Meat substitute ingredients

Abstract

[Problem] To provide a meat substitute composition having texture and functional characteristics that are closer to those of real meat than conventional meat substitute compositions. [Solution] Provided is a meat substitute composition containing a polypeptide that satisfies condition (1) or condition (2). (1) The number of amino acid residues is 150 or more, the content of alanine residues is 12-40%, and the content of glycine residues is 11-55%. (2) The sum of the content of residues of at least one type of amino acid selected from the group consisting of serine, threonine and tyrosine, the content of alanine residues and the content of glycine residues is 56% or more.

Description

[Technical Field]

[0001] The present invention relates to meat substitute compositions. [Background technology]

[0002] In recent years, there has been active development of meat substitute compositions that artificially reproduce the taste and texture of real meat, with the aim of addressing food issues, providing vegan food, etc. Known meat substitute compositions include, for example, cultured meat produced by culturing cells collected from animals, and meat substitutes made from plant proteins such as soybeans, peas, and wheat (e.g., Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-520554 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-517273 [Patent Document 3] Japanese Patent Application Laid-Open No. 2009-537177 Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, several meat substitutes made from plant proteins are commercially available, but they have yet to fully reproduce the texture (appearance, etc.) and sensory characteristics (flavor and texture, etc.) of real meat.

[0005] The present invention aims to provide a meat substitute composition that has texture and organoleptic properties that more closely resemble those of real meat compared to conventional meat substitute compositions. [Means for solving the problem]

[0006] The present inventors have discovered that by adding a polypeptide (e.g., a structural protein) having a specific amino acid residue composition to a meat substitute composition that uses conventional plant proteins, it is possible to reproduce the texture and sensory characteristics of real meat more closely. The present invention is based on this discovery.

[0007] That is, the present invention relates to, for example, the following inventions. [1] A meat substitute composition comprising a polypeptide that satisfies the following (1) or (2): (1) The polypeptide has 150 or more amino acid residues, an alanine residue content of 12 to 40%, and a glycine residue content of 11 to 55%; (2) The total content of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine, the alanine residue content, and the glycine residue content is 56% or more. [2] The meat substitute composition according to [1], wherein the polypeptide satisfies both (1) and (2) above. [3] The meat substitute composition according to [1] or [2], wherein the polypeptide is a recombinant polypeptide. [4] The meat substitute composition according to any of [1] to [3], wherein the polypeptide has a plurality of repeat sequence units, and each repeat sequence unit has 6 to 200 amino acid residues. [5] The polypeptide is (A) n The meat substitute composition according to any one of [1] to [4], comprising a motif. [6] The meat substitute composition according to any one of [1] to [5], wherein the polypeptide is a structural protein. [7] The meat substitute composition according to any one of [1] to [6], wherein the polypeptide is fibroin. [8] The meat substitute composition according to any one of [1] to [7], wherein the polypeptide is in the form of fiber. [9] The meat substitute composition according to any one of [1] to [8], further comprising a plant protein.

[10] An artificial meat product comprising the meat substitute composition according to any one of [1] to [9], or a processed product thereof.

[11] A method for producing an artificial meat dish, comprising the step of cooking the meat substitute composition according to any one of [1] to [9], or the artificial meat product according to

[10] . [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a meat substitute composition that has texture and sensory properties that are closer to those of real meat than conventional meat substitute compositions. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a photograph showing the appearance of a hamburger steak prepared using the meat substitute composition prepared in Test Example 1. [Figure 2] 1 is a photograph showing the appearance of a hamburger steak prepared using the meat substitute composition prepared in Test Example 1. [Figure 3] 1 is a photograph showing the appearance of a hamburger steak prepared using the meat substitute composition prepared in Test Example 1. [Figure 4] FIG. 1 is an explanatory diagram schematically illustrating an example of a spinning apparatus for producing polypeptide fibers. [Figure 5] 1 is a photograph showing the appearance of a hamburger steak prepared using the meat substitute composition prepared in Test Example 2. [Figure 6] 1 is a photograph showing the appearance of a hamburger steak prepared using the meat substitute composition prepared in Test Example 2. [Figure 7] 1 is a graph showing the change in appearance (diameter of the hamburger steak) that occurred during cooking of the hamburger steak prepared using the meat or meat substitute composition prepared in Test Example 3. [Figure 8] FIG. 10 is a schematic diagram showing the data processing of the texture profile carried out in Test Example 4. [Figure 9] 1 is a graph showing the "hardness" of hamburger steaks prepared using meat or meat substitute compositions prepared in Test Example 4. [Figure 10] 1 is a graph showing the "cohesiveness" of hamburger steaks prepared using meat or meat substitute compositions prepared in Test Example 4. [Figure 11] 1 is a graph showing the "elasticity" of hamburger steaks prepared using meat or meat substitute compositions prepared in Test Example 4. [Figure 12] 1 is a graph showing the "chewing properties" of hamburger steaks prepared using meat or meat substitute compositions prepared in Test Example 4. [Figure 13]1 is a photograph showing the appearance of a hamburger steak prepared using meat prepared in Test Example 5. [Figure 14] 1 is a photograph showing the appearance of a hamburger steak prepared using the meat substitute composition prepared in Test Example 5. [Figure 15] 1 is a graph showing the change in appearance (diameter of the hamburger steak) that occurred during cooking of the hamburger steak prepared using the meat or meat substitute composition prepared in Test Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0011] [Meat Substitute Composition] The meat substitute composition according to this embodiment comprises a polypeptide having a specific amino acid residue composition.

[0012] As used herein, the term "meat substitute composition" refers to a meat substitute that artificially reproduces the chemical properties (nutritional composition, etc.) or quality (taste, flavor, texture, appearance, etc.) of meat without relying on livestock-derived meat. Meat substitute compositions also include so-called cultured meat and meat substitutes. "Cultured meat," which is produced in a lab by extracting tissues or cells from animals and culturing those cells, is also called "lab-meat," "in vitro meat," "clean meat," etc. "Meat substitutes" made from non-animal-derived ingredients such as plants are also called "imitation meat," "fake meat," "vegan meat," "plant-based meat," "artificial meat," etc.

[0013] The polypeptide according to this embodiment may satisfy either of the following conditions (1) or (2): (1) It has 150 or more amino acid residues, an alanine residue content of 12 to 40%, and a glycine residue content of 11 to 55%; (2) The total content of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine, the alanine residue content, and the glycine residue content is 56% or more.

[0014] As used herein, the "alanine residue content" is a value represented by the following formula: alanine residue content = (number of alanine residues contained in a polypeptide / total number of amino acid residues in the polypeptide) × 100 (%). The glycine residue content, serine residue content, threonine residue content, tyrosine residue content, glutamine residue content, and lysine residue content are synonymous with the above formula, with alanine residue replaced with glycine residue, serine residue, threonine residue, tyrosine residue, glutamine residue, and lysine residue, respectively.

[0015] A polypeptide satisfying (1) may have 150 or more amino acid residues. The number of amino acid residues may be, for example, 200 or more or 250 or more, and is preferably 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more.

[0016] A polypeptide satisfying (1) may have an alanine residue content of 12 to 40%, for example, 15 to 40%, 18 to 40%, 20 to 40%, or 22 to 40%.

[0017] A polypeptide satisfying (1) may have a glycine residue content of 11 to 55%. The glycine residue content may be, for example, 11% to 55%, 13% to 55%, 15% to 55%, 18% to 55%, 20% to 55%, 22% to 55%, or 25% to 55%.

[0018] Polypeptides that satisfy (1) contain relatively large amounts of alanine and glycine residues. Because alanine and glycine residues are nonpolar amino acids, they are arranged so that their side chains face inward during the folding process of polypeptide production, and tend to adopt an α-helical or β-sheet structure. Therefore, when a meat substitute composition contains a polypeptide that satisfies (1), the meat substitute composition is less likely to crumble during and after cooking, and its chewiness increases, thereby improving the texture (appearance) and sensory characteristics (feel). Furthermore, when a polypeptide that satisfies (1) is formed into, for example, a fiber, sponge, gel, or film, these secondary structures can exhibit high strength and toughness, thereby more significantly demonstrating the above-mentioned effects.

[0019] In addition, glycine is said to have a detoxifying effect by lowering blood cholesterol and excreting harmful substances from the body. Alanine is said to have the function of converting alcohol into an energy source and promoting alcohol metabolism. Therefore, by containing a polypeptide that satisfies (1), the meat substitute composition of this embodiment is expected to exhibit these nutritional functions.

[0020] A polypeptide satisfying (2) may have a combined content (total content) of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine (i.e., any of the following: serine residue content, threonine residue content, tyrosine residue content, the sum of serine residue content and threonine residue content, the sum of serine residue content and tyrosine residue content, the sum of threonine residue content and tyrosine residue content, or the sum of serine residue content, threonine residue content, and tyrosine residue content), alanine residue content, and glycine residue content (total content) of 56% or more. This combined content may be, for example, 57% or more, 58% or more, 59% or more, or 60% or more. There is no particular upper limit to this combined content, but it may be, for example, 90% or less, 85% or less, or 80% or less.

[0021] In one embodiment, the polypeptide satisfying (2) may have a total serine residue content, threonine residue content, and tyrosine residue content of 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, or 7% or more. The total serine residue content, threonine residue content, and tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0022] A polypeptide satisfying condition (2) contains a relatively large amount of one amino acid residue selected from the group consisting of serine, threonine, and tyrosine, as well as alanine and glycine residues. Serine, threonine, and tyrosine residues are relatively hydrophilic amino acid residues, while alanine and glycine residues are relatively hydrophobic amino acid residues. Therefore, the water retention and / or oil absorption properties of a polypeptide satisfying condition (2) can be manipulated as desired by changing the ratio of the content of one amino acid residue selected from the group consisting of serine, threonine, and tyrosine to the content of alanine and glycine residues. The water retention and / or oil absorption properties of the polypeptide increase its affinity with aqueous and / or oily components, thereby improving the texture (appearance) and sensory properties (flavor, texture) of the meat substitute composition.

[0023] In addition, serine is said to lower blood cholesterol and have a detoxifying effect, excreting harmful substances from the body. Tyrosine, which is involved in the synthesis of tyrosine, has been reported to have preventive effects against dementia and Parkinson's disease. Therefore, by including a polypeptide that satisfies (2), the meat substitute composition of this embodiment is expected to exhibit these nutritional functions.

[0024] The polypeptide according to this embodiment preferably satisfies both of the above (1) and (2), thereby enabling the effects of the present invention to be more pronounced.

[0025] The polypeptide of this embodiment has an even distribution of serine residues, threonine residues, or tyrosine residues, and the total content of serine residues, threonine residues, and tyrosine residues among any 20 consecutive amino acid residues may be 5% or more, 10% or more, or 15% or more, or may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0026] A polypeptide according to one embodiment may have a repeat sequence. That is, the polypeptide according to this embodiment may have a plurality of amino acid sequences (repeat sequence units) with high sequence identity within the polypeptide. The amino acid sequence of the repeat sequence units is not particularly limited, as long as the polypeptide as a whole satisfies the above-mentioned (1) or (2). The number of amino acid residues in the repeat sequence units is preferably 6 to 200. Furthermore, the sequence identity between the repeat sequence units may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0027] A polypeptide according to one embodiment may contain glutamine residues and / or lysine residues. By containing glutamine residues and / or lysine residues, intramolecular or intermolecular crosslinking of the polypeptide occurs in the presence of an edible crosslinking agent such as transglutaminase. The content of glutamine residues may be, for example, 0% to 30%, 0% to 25%, 0% to 20%, 5% to 20%, 10% to 20%, or 15% to 20%. The content of lysine residues may be, for example, 5% or more, 10% or more, 25% or less, 20% or less, 15% or less, or 10% or less.

[0028] In one embodiment, the polypeptide comprises: (A) n In the present specification, (A) nThe motif refers to an amino acid sequence consisting mainly of alanine residues. (A) n The number of amino acid residues in the motif may be 2 to 27, or may be an integer of 2 to 20, 2 to 16, or 2 to 12. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).

[0029] In one embodiment, (A) n The motif may be contained within a repeat unit. (A) n The motif contains mainly alanine residues and therefore tends to adopt an α-helix or β-sheet structure. (A) n The inclusion of the motif in the repeating sequence unit results in the polypeptide of this embodiment having a repeating secondary structure, which makes the meat substitute composition less likely to crumble during and after cooking and more chewy, thereby improving the texture (appearance) and sensory properties (feel). Furthermore, when the polypeptide is in the form of, for example, fiber, sponge, gel, or film, these secondary structures can provide high strength and toughness, thereby more significantly demonstrating the above-mentioned effects.

[0030] The polypeptide according to this embodiment may be, for example, a structural protein. A structural protein refers to a protein involved in the structure of a living organism, a protein constituting a structure produced by a living organism, or a protein derived therefrom. Examples of structural proteins include fibroin, keratin, collagen, elastin, and resilin.

[0031] The structural protein is not particularly limited as long as it satisfies the above (1) or (2), but is preferably fibroin, such as silk fibroin, spider silk fibroin, and hornet silk fibroin, and proteins derived therefrom.

[0032] Examples of fibroin include naturally occurring fibroin, such as fibroin produced by insects or arachnids.

[0033] Examples of fibroin produced by insects include silk fibroin produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, as well as hornet silk fibroin excreted by larvae of the Japanese hornet (Vesp simillima xanthoptera).

[0034] A more specific example of fibroin produced by insects is silkworm fibroin L chain (GenBank accession numbers M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

[0035] Examples of fibroin produced by spiders include spider silk proteins produced by spiders belonging to the order Araneae.More specifically, spiders belonging to the Araneus genus, such as the orb spider, the garden spider, the red orb spider, the green orb spider, and the bean orb spider; spiders belonging to the Neoscona genus, such as the mountain orb spider, the house orb spider, the dun orb spider, and the Satsuma orb spider; spiders belonging to the Pronus genus, such as the little orb spider; spiders belonging to the Cyrtarachne genus, such as the Japanese orb spider and the large orb spider; spiders belonging to the Gasteracantha genus, such as the spiny orb spider and the Japanese orb spider; spiders belonging to the Ordgarius genus such as the Orb-weaver spider and the Black-legged spider; spiders belonging to the Argiope genus such as the Orb-weaver spider, the Orb-weaver spider and the Long-legged spider; spiders belonging to the Arachnura genus such as the Orb-weaver spider; spiders belonging to the Acusilas genus such as the Siberian shrike spider; spiders belonging to the Cytophora genus such as the Orb-weaver spider, the Orb-weaver spider and the Long-legged shrike spider; spiders belonging to the Poltys genus such as the House spider; dust spiders; Spider silk proteins produced by spiders of the genus Cyclosa, such as the four-headed dung bean spider, the round dung bean spider, and the black dung bean spider, and spiders of the genus Chorizopes, such as the Japanese canary spider, as well as spiders of the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the broad-legged spider, and the scaly canary spider, spiders of the genus Leucauge, such as the large white weaver spider, the medium-sized weaver spider, and the small white weaver spider, and spiders of the genus Orb spider, such as the orb spider and the giant orb spider. Examples include spider silk fibroin produced by spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as golden spiders, spiders belonging to the genus Dyschiriognatha such as small reed spiders, spiders belonging to the genus Latrodectus such as black widow spiders, redback spiders, grey widow spiders and three-spotted widow spiders, and spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Euprosthenops.Examples of spider silk fibroin include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, and Flag.

[0036] More specific examples of spider silk fibroins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), and dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession numbers CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephila clavipes] cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

[0037] More specific examples of naturally occurring fibroins include those whose sequence information is registered in NCBI GenBank. For example, these can be confirmed by extracting, from among the sequences registered in NCBI GenBank that contain INV as the division, sequences with spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" as keywords in the definition, sequences with a specific product character string in the CDS, and sequences with a specific character string in the tissue type field in the source.

[0038] The fibroin may be a modified fibroin. As used herein, the term "modified fibroin" refers to artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin whose domain sequence is identical to the amino acid sequence of naturally occurring fibroin. The modified fibroin is a fibroin represented by Formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n The modified fibroin is a protein containing a domain sequence represented by a motif. The modified fibroin may have further amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeats of the amino acid motif characteristic of fibroin and consist of about 100 amino acid residues.

[0039] The modified fibroin may be one that uses the amino acid sequence of naturally occurring fibroin as is, or one that has had its amino acid sequence modified based on the amino acid sequence of naturally occurring fibroin (for example, one that has had its amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), or one that has been artificially designed and synthesized without relying on naturally occurring fibroin (for example, one that has the desired amino acid sequence obtained by chemically synthesizing a nucleic acid that encodes a designed amino acid sequence).

[0040] As used herein, the term "domain sequence" refers to a crystalline region specific to fibroin (typically, the amino acid sequence (A) n It is an amino acid sequence that generates a region (corresponding to a motif) and an amorphous region (typically corresponding to an REP in an amino acid sequence), and is represented by formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n The amino acid sequence represented by the motif (A) n The motif shows an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is 2 to 27. (A) n The number of amino acid residues in the motif may be an integer of 2 to 20, 2 to 16, or 2 to 12, or may be an integer of 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the motif is composed of only alanine residues). nAt least seven of the motifs may be composed of only alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may also be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. (A) n The motifs may have the same or different amino acid sequences. Multiple REPs may have the same or different amino acid sequences. The total content of serine, threonine, and tyrosine residues in each REP may be 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, or 15% or more, or may be 50% or less, 40% or less, 30% or less, or 20% or less. Furthermore, the total content of serine residues, threonine residues, and tyrosine residues in all REPs (the total content of serine residues, threonine residues, and tyrosine residues when all REPs are considered as one polypeptide) may be 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, or 15% or more, and may be 50% or less, 40% or less, 30% or less, or 20% or less. The content of glutamine residues in all REPs may be 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, or 15% or more, and may be 30% or less, or 20% or less. The number of amino acid residues in the domain sequence may be, for example, 6 to 300, 6 to 250, or 6 to 200.

[0041] The modified fibroin according to this embodiment can be obtained, for example, by modifying the amino acid sequence of a cloned gene sequence of naturally occurring fibroin, for example, by substituting, deleting, inserting, and / or adding one or more amino acid residues. The substitution, deletion, insertion, and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis. Specifically, this can be performed in accordance with the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology 100, 448 (1983).

[0042] The fibroin is not particularly limited as long as it satisfies the above (1) or (2). Specific examples of fibroin include the fibroins (modified fibroins) shown in Table 1 below.

[0043] [Table 1]

[0044] The polypeptide according to one embodiment may be a purified polypeptide. A purified polypeptide preferably contains only the polypeptide, but may contain unavoidable contaminating impurities.

[0045] The polypeptide according to one embodiment may be a recombinant polypeptide. A recombinant polypeptide refers to a polypeptide produced using genetic recombination technology. The recombinant polypeptide may be isolated from a non-animal-derived genetically modified organism, depending on the intended use of the meat substitute composition.

[0046] In one embodiment, the polypeptide may be derived from a non-plant source, specifically, for example, a polypeptide purified from a non-plant, or a polypeptide produced using recombinant genetic technology from a gene isolated from a non-plant.

[0047] In one embodiment, the polypeptide may be antibacterial. Specifically, the polypeptide may have an antibacterial amino acid sequence or an antibacterial protein motif. Compared to conventional meat substitute compositions, the polypeptide may have a longer shelf life, particularly, it may be stored or transported at room temperature.

[0048] The polypeptide according to this embodiment may be in the form of, for example, a shaped article (e.g., fiber, gel, film, porous body (sponge), particle, molded article) containing the polypeptide or consisting of the polypeptide. The polypeptide according to this embodiment is preferably in the form of fiber (polypeptide fiber), as this allows for a more pronounced effect of the present invention, namely, having a texture and sensory properties closer to those of real meat, compared to conventional meat substitute compositions.

[0049] The polypeptide fibers may be long fibers or short fibers. The length of the short fibers may be, for example, 1 to 20 mm, 1 to 15 mm, 1 to 10 mm, or 1 to 5 mm.

[0050] The polypeptide fiber may be a filament yarn (multifilament, monofilament, etc.), a spun yarn, a twisted yarn, a false-twisted yarn, a textured yarn, a blended yarn, a mixed yarn, or the like.

[0051] Polypeptide fibers can be produced by known spinning methods. For example, the polypeptide according to the present invention is first added to a solvent or solution such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), hydrochloric acid, formic acid, or hexafluoroisopropanol (HFIP) together with an inorganic salt as a solubilizer to prepare a dope solution. This dope solution can then be spun by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain the desired polypeptide fiber.

[0052] Fig. 4 is an explanatory diagram schematically illustrating an example of a spinning apparatus for producing polypeptide fibers. The spinning apparatus 10 shown in Fig. 4 is an example of a spinning apparatus for dry / wet spinning, and includes an extrusion unit 1, an undrawn yarn production unit 2, a wet heat drawing unit 3, and a drying unit 4.

[0053] A spinning method using a spinning apparatus 10 will be described. First, a dope solution 6 stored in a storage tank 7 is extruded from a spinneret 9 by a gear pump 8. Next, the extruded dope solution 6 passes through an air gap 19 and is supplied into a coagulation liquid 11 in a coagulation liquid tank 20, where the solvent is removed and the polypeptide is coagulated to form a fibrous coagulate. Next, the fibrous coagulate is supplied into warm water 12 in a drawing bath 21 and drawn. The drawing ratio is determined by the speed ratio between a supply nip roller 13 and a take-up nip roller 14. Thereafter, the drawn fibrous coagulate is supplied to a drying device 4 and dried in a yarn path 22, whereupon a polypeptide fiber 36 is obtained as a wound yarn 5. Reference numerals 18a to 18g denote yarn guides.

[0054] The coagulation liquid 11 may be any solvent or solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, and 2-propanol, as well as acetone, sodium hydroxide, sodium carbonate, and sodium bicarbonate. The coagulation liquid 11 may contain water as appropriate. The temperature of the coagulation liquid 11 is preferably 0 to 30°C. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinneret 9, the extrusion rate is preferably 0.2 to 6.0 ml / hour per hole, and more preferably 1.4 to 4.0 ml / hour per hole. The distance over which the coagulated polypeptide passes through the coagulation liquid 11 (effectively, the distance from the yarn guide 18a to the yarn guide 18b) may be any length that allows efficient desolvation, and is, for example, 200 to 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Furthermore, stretching (pre-stretching) may be performed in the coagulation liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage as necessary.

[0055] The stretching carried out when obtaining polypeptide fibers may be, for example, the above-mentioned pre-stretching carried out in the coagulation liquid bath 20 and wet heat stretching carried out in the stretching bath 21, as well as dry heat stretching.

[0056] The wet heat drawing can be carried out in warm water, in a solution of warm water with an organic solvent added, or under steam heating. The temperature may be, for example, 50 to 90° C., and preferably 75 to 85° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.

[0057] Hot drawing can be carried out using an electric tubular furnace, a hot plate, etc. The temperature may be, for example, 140°C to 270°C, and preferably 160°C to 230°C. In hot drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.

[0058] The wet heat stretching and the dry heat stretching may be carried out independently, or may be carried out in multiple stages or in combination. That is, the wet heat stretching and the dry heat stretching may be appropriately combined, for example, by carrying out wet heat stretching in the first stage and dry heat stretching in the second stage, or by carrying out wet heat stretching in the first stage, wet heat stretching in the second stage, and dry heat stretching in the third stage.

[0059] The lower limit of the final draw ratio relative to the undrawn yarn (or pre-drawn yarn) is preferably any one of more than 1, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more, and the upper limit is preferably 40 or less, 30 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less. Drawn yarns can exhibit higher strength, and therefore can exhibit the effects of the present invention more significantly.

[0060] The polypeptide fiber according to this embodiment may have a stress of 0.5 gf / d or more. The stress is preferably 0.8 gf / d or more, and more preferably 1 gf / d or more. A stress within this range has the effect of further improving the yield in the production of the meat substitute composition. The strength is a value determined by a standard tensile test of a multifilament yarn.

[0061] The diameter of the polypeptide fiber according to this embodiment may be less than 50 μm. The diameter of the protein fiber is, for example, preferably less than 45 μm or less than 40 μm, more preferably 35 μm or less, 32 μm or less, or 30 μm or less, and even more preferably 15 μm or less or 10 μm or less. Using protein fibers with a smaller diameter results in a more realistic appearance and a mouthfeel and texture closer to the real thing. The diameter of the protein fiber may be, for example, 5 μm or more, 10 μm or more, 15 μm or more, 18 μm or more, 20 μm or more, 22 μm or more, 25 μm or more, 30 μm or more, 32 μm or more, 35 μm or more, 40 μm or more, or 45 μm or more. The diameter of the protein fibers may be, for example, 5 to 50 μm, 10 to 50 μm, 15 to 50 μm, 5 to 45 μm, 5 to 40 μm, 5 to 35 μm, 5 to 32 μm, 5 to 50 μm, 10 to 45 μm, 10 to 40 μm, 10 to 35 μm, 10 to 32 μm, or 10 to 30 μm.

[0062] The polypeptide according to this embodiment may be in the form of a molded article (polypeptide resin). By using a polypeptide molded article, it is possible to reproduce real meat with bones.

[0063] The meat substitute composition according to this embodiment may contain one or more of the above-mentioned polypeptides.

[0064] The meat substitute composition of this embodiment may contain protein components other than the above-mentioned polypeptides. Such protein components are preferably protein components derived from non-animal sources that are used in conventional meat substitute compositions, such as proteins derived from soybeans, peas, wheat, oats, rye, barley, canola, sunflower, sorghum, rice, amaranth, potato, tapioca, arrowroot, canna, lupin, rapeseed, algae, edible filamentous fungi, and mixtures thereof.

[0065] The content of polypeptide in the meat substitute composition according to this embodiment, based on the total amount of the meat substitute composition, may be, for example, 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, 0.4% by weight or more, 0.5% by weight or more, 0.6% by weight or more, 0.7% by weight or more, 0.8% by weight or more, 0.9% by weight or more, 1% by weight or more, 1.5% by weight or more, 2% by weight or more, 2.5% by weight or more, 3% by weight or more, 3.5% by weight or more, 4% by weight or more, 4.5% by weight or more, 5% by weight or more, 10% by weight or more, 15% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, or 50% by weight or more. The polypeptide content in the meat substitute composition according to this embodiment, based on the total amount of the meat substitute composition, may be, for example, 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, or 4.5% by weight or less. , may be 4% by weight or less, may be 3.5% by weight or less, may be 3% by weight or less, may be 2.5% by weight or less, may be 2% by weight or less, may be 1.5% by weight or less, may be 1% by weight or less, may be 0.9% by weight or less, may be 0.8% by weight or less, may be 0.7% by weight or less, may be 0.6% by weight or less, may be 0.5% by weight or less, may be 0.4% by weight or less, may be 0.3% by weight or less, may be 0.2% by weight or less.The content of the polypeptide in the meat substitute composition according to this embodiment may be, for example, 0.1 to 10% by weight, 0.2 to 10% by weight, 0.3 to 10% by weight, 0.4 to 10% by weight, 0.5 to 10% by weight, 0.6 to 10% by weight, 0.7 to 10% by weight, 0.8 to 10% by weight, 0.9 to 10% by weight, 1 to 10% by weight, 0.1 to 9% by weight, 0.1 to 8% by weight, 0.1 to 7% by weight, 0.1 to 6% by weight, 0.1 to 5% by weight, 0.5 to 9% by weight, 0.5 to 8% by weight, 0.5 to 7% by weight, or 0.5 to 6% by weight, based on the total amount of the meat substitute composition. , may be 0.5 to 5% by weight, may be 1 to 9% by weight, may be 1 to 8% by weight, may be 1 to 7% by weight, may be 1 to 6% by weight, may be 1 to 5% by weight, may be 1 to 4% by weight, may be 1.5 to 10% by weight, may be 1.5 to 9% by weight, may be 1.5 to 8% by weight, may be 1.5 to 7% by weight, may be 1.5 to 6% by weight, may be 1.5 to 5% by weight, may be 2 to 10% by weight, may be 2 to 9% by weight, may be 2 to 8% by weight, may be 2 to 7% by weight, may be 2 to 6% by weight, may be 2 to 5% by weight, may be 3 to 10% by weight, may be 3 to 9% by weight, may be 3 to 8% by weight, may be 3 to 7% by weight, may be 3 to 6% by weight, or may be 3 to 5% by weight.

[0066] The content of polypeptide in the meat substitute composition according to this embodiment, based on the total amount of the meat substitute composition, may be, for example, 0.1% by volume or more, 0.2% by volume or more, 0.3% by volume or more, 0.4% by volume or more, 0.5% by volume or more, 0.6% by volume or more, 0.7% by volume or more, 0.8% by volume or more, 0.9% by volume or more, 1% by volume or more, 1.5% by volume or more, 2% by volume or more, 2.5% by volume or more, or It may be % by volume or more, may be 3% by volume or more, may be 3.5% by volume or more, may be 4% by volume or more, may be 4.5% by volume or more, may be 5% by volume or more, may be 10% by volume or more, may be 15% by volume or more, may be 20% by volume or more, may be 30% by volume or more, may be 40% by volume or more, or may be 50% by volume or more. The polypeptide content in the meat substitute composition according to this embodiment, based on the total amount of the meat substitute composition, may be, for example, 90% by volume or less, 80% by volume or less, 70% by volume or less, 60% by volume or less, 50% by volume or less, 40% by volume or less, 30% by volume or less, 20% by volume or less, 15% by volume or less, 10% by volume or less, 5% by volume or less, 4.5% by volume or less, 4% by volume or less, 3.5% by volume or less, 3% by volume or less, 2.5% by volume or less, 2% by volume or less, 1.5% by volume or less, 1% by volume or less, or 0.5% by volume or less. The content of the polypeptide in the meat substitute composition according to this embodiment may be, for example, 0.1 to 10% by volume, 0.5 to 10% by volume, 1 to 10% by volume, 1.5 to 10% by volume, 2.5 to 10% by volume, 0.1 to 5% by volume, 0.1 to 4% by volume, 0.1 to 3% by volume, 0.1 to 2.5% by volume, 5 to 10% by volume, 0.5 to 8% by volume, 0.5 to 5% by volume, 0.5 to 4% by volume, 0.5 to 3.5% by volume, 0.5 to 3% by volume, 1 to 8% by volume, 1 to 5% by volume, 1.5 to 5% by volume, 2.5 to 5% by volume, 3 to 8% by volume, or 0.5 to 2.5% by volume.

[0067] The polypeptide content in the meat substitute composition of this embodiment may be, for example, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, or 50% by weight or more, based on the total amount of protein components contained in the meat substitute composition. The polypeptide content in the meat substitute composition of this embodiment may be, for example, 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, or 20% by weight or less, based on the total amount of protein components contained in the meat substitute composition.

[0068] The protein content of the meat substitute composition according to this embodiment, based on the total weight of the meat substitute composition, may be, for example, 0.5% by weight or more, 1% by weight or more, 2% by weight or more, 3% by weight or more, 4% by weight or more, 5% by weight or more, 6% by weight or more, 7% by weight or more, 8% by weight or more, 9% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, or 50% by weight or more. The protein content of the meat substitute composition according to this embodiment, based on the total weight of the meat substitute composition, may be, for example, 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, or 10% by weight or less.

[0069] The meat substitute composition according to this embodiment may contain, for example, water, vegetable oil, sugars, salts, minerals, coloring agents, antioxidants, thickening agents, dietary fiber, flavorings, edible cross-linking agents, etc.

[0070] Examples of vegetable oils include corn oil, olive oil, soybean oil, peanut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil, linseed oil, palm oil, walnut oil, algae oil, coconut oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, and oils produced by bacteria, algae, archaea, fungi, or genetically modified bacteria, algae, archaea, or fungi. Examples of sugars include oligosaccharides, sugars, and starches derived from plants such as arrowroot, corn starch, dogtooth starch, potato starch, sago, and tapioca. Examples of salts include sodium chloride, potassium chloride, glutamates (e.g., monosodium glutamate), glycinates, guanylates, inosinates, and 5'-ribonucleotide salts. Minerals include, for example, aluminum, ammonium, calcium, magnesium, and potassium salts. Antioxidants may be natural or synthetic and include, for example, those that prevent discoloration of colored vegetable proteins due to oxidation. Thickening stabilizers include, for example, alginic acid and its salts, agar, carrageenan and its salts, modified Eucheuma algae, gums (carob bean, guar, tragacanth, and xanthan), pectin, and sodium carboxymethylcellulose. Dietary fibers include, for example, soybean fiber and gluten filaments. Flavorings include, for example, spice extracts, spice oils, natural liquid smoke, natural smoke extracts, yeast extracts, shiitake mushroom extracts, and flavoring agents such as onion flavor, garlic flavor, and herb flavor. Examples of edible cross-linking agents include those that can promote filament formation, such as edible cross-linking agents such as konjac glucomannan (KGM) powder and transglutaminase, beta-glucans such as Pureglucan (registered trademark, manufactured by Takeda), calcium salts, and magnesium salts.

[0071] The meat substitute composition of this embodiment can be obtained according to conventional methods, except for the addition of the polypeptide of the present invention. The meat substitute composition of this embodiment can be obtained, for example, by uniformly mixing the polypeptide of the present invention with the various components described above, as necessary, and molding the mixture into a desired shape. The meat substitute composition of this embodiment can also be obtained, for example, by uniformly mixing the various components described above other than the polypeptide of the present invention, adding the polypeptide of the present invention to the mixture, mixing the mixture uniformly, and molding the mixture into a desired shape. Furthermore, when the polypeptide of the present invention is in the form of a fiber, the meat substitute composition of this embodiment can also be obtained by uniformly mixing the various components described above other than the polypeptide of the present invention, and then molding the mixture into a desired shape through processes such as stitching, weaving, braiding, knitting, and needle punching.

[0072] The present invention also relates to artificial meat products comprising the meat substitute composition of the present invention or processed products thereof. Processed meat substitute compositions include, for example, heat-treated meat substitute compositions, seasoned meat substitute compositions, and meat substitute compositions cooked with other ingredients (including cooked and semi-cooked products) as needed. The meat substitute compositions can be processed into various foods for either human or animal consumption. For example, the final product can be a meat substitute composition for human consumption that imitates a ground meat product, a steak product, a sirloin tip product, a kebab product, a shredded product, a diced meat product, or a nugget product. Any of the above products can be placed on a tray and covered with a package, vacuum-packed, placed in a retort can or bag, or frozen.

[0073] The present invention also relates to a method for producing an artificial meat dish, which comprises the step of cooking the meat substitute composition or artificial meat product of the present invention. Cooking the meat substitute composition or artificial meat product can be carried out according to conventional methods. [Example]

[0074] The present invention will be described in more detail below based on test examples, although the present invention is not limited to the following test examples.

[0075] Test Example 1: Preparation and Evaluation of Meat Substitute Compositions Comparative Example 1 Each ingredient listed in Table 2 was added to commercially available textured vegetable protein (TVP, manufactured by Bob's Red Mill) in the weight percentages shown in Table 2 (weight percentages relative to the total weight of the final meat substitute composition of Comparative Example 1) and mixed uniformly. After mixing, the mixture was heated in a household microwave oven at 700 W for 2 minutes. After allowing to cool to room temperature, 7 g of water equivalent to the amount of evaporated liquid was added.

[0076] [Table 2]

[0077] Next, each component listed in Table 3 was added to the mixture obtained above in the weight percentage shown in Table 3 (weight percentage relative to the total weight of the final meat substitute composition of Comparative Example 1) and mixed uniformly. [Table 3]

[0078] To the resulting mixture, each of the components listed in Table 4 was added in the weight percentages shown in Table 4 (weight percentages relative to the total weight of the final meat substitute composition of Comparative Example 1), and mixed uniformly. Through the above process, the meat substitute composition of Comparative Example 1 was obtained. [Table 4]

[0079] 60 g of the meat substitute composition of Comparative Example 1 was taken by hand, molded into the shape of a hamburger steak, cooked in a frying pan, and subjected to a sensory evaluation.

[0080] Comparative Example 2 60 g of the meat substitute composition of Comparative Example 1 was mixed with approximately 3 g of salad oil to obtain a meat substitute composition of Comparative Example 2. This was taken by hand, formed into the shape of a hamburger steak, cooked in a frying pan, and subjected to a sensory evaluation.

[0081] Example 1 Several hundred 3.6 m long fibers were obtained from silk bobbins (derived from silkworms) using a small warping machine (SW550, manufactured by CCI TECH INC.). The obtained long fibers were cut using a tabletop high-power fiber cutter (NP-300, manufactured by INTEC CO. LTD.) to obtain 3 mm long silk staple fibers (approximately 100 g).

[0082] 60 g of the meat substitute composition of Comparative Example 1 was mixed with approximately 3 g of the silk short fibers obtained above to obtain the meat substitute composition of Example 1. This was taken by hand, formed into the shape of a hamburger patty, cooked in a frying pan, and subjected to sensory evaluation.

[0083] Example 2 Approximately 3 g of the silk staple fibers obtained in Example 1 were immersed in hot water above 80°C for 1 minute and then filtered. After filtration, approximately 10 g of salad oil was added to the silk staple fibers and uniformly dispersed. 60 g of the meat substitute composition of Comparative Example 1 was mixed with the obtained silk staple fiber dispersion to obtain the meat substitute composition of Example 2. This was taken by hand, formed into the shape of a hamburger patty, cooked in a frying pan, and subjected to sensory evaluation.

[0084] (Sensory Evaluation) The hamburger steaks obtained by cooking the meat substitute compositions of Comparative Examples 1 and 2 and Examples 1 and 2 were subjected to a sensory evaluation of appearance, taste, aroma (fragrance), and texture. The sensory evaluation was carried out by one panelist, and each item was rated on a four-point scale from 1 to 4 (1: poor, 2: average, 3: somewhat good, 4: good). The results are shown in Table 5.

[0085] [Table 5]

[0086] 1 to 3 show photographs of the hamburger steaks of Comparative Examples 1 and 2 and Examples 1 and 2.

[0087] The appearance (including texture) of the hamburger steaks of Comparative Example 1 and Comparative Example 2 was similar, but the hamburger steak of Comparative Example 2 had a slightly stronger meaty aroma due to the addition of salad oil.

[0088] The hamburger steak of Example 1 was able to maintain its shape and its appearance was improved by adding short silk fibers. It also had a "meat-like sinew" texture. On the other hand, the added short silk fibers floated to the surface. It was shown (Figure 1, etc.).

[0089] The hamburger steak of Example 2 was able to maintain its shape and be properly grilled (see Figure 2, etc.), and the appearance was significantly improved. In addition, the short silk fibers acted as connective tissue, giving the meat more cohesiveness compared to Comparative Example 2 and Example 1. As a result, the appearance and texture were much closer to meat. In addition, the short silk fibers absorbed the oil, making the meat feel soft overall. The aroma of the oil gave it a meaty flavor.

[0090] [Test Example 2: Preparation and evaluation of meat substitute composition] <Example 3> The nucleotide sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415) were obtained from the GenBank web database, and then amino acid residues were substituted, inserted, and deleted to improve productivity. Furthermore, a tag sequence and hinge sequence were added to the N-terminus to design a recombinant fibroin having the amino acid sequence shown in SEQ ID NO: 1 (hereinafter also referred to as "PRT799").

[0091] Nucleic acids encoding the designed recombinant fibroins were synthesized. An NdeI site was added to the 5' end of each nucleic acid, and an EcoRI site was added downstream of the stop codon. These nucleic acids were cloned into a cloning vector (pUC118), excised using restriction enzymes NdeI and EcoRI, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

[0092] The resulting expression vector was used to transform E. coli BLR (DE3). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture was then transferred to 100 mL of seed culture medium (Table 6) containing ampicillin at OD 200. 600 The culture temperature was kept at 30°C, and the OD 600 The flask culture was continued until the fertilization rate reached 5 (about 15 hours), and a seed culture solution was obtained.

[0093] [Table 6]

[0094] The seed culture solution was added to a jar fermenter containing 500 ml of production medium (Table 7) and the OD 600 The culture temperature was maintained at 37°C and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration.

[0095] [Table 7]

[0096] Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g glucose / 1 L, 120 g yeast extract / 1 L) was added at a rate of 1 mL / min. The culture temperature was maintained at 37°C, and the pH was controlled to a constant 6.7. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours.

[0097] Subsequently, 1M isopropyl-β-thiogalactopyranoside (IPTG) aqueous solution was added to the culture medium to a final concentration of 1 mM to induce expression of the desired recombinant fibroin. 20 hours after IPTG addition, the culture medium was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture medium before and after IPTG addition, and the appearance of a band of the size corresponding to the desired recombinant fibroin, which was dependent on IPTG addition, confirmed the expression of the desired recombinant fibroin.

[0098] The cells were harvested 24 hours after the addition of IPTG and washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM phenylmethylsulfonyl fluoride (PMSF) and disrupted three times using a high-pressure homogenizer (Panda Plus 2000, GEA Niro Saovi). The disrupted cells were centrifuged in a centrifuge (Model 7000, Kubota) at 11,000 g for 10 minutes at room temperature to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) or 3% SDS buffer (pH 3.0) until highly purified. The washed precipitate was diluted to a concentration of 100 mg / mL with 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM The protein was suspended in Tris-HCl (pH 7.0) and stirred at 60°C for 30 minutes to dissolve. After dissolution, the protein was dialyzed against water using a dialysis tube (Cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation and the water was removed using a freeze-dryer to obtain a freeze-dried powder of recombinant fibroin.

[0099] A spinning solution (dope solution) was prepared using the freeze-dried powder of recombinant fibroin obtained above. 99% formic acid was added to the freeze-dried powder so that the concentration of the freeze-dried powder was 24% by mass. After dissolving for 14 hours on a rotator, dust and bubbles were removed. This was used as the spinning solution (dope solution).

[0100] Using the spinning apparatus shown in Figure 4, the dope solution was discharged into the coagulation solution (methanol) using a nitrogen air pump. The wet spinning conditions were as follows. As a result, recombinant fibroin fibers were obtained. <Wet spinning conditions> Dope solution temperature: 25°C Coagulation solution (methanol) 1 temperature: 5°C Coagulation solution (methanol) 2 temperature: 25°C Water bath stretching tank temperature: 25°C Hot roller (HR) temperature: 60°C

[0101] Using a small warping machine (SW550, manufactured by CCI TECH INC.), several hundred 3.6 m long fibers were obtained from a bobbin (derived from recombinant fibroin). The obtained long fibers were cut using a tabletop high-power fiber cutter (NP-300, manufactured by INTEC CO. LTD.) to obtain recombinant fibroin short fibers (approximately 100 g) with a length of 2 mm.

[0102] The resulting recombinant fibroin short fibers were dispersed in sodium bicarbonate water for 10 minutes, filtered, and then washed several times with ion-exchanged water.

[0103] 60 g of the meat substitute composition of Comparative Example 1, approximately 3 g of salad oil, and 0.6 g of washed recombinant fibroin short fibers were mixed to obtain the meat substitute composition of Example 3. This was taken by hand, formed into the shape of a hamburger patty, cooked in a frying pan, and subjected to sensory evaluation.

[0104] Example 4 The meat substitute composition of Example 4 was obtained in the same manner as in Example 3, except that the amount of washed recombinant fibroin short fibers was changed from 0.6 g to 3 g. The composition was then taken by hand, formed into the shape of a hamburger patty, cooked in a frying pan, and subjected to sensory evaluation.

[0105] Comparative Example 3 A meat substitute composition of Comparative Example 3 was obtained in the same manner as in Comparative Example 2 of Test Example 1. This was taken by hand, formed into the shape of a hamburger steak, cooked in a frying pan, and subjected to a sensory evaluation.

[0106] <Comparative Example 4> The meat substitute composition of Comparative Example 3 was taken by hand, formed into the shape of a hamburger patty, and cooked in a frying pan for a longer time (twice as long) than in Examples 3 to 4 and Comparative Example 3, and then subjected to sensory evaluation.

[0107] (Sensory Evaluation) The hamburger steaks of Comparative Examples 3 and 4 and Examples 3 and 4 were subjected to a sensory evaluation of appearance, taste, aroma, texture, and overall evaluation (overall meatiness). The sensory evaluation was carried out by two panelists, and each item was rated on a four-point scale from 1 to 4 (1: poor, 2: average, 3: somewhat good, 4: good). The results are shown in Table 8.

[0108] [Table 8]

[0109] Photographs of the hamburger steaks of Comparative Examples 3 and 4 and Examples 3 and 4 are shown in FIGS.

[0110] The hamburger steaks of Examples 3 and 4 maintained their appearance and overall shape due to the addition of recombinant fibroin fiber, and there were no cracks in the sear marks. Surprisingly, differences in the sear marks on the surface were observed between the hamburger steaks of Comparative Example 3 and Examples 3 and 4, even though they were cooked for the same amount of time (FIG. 5). The hamburger steak of Comparative Example 4 was not able to achieve the same sear marks as the hamburger steak of Example 4, even though it was cooked for twice as long as the hamburger steaks of Comparative Example 3 and Examples 3 and 4 (FIG. 6). The hamburger steaks of Examples 3 and 4 had an improved texture and were less likely to fall apart when cooked compared to the hamburger steaks of Comparative Examples 3 and 4.

[0111] Regarding the taste of the hamburgers of Examples 3 and 4, the powdery texture of the ingredients felt in the hamburger of Comparative Example 3 disappeared, and while "meat-like sinews" could be felt, the final fibrous texture was not felt. Furthermore, the hamburgers of Examples 3 and 4 had a meat-like taste that was different from the hamburger of Comparative Example 3. The hamburgers of Examples 3 and 4 were evaluated similarly to the hamburger of Example 3, but no recombinant fibroin short fibers were observed to be raised on the surface.

[0112] The hamburger steak of Example 4, which contained 5% by weight of recombinant fibroin staple fibers, was more flavorful, more intact (no cracks on the surface), and less powdery in texture.

[0113] Test Example 3: Preparation and Evaluation of Meat Substitute Compositions Materials (Meat Raw Materials and Meat Substitute Composition Raw Materials) Two types of meat raw materials or meat substitute composition raw materials were prepared: ground pork and OmniPork (manufactured by OmniFoods). OmniPork ingredient list: water, protein blend (soy protein concentrate, soy protein isolate, shiitake mushroom fermented pea & rice protein), thickeners (methylcellulose, maltodextrin), yeast extract, palm oil, potato starch, cane sugar, salt, natural flavors (canola and sunflower oil), barley malt extract, color (beet red), dextrose, anti-caking agent (silicon dioxide).

[0114] (Polypeptide fiber) The freeze-dried powder of artificial fibroin PRT799 obtained in Example 3 was added to formic acid (manufactured by Asahi Chemical Co., Ltd.) to a concentration of 31% by mass, and then dissolved at 80° C. After that, dust and bubbles were removed to obtain spinning solution 1 (dope solution 1).

[0115] To improve hydrophobicity, we designed an amino acid sequence in which all QQs in the amino acid sequence shown in SEQ ID NO: 1 were replaced with VFs and the remaining Qs were replaced with Is (amino acid sequence PRT966 shown in SEQ ID NO: 2).

[0116] A spinning solution was prepared using the freeze-dried powder of recombinant fibroin PRT966 obtained in the same manner as in Example 3. 99% formic acid was added to the freeze-dried powder so that the concentration of the freeze-dried powder became 26% by mass. After dissolving at 40°C, dust and bubbles were removed. This was designated as spinning solution 2 (dope solution 2).

[0117] Similarly, the freeze-dried powder of recombinant fibroin PRT966 obtained above was added to formic acid to a concentration of 30% by mass, and then dissolved at 40° C. After that, dust and bubbles were removed to obtain spinning solution 3 (dope solution 3).

[0118] The prepared dope solution 1, dope solution 2, or dope 3 was filled into a reserve tank. Using an inert gas (nitrogen), the spinning solution was discharged from a needle with an inner diameter of 0.20 mm into a coagulation solution (methanol) bath. After the protein was coagulated, the fibers were washed and stretched in a methanol washing bath and a water washing bath, and then dried using a hot plate to obtain recombinant fibroin fibers (raw yarns) 1 to 3. The wet spinning conditions were as follows: Discharge pressure: 0.5 bar, Stretch ratio: 5 to 7 times, Coagulation bath solution temperature: 5 to 20°C, Drying temperature: 60°C.

[0119] The fiber diameter was determined using an optical microscope for measuring physical properties. The stress and elongation of the protein fibers were measured using a tensile tester (Shimadzu EZ-S small tabletop tester) at an ambient temperature of 20°C and 60% relative humidity, and the toughness was calculated. The sample was attached to a cardboard mold, and the distance between the grippers was 20 mm and the tensile speed was 10 mm / min. The load cell capacity was 1 N and the gripping jig was a clip type. The toughness was calculated based on the following formula: [E / (r2 × π × L) × 1000] (unit: MJ / m3), where E is the fracture energy (unit: J), r is the fiber radius (unit: mm), π is the circumference ratio, and L is the gripper distance during the tensile test: 20 mm. The elongation and fiber diameter were evaluated, and the measured values were converted by setting the physical property values of recombinant fibroin fiber 1 at 100%, and the results are shown in Table 9. Similarly, the stress, elongation, and fiber diameter of commercially available soybean fiber and naturally derived silk fiber were also evaluated, and the results are shown in Table 9.

[0120] [Table 9]

[0121] The long fibers were cut into 3 mm lengths using a tabletop heavy-duty fiber cutter (NP-300, manufactured by INTEC CO. LTD.), yielding approximately 20 g of 3 mm recombinant fibroin short fibers 1 to 3.

[0122] (Preparation of Hamburger Steak Filling) As shown in Table 10, the resulting short fibers were added to the meat raw material or meat substitute composition (Examples 5 to 8, Comparative Examples 5 to 8), and the mixture was kneaded for several minutes to uniformly disperse the fibers. 100 g of the meat substitute composition was placed in a circular mold 9 cm in diameter and 1.8 cm thick, and molded into the shape of a hamburger steak filling. The mixture was then covered and stored at 4°C.

[0123] (Cooking and Evaluation of Hamburger Steaks) After 16 hours, an electric grill was set to 180°C and the hamburger steaks were cooked on a cooking sheet for 7 minutes on each side.

[0124] [Table 10]

[0125] The change in appearance (hamburger steak diameter) that occurred during the cooking process was evaluated, and the change rate in Comparative Example 6 was set to 100%, and each measurement value was converted. The results are shown in Figure 7. The change in hamburger steak diameter before and after cooking was calculated using the following formula: Diameter reduction rate (%) = [(raw meat diameter) - (cooked meat diameter)] / (raw meat diameter) x 100%

[0126] When meat ingredients were used (Comparative Example 5), the animal meat proteins were denatured and rapidly shrunk during cooking. Because the textured vegetable proteins in the meat substitute composition were already denatured proteins, when the meat substitute composition was used (Comparative Example 6), the shrinkage phenomenon during the cooking process was not significant. Adding 1 wt% protein fiber to the meat substitute composition did not result in a decrease in diameter, but adding 5 wt% recombinant fibroin fiber 2 (Example 8) increased the rate of diameter reduction of the hamburger, resulting in a more realistic appearance during cooking.

[0127] [Test Example 4: Preparation and evaluation of meat substitute composition] (Preparation, cooking, and evaluation of hamburger patty) As shown in Table 11, the short fibers obtained above were added to the meat raw material or the meat substitute composition (Examples 9 to 13, Comparative Examples 9 to 12), and hamburger patty was prepared as in Test Example 3 above, cooked, stored at 4°C and 60% relative humidity, and subjected to texture profile analysis.

[0128] [Table 11]

[0129] For Examples 9 to 13 and Comparative Examples 9 to 12, texture profile analysis of the cooked hamburger steaks was performed using a Universal Testing Instrument EZ Test (Shimadzu Corporation). Cooked hamburger steak samples were cut to the original height x 2.5 cm x 2.5 cm and compressed to 50% of the original height using a 500 N load cell. The crosshead speed was 300 mm / min, and the time interval between two compressions was set to 2 seconds. Each sample was measured nine times to evaluate the overall texture profile, including "hardness," "cohesiveness," "springiness," and "chewiness." "Hardness," "cohesiveness," "springiness," and "chewiness" were calculated based on Figure 8 and the following formula. The measured values for Comparative Example 10, which did not contain short fibers, were set to 100%, and the respective measured values were converted. The evaluation results are shown in Table 12 and Figures 9 to 12. Hardness (H) Maximum test force when food is loaded with a plunger Cohesiveness: A2 / A1 When food is subjected to stress, it can become deformed or damaged. The load is applied twice in succession, and the ratio of the load area (energy) between the first and second times is calculated. Spriginess: T2 / T1 The plunger is used to apply two successive loads to the food, and the ratio of the "depression and displacement" is measured. Chewiness: H×A2 / A1×T2 / T1 Hardness x Elasticity x Cohesiveness...Solid food

[0130] [Table 12]

[0131] When 1.5% by volume of fiber was added to the meat substitute composition, the addition of soy fiber (Comparative Example 11) and naturally derived silk fiber (Comparative Example 12) increased the firmness of the hamburger steak (FIG. 9), but significantly decreased the elasticity and cohesive strength of the hamburger steak (FIGS. 10 and 11). On the other hand, the addition of recombinant fibroin fiber (Examples 9, 11, and 13) increased the firmness of the hamburger steak (FIG. 9) without significantly decreasing the elasticity and cohesive strength of the hamburger steak (FIGS. 10 and 11).

[0132] When two types of proteins with different hydrophobicity were used, and recombinant fibroin fibers with high hydrophobicity (Examples 10 to 12) were used, the hardness could be increased more than that of recombinant fibroin fibers with low hydrophobicity (Example 9), resulting in a better chewability.

[0133] Two types of proteins with different mechanical properties were used, and when recombinant fibroin fiber 3 with a fiber diameter of approximately 30 μm was used (Example 13), a more realistic texture was observed compared to when recombinant fibroin fiber 2 with a diameter of approximately 10 μm was used (Example 11).

[0134] When the amount of recombinant protein added was increased within the range of 0.5 to 2.5% by volume (Examples 10 to 12), the hardness of the hamburger steak could be increased without significantly reducing its elasticity and cohesive strength, resulting in a more authentic texture.

[0135] Test Example 5: Preparation and evaluation of meat substitute composition (Preparation, cooking, and evaluation of hamburger steak filling) As shown in Table 13, the short fibers obtained above were added to the meat raw material or the meat substitute composition (Example 14, Comparative Examples 13 to 15), and hamburger steak filling was prepared and cooked as in Test Example 3. The change in appearance when the first side was cooked is shown in Figures 13 and 14.

[0136] [Table 13]

[0137] When meat ingredients were used (Comparative Example 13), the moisture and fat flowed downward and were discharged during cooking, but when a meat substitute composition was used (Comparative Example 14), some of the moisture was discharged to the upper surface, and water droplets formed on the surface. By adding recombinant fibroin fiber to the meat substitute composition (Example 14), the formation of water droplets was barely observed, as in the case of using meat ingredients, and the product showed a more realistic appearance change during cooking.

[0138] [Test Example 6: Preparation and evaluation of meat substitute composition] (Preparation, cooking and evaluation of hamburger patty) As shown in Table 14, the short fibers obtained above were added to the meat raw material or the meat substitute composition (Examples 15 to 13, Comparative Examples 15 to 12), and hamburger patty was prepared and cooked as in Test Example 3 above.

[0139] [Table 14]

[0140] The change in appearance (hamburger steak diameter) that occurred during the cooking process was evaluated, and the change rate of Comparative Example 16 was set to 100%, and each measurement value was converted. The results are shown in Figure 15. The change in hamburger steak diameter before and after cooking was calculated using the following formula: Diameter reduction rate (%) = [(raw meat diameter) - (cooked meat diameter)] / (raw meat diameter) x 100%

[0141] When meat ingredients were used (Comparative Example 15), the animal meat proteins were denatured and rapidly shrunk during cooking. When a meat substitute composition was used (Comparative Example 16), the shrinkage phenomenon during the cooking process was not significant. Adding soybean fiber (Comparative Example 17) or naturally derived silk fiber (Comparative Example 18) to the meat substitute composition did not result in a decrease in diameter. However, adding 5% by weight of each of the recombinant fibroin fibers 1 to 3 (Examples 15 to 17) increased the rate of decrease in diameter of the hamburger, resulting in a more realistic appearance during cooking.

[0142] [Explanation of symbols]

[0143] 1...extrusion device, 2...undrawn yarn manufacturing device, 3...moist heat drawing device, 4...drying device, 6...dope solution, 10...spinning device, 20...coagulation liquid tank, 21...drawing bath, 36...polypeptide fiber.

Claims

1. A meat substitute composition comprising a polypeptide that satisfies either (1) or (2) below: (1) The polypeptide has 150 or more amino acid residues, an alanine residue content of 12 to 40%, and a glycine residue content of 11 to 55%; or (2) The total content of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine, the alanine residue content, and the glycine residue content is 56% or more.

2. The meat substitute composition of claim 1, wherein the polypeptide satisfies both (1) and (2).

3. 3. The meat substitute composition of claim 1 or 2, wherein the polypeptide is a recombinant polypeptide.

4. The meat substitute composition according to any one of claims 1 to 3, wherein the polypeptide has a plurality of repeating sequence units, and the number of amino acid residues in the repeating sequence units is 6 to 200.

5. The polypeptide comprises: (A) ｎ A meat substitute composition according to any one of claims 1 to 4, comprising a motif.

6. A meat substitute composition according to any one of claims 1 to 5, wherein the polypeptide is a structural protein.

7. A meat substitute composition according to any one of claims 1 to 6, wherein the polypeptide is fibroin.

8. 8. The meat substitute composition of claim 1, wherein the polypeptide is in the form of a fiber.

9. 9. The meat substitute composition of claim 1, further comprising a plant protein.

10. An artificial meat product comprising the meat substitute composition according to any one of claims 1 to 9 or a processed product thereof.

11. A method for producing an artificial meat dish, comprising the step of cooking the meat substitute composition according to any one of claims 1 to 9 or the artificial meat product according to claim 10.

Images