coconut oil or palm oil.

[0195] 80. The composition as described in any one of the three preceding embodiments, wherein the red pigment is obtained from a plant, preferably from beetroot or amaranth grain.

[0196] 81. The composition as described in any one of the four preceding embodiments, wherein the red pigment is betaine or amaranth pigment, preferably betaine obtained from beet root or amaranth pigment obtained from amaranth grain.

[0197] 82. The composition as described in any one of the five preceding embodiments, wherein the red pigment is betaine juice or betaine powder, preferably betaine in the form of betaine powder.

[0198] 83. The composition as described in any one of the preceding six embodiments, wherein the enzyme having laccase activity is isolated.

[0199] 84. The composition as described in any one of the seven preceding embodiments, wherein the enzyme having laccase activity is purified.

[0200] 85. The composition as described in any one of the preceding eight embodiments, wherein the enzyme having laccase activity is laccase, preferably laccase classified as EC1.10.3.2.

[0201] 86. A composition comprising an enzyme having laccase activity, the enzyme having laccase activity being embedded in a fat at a melting temperature of at least 25°C, preferably 25°C-60°C, more preferably 25°C-40°C, and most preferably 30°C-40°C.

[0202] 87. The composition as described in the preceding embodiment, wherein the fat is a plant-derived fat, preferably coconut oil or palm oil.

[0203] 88. The composition as described in any one of the two preceding embodiments, wherein the enzyme having laccase activity is isolated.

[0204] 89. The composition as described in any one of the three preceding embodiments, wherein the enzyme having laccase activity is purified.

[0205] 90. The composition as described in any one of the four preceding embodiments, wherein the enzyme having laccase activity is laccase, preferably a laccase classified as EC1.10.3.2.

[0206] 91. The artificial meat product, method, use, material or composition as described in any of the foregoing embodiments, wherein the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of WYHXHX(3)-QYXDGX(2)-G, such as a prosite sequence, wherein X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0207] 92. The artificial meat product, method, use, material or composition as described in any of the foregoing embodiments, wherein the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of GTXWYHX(5)-QY, such as a prosite sequence, wherein X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0208] 93. The artificial meat product, method, use, material, or composition as described in any of the foregoing embodiments, wherein the amino acid sequence of the enzyme having laccase activity comprises a sequence consisting of [IL]-XD-[YW]-[YFNM]-[YHG], such as a prosite sequence, wherein [IL] indicates that the amino acid can be I or L, [YW] indicates that the amino acid can be Y or W, [YFNM] indicates that the amino acid can be Y, F, N, or M, [YHG] indicates that the amino acid can be Y, H, or G, and X can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

[0209] 94. The artificial meat product, method, use, material, or composition as described in any of the foregoing embodiments, wherein the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 any one of SEQ ID NO:1-8.

[0210] 95. The artificial meat product, method, use, material, or composition as described in any of the foregoing embodiments, wherein the enzyme having laccase activity has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 polypeptide of any one of SEQ ID NO:1-4.

[0211] 96. The artificial meat product, method, use, material or composition as described in any of the foregoing embodiments, wherein the enzyme having laccase activity differs from the mature polypeptide of any one of SEQ ID NO:1-8 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0212] 97. The artificial meat product, method, use, material or composition as described in any of the foregoing embodiments, wherein the enzyme having laccase activity differs from the polypeptide of any one of SEQ ID NO:1-4 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0213] Example

[0214] Example 1

[0215] Beetroot juice is converted from red to brown using an enzymatic process.

[0216] Beetroot juice (1%) was prepared by dissolving beetroot powder (1%) in 50 mM MES buffer (pH 6.3). 10 mg / L of the enzymes shown in Table 1 was added to the beetroot juice and incubated for 10 minutes. The prepared MtL laccase was a laccase derived from *Heterotrophus thermophilus*, prepared in 95% polyol. Absorbance was measured at 350 and 530 nm using a spectrophotometer, and the decrease in the 530 / 350 nm ratio was used as a measure of browning.

[0217] Table 1

[0218]

[0219] All laccases tested showed the ability to turn beetroot juice from red to brown. The last four enzymes in the table are non-laccase oxidases, including monophenol monooxidase and tyrosinase, which do not appear to have this ability.

[0220] Example 2

[0221] Low oxidation levels at 4°C may lead to undesirable browning.

[0222] Although the optimal temperature for laccase is 50°C-60°C, the low oxidation levels at 4°C during refrigeration before cooking can lead to undesirable browning. Plant-based hamburger patties were prepared by mixing 50g of textured pea protein (extruded yellow pea protein isolate) pre-soaked in 90g of water, 2.4g of potato starch, 1.4g of salt, and 10g of beetroot powder. For each patty, 20g was weighed and formed into patties with a diameter of 4.5cm. Each patty was prepared either without laccase or with 14.3mg EP / kg or 1.24mg EP / kg of MtL laccase. The patties were stored at 4°C for 24 hours, and browning was visually inspected. Some browning was observed at low doses, and significant browning was observed at high doses.

[0223] Table 2

[0224] Added laccase (mg EP / kg) 0 1.24 14.3 browning degree - + +++

[0225] Example 3

[0226] Laccase was formulated from plant-derived fats.

[0227] To ensure that browning occurs only during cooking and not during storage, laccase is prepared in fats with a melting temperature of 30°C–40°C. This ensures that laccase-catalyzed browning occurs only within the appropriate temperature range (40°C–75°C) where the laccase is released into the hamburger patty. Coconut oil with a melting point of approximately 30°C is melted at 40°C, and 0.3 mg EP / ml MtL laccase is mixed with the liquid oil. 50 μl of the cured oil, with or without laccase, is aliquoted into Eppendorf tubes, 1 ml of beetroot juice (1%) is added, and the mixture is incubated at the stated temperature for 50 min.

[0228] Table 3

[0229] Temperature (°C) Enzyme-free (530 / 350nm) Laccase (530 / 350nm) 15 1.6 1.6 25 1.5 1.5 35 1.5 1.5 45 1.5 1.4 55 1.3 0.5 65 1.0 0.6 75 0.7 0.6 85 0.4 0.4 95 0.2 0.2

[0230] An increase in enzyme-catalyzed browning was observed between 45°C and 75°C.

[0231] Example 4

[0232] Formulate laccase relative to unformulated laccase in plant-derived fats.

[0233] Coconut oil was melted at 40°C, and 0.3 mg EP / ml MtL laccase was mixed with the liquid oil. 50 μl of solidified fat in 10 x 5 μl spheres was added. After solidification, 1 ml of 1% beetroot juice was added, and the mixture was incubated at 4°C or 60°C. In the control group, a similar concentration of laccase was added directly to the tubes. At 65°C, the browning rate of laccase prepared in 5 μl droplets was similar to that of laccase added directly to beetroot juice; therefore, preparation in fat had only a small negative impact on catalytic efficiency at cooking temperatures. Conversely, at a pre-cooking refrigeration temperature (4°C), the browning rate was significantly lower when laccase was prepared in fat.

[0234] Table 4: Browning rate at 65℃

[0235]

[0236] Table 4 shows that the browning rate at 65°C is similar for laccase that is added directly and laccase that is formulated in fat.

[0237] Table 5 Browning rate at 4℃

[0238]

[0239] Table 5 shows that at 4°C (refrigeration temperature), when laccase was added directly, the 530 / 350nm ratio decreased to 0.5 after 24 hours, while when laccase was prepared in fat, the 530 / 350nm ratio decreased to 0.5 after 48-72 hours. That is, when laccase was prepared in fat, the browning rate was significantly lower.

[0240] Example 5

[0241] Hamburger patties containing laccase formulated with plant-based fats

[0242] Plant-based hamburger patties were prepared by mixing 50g of textured pea protein (extruded yellow pea protein isolate) pre-soaked in 90g of water, 2.4g of potato starch, 1.4g of salt, and 10g of beetroot powder. For each patty, 20g was weighed out and patties were formed with a diameter of 4.5cm. Each patty was prepared with or without laccase, or with 14.3mg EP / kg, 7.15mg EP / kg, or 1.24mg EP / kg MtL of laccase. The patties were fried to a core temperature of 75°C–80°C, allowed to cool, and then cut in half lengthwise.

[0243] Significant browning was observed in the center of the meat patties at doses of 14.3 mg EP / kg and 7.15 mg EP / kg.

[0244] Table 6

[0245] Laccase (mg EP / kg) 0 1.24 7.15 14.3 browning degree - + ++ +++

[0246] Browning was also observed during cooking when 14.3 mg EP / kg laccase was mixed with coconut oil before preparing hamburger patties.

[0247] Table 7

[0248] Laccase in fat (mg EP / kg) 0 14.3 browning degree - ++

[0249] Example 6

[0250] Copper oxidase phylogenetic tree and prosite sequence pattern

[0251] Laccase phylogenetic tree

[0252] Phylogenetic trees were constructed from laccase peptide sequences containing the AA1 domain as defined in CAZY (Lombard, Henrissat et al., 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, http: / / www.cazy.org / ). Phylogenetic trees were constructed from multiple alignments of mature peptide sequences containing at least one AA1 domain. The sequences were aligned using MUSCLE algorithm version 3.8.31 (Edgar, 2004. Nucleic Acids Research 32(5):1792-1797), and the tree was constructed using FastTree version 2.1.8 (Price et al., 2010. PloSone 5(3)) and visualized using iTOL (Letunic and Bork, 2007. Bioinformatics 23(1):127-128).

[0253] A subset of peptides containing the AA1 domain also contains three Cu-oxidase domains, such as those defined by Pfam domains IDPF00394 (Cu-oxidase), PF07731 (Cu-oxidase_2), and PF07732 (Cu-oxidase_3) (The Pfam protein families database: towards a more sustainable future: RD Finn, P. Coggill, RY Eberhardt, SREddy, J. Mistry, A. L. Mitchell, SCPotter, M. Punta, M. Qureshi, A. Sangrador-Vegas, GASalazar, J. Tate, A. Bateman, Nucleic Acids Research (2016) Database Issue 44: D279-D285; Pfam database version 32). All laccase peptides in Table 1 (Example 1) and Table 9 (Example 7) contain an AA1 domain and three Pfam domains. As an example, in SEQ ID NO:5 from *Thermophilus thymol* (identical to SEQ ID NO:1 except for including a signal peptide), the AA1 domain is located at positions 82 to 597, and the Pfam domains are located at positions 96 to 206 (PF07732), 216 to 362 (PF00394), and 439 to 569 (PF07731).

[0254] Prosite sequence mode

[0255] A subset of peptides containing the AA1 domain may contain one or more prosite sequence patterns (De Castro et al., ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. 2006 July 1; 34). An example is the conserved prosite pattern disclosed in WO 2016 / 097270, WYHXHX(3)-QYXDGX(2)-G, where X is an amino acid residue A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. This prosite sequence pattern is located at positions 183 to 198 of SEQ ID NO:5 (MtL) of this application.

[0256] All laccase peptides in Table 1 (Example 1) contain the conserved prosite pattern WYHXHX(3)-QYXDGX(2)-G. The Aspergillus niger laccase peptides in Table 9 (Example 7) do not contain this prosite pattern.

[0257] Laccases with a prosite pattern according to the sequence pattern WYHXHX(3)-QYXDGX(2)-G were found to effectively convert the red color of, for example, beetroot juice to brown. Two laccases from Aspergillus niger (shown as inactive in Example 7) did not contain such a prosite pattern. Therefore, laccases with this prosite pattern effectively convert the red color of, for example, beetroot juice.

[0258] Examples of laccases with the prosite sequence WYHXHX(3)-QYXDGX(2)-G are shown in Table 8.

[0259] Table 8

[0260]

[0261]

[0262] SEQ ID NO:5 from *Thermophilus thymol* is identical to SEQ ID NO:1 except that it includes a signal peptide.

[0263] SEQ ID NO:6 from Rhizoctonia solani is identical to SEQ ID NO:2 except that it includes a signal peptide.

[0264] SEQ ID NO:7 from Coprinus gracilistylus is identical to SEQ ID NO:3 except that it includes a signal peptide.

[0265] SEQ ID NO:8 from Pistia sp. is identical to SEQ ID NO:4 except that it includes a signal peptide.

[0266] Other laccases with the prosite sequence WYHXHX(3)-QYXDGX(2)-G are shown in WO2016 / 097270, for example, in Table 1 and on page 5. Among these, examples from WO 2016 / 097270 show that laccases from *T. hirsuta*, *P. brumalis*, *P. radiata*, and *C. subvemispora* can convert annatto red in whey.

[0267] WO 2016 / 097270 discloses laccases with the proximity pattern WYHXHX(3)-QYXDGX(2)-G exhibiting site structures with higher activity compared to laccases lacking this proximity pattern. Examples of laccases with the proximity pattern and this open structure can be found in PDB identifiers: 1KYA, 3V9C, 3FPX, and 2XYB.

[0268] We further discovered that the characteristic of red laccases that effectively convert, for example, beetroot juice is the presence of a conserved prosite pattern according to the amino acid sequence GTXWYHX(5)-QY. Neither of the two ineffective Aspergillus niger laccases contains this prosite pattern. As an example, this prosite sequence pattern is located at positions 180 to 192 of SEQ ID NO:5(MtL) of this application.

[0269] We further discovered that laccases that efficiently convert red substances, such as beetroot juice, are characterized by a conserved prosite pattern based on the amino acid sequence [IL]-XD-[YW]-[YFNM]-[YHG]. Two inactive Aspergillus niger laccases do not contain this prosite pattern. [IL] indicates that the amino acid can be I or L, [YW] indicates that the amino acid can be Y or W, and so on. X can be any of the 20 amino acids.

[0270] Example 7

[0271] The two laccases from Aspergillus niger, lacking the prosite pattern identified in Example 6, could not convert the red color of beetroot juice to brown.

[0272] Beetroot juice (1%) was prepared by dissolving beetroot powder (1%) in 50 mM MES buffer (pH 6.3). 10 mg / L of the enzymes shown in Table 9 was added to the beetroot juice and incubated at 50 °C for 10 min. Absorbance was measured at 350 and 530 nm using a spectrophotometer, and the decrease in the 530 / 350 nm ratio was used as a measure of browning.

[0273] Table 9

[0274]

[0275] The two laccases from Aspergillus niger tested in WO 2016 / 097270 have been heterologously expressed in Aspergillus oryzae.

[0276] To produce purified laccase, a single-sequence-verified expression clone has been isolated.

[0277] The conclusion drawn was that Aspergillus niger laccases that did not contain the prosite pattern identified in Example 6 did not show browning activity in the above assays.

Claims

1. An artificial meat product comprising at least 50% w / w plant protein in total protein, the artificial meat product comprising a red pigment that changes color upon oxidation and an enzyme having laccase activity, wherein the amino acid sequence of the enzyme having laccase activity comprises the sequence WYHXHX(3)-QYXDGX(2)-G, wherein X is any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, wherein the red pigment is betaine or amaranthin, and wherein the enzyme having laccase activity is laccase.

2. A method for producing cultured meat products, the method comprising: a) Provide materials containing at least 50% w / w plant protein in total protein; as well as b) Add a red pigment that changes color upon oxidation and an enzyme with laccase activity to the material, wherein the amino acid sequence of the enzyme with laccase activity comprises a sequence consisting of WYHXHX(3)-QYXDGX(2)-G, wherein X is any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, wherein the red pigment is betaine or amaranthin, and wherein the enzyme with laccase activity is laccase.

3. Use of an enzyme with laccase activity for the controlled enzymatic browning reaction of a cultured meat product containing at least 50% w / w plant protein in total protein and containing a red pigment that changes color upon oxidation, wherein the amino acid sequence of the enzyme with laccase activity comprises the sequence WYHXHX(3)-QYXDGX(2)-G, wherein X is any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, wherein the red pigment is betaine or amaranthin, and wherein the enzyme with laccase activity is laccase.

4. The cultured meat product of claim 1 or the use of claim 3, wherein the cultured meat product contains at least 70% w / w plant protein in total protein.

5. The cultured meat product of claim 1 or the use of claim 3, wherein the cultured meat product contains at least 90% w / w plant protein in total protein.

6. The cultured meat product of claim 1 or the use of claim 3, wherein the cultured meat product contains at least 95% w / w plant protein in total protein.

7. The cultured meat product of claim 1 or the use of claim 3, wherein the cultured meat product contains at least 98% w / w plant protein in total protein.

8. The cultured meat product of claim 1 or the use of claim 3, wherein the cultured meat product contains at least 100% w / w plant protein in total protein.

9. The method of claim 2, wherein the method comprises providing material containing at least 70% w / w plant protein in the total protein.

10. The method of claim 2, wherein the method comprises providing material containing at least 90% w / w plant protein in the total protein.

11. The method of claim 2, wherein the method comprises providing material containing at least 95% w / w plant protein in the total protein.

12. The method of claim 2, wherein the method comprises providing material containing at least 98% w / w plant protein in total protein.

13. The method of claim 2, wherein the method comprises providing material containing at least 100% w / w plant protein in total protein.

14. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the enzyme having laccase activity is embedded in fat with a melting temperature of at least 25°C.

15. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of 25°C-60°C.

16. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of 25°C-40°C.

17. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the enzyme having laccase activity is embedded in fat at a melting temperature of 30°C-40°C.

18. The artificial meat product, method, or use as described in claim 14, wherein the fat is plant-based fat.

19. The artificial meat product, method, or use as described in claim 14, wherein the fat is coconut oil or palm oil.

20. The artificial meat product of claim 1 or the method of claim 2, wherein the red pigment is obtained from plants.

21. The artificial meat product of claim 1 or the method of claim 2, wherein the red pigment is obtained from beetroot or amaranth grains.

22. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the red pigment is betaine in the form of beetroot juice or beetroot powder.

23. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the red pigment is betaine in the form of beetroot powder.

24. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the artificial meat product is selected from artificial ground meat products, burger patties, sausages, artificial meatball products, and artificial gullasch meat products.

25. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein at least 50% w / w of the plant protein is obtained from cereals; false cereals; tubers; oilseeds; legumes; or leaves.

26. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein at least 70% w / w of the plant protein is obtained from cereals; false cereals; tubers; oilseeds; legumes; or leaves.

27. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein at least 90% w / w of the plant protein is obtained from cereals; false cereals; tubers; oilseeds; legumes; or leaves.

28. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein at least 95% w / w of the plant protein is obtained from cereals; false cereals; tubers; oilseeds; legumes; or leaves.

29. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein at least 98% w / w of the plant protein is obtained from cereals; false cereals; tubers; oilseeds; legumes; or leaves.

30. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein 100% w / w of the plant protein is obtained from cereals; false cereals; tubers; oilseeds; legumes; or leaves.

31. The artificial meat product, method, or use as described in claim 25, wherein the grain is selected from rice, corn, wheat, and oats; the false grain is selected from buckwheat and quinoa; the tuber is potato; the oilseed is selected from soybean and sunflower; the legume is selected from pea, chickpea, lentil, and lupin; and / or the leafy plant is grass.

32. The cultured meat product, method, or use as described in claim 25, wherein at least 50% w / w of the plant protein is obtained from peas, soybeans, or wheat.

33. The cultured meat product, method, or use as described in claim 25, wherein at least 70% w / w of the plant protein is obtained from peas.

34. The cultured meat product, method, or use as described in claim 25, wherein at least 90% w / w of the plant protein is obtained from yellow or green peas.

35. The cultured meat product, method, or use as described in claim 25, wherein at least 95% w / w of the plant protein is obtained from yellow or green peas.

36. The cultured meat product, method, or use as described in claim 25, wherein at least 98% w / w of the plant protein is obtained from yellow or green peas.

37. The cultured meat product, method, or use as described in claim 25, wherein at least 100% w / w of the plant protein is obtained from yellow peas or green peas.

38. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the plant protein has been extruded.

39. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the plant protein has been extruded and chopped.

40. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the enzyme having laccase activity is isolated.

41. The artificial meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the enzyme having laccase activity is classified as laccase of EC 1.10.3.

2.

42. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the concentration of the enzyme with laccase activity is 0.1-100 mg EP / kg cultured meat product, wherein EP is an enzyme protein.

43. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the concentration of the enzyme with laccase activity is 0.5-50 mg EP / kg cultured meat product, wherein EP is an enzyme protein.

44. The cultured meat product of claim 1, the method of claim 2, or the use of claim 3, wherein the concentration of the enzyme with laccase activity is 1-20 mg EP / kg cultured meat product, wherein EP is an enzyme protein.

Images