Composition for substituting egg white

Abstract

Provided is a technology for substituting egg white in a food. A serpin derived from a plant or a fungus is used to substitute egg white in a food.

Description

Egg white substitute composition

[0001] In one aspect, the present invention relates to a technology for replacing egg white in foods.In one aspect, the present invention relates to a technology for binding food ingredients.In one aspect, the present invention relates to a technology for emulsifying oils and fats in food ingredients.

[0002] Egg white has the ability to solidify when heated (thermal gelling ability) and is used, for example, as a binder for food ingredients.

[0003] Methylcellulose and thickening polysaccharides are known as substitutes for egg white, but no protein with the same thermal gelling ability as egg white is known.

[0004] Serpins are proteins discovered as serine protease inhibitors (Non-Patent Document 1). Serpins are found widely throughout the plant kingdom, for example. Approximately 50,000 structural homologs of serpins are registered in protein databases.

[0005] Nardy Lampl et al., Arabidopsis AtSerpin1, Crystal Structure and in Vivo Interaction with Its Target Protease RESPONSIVE TO DESICCATION-21 (RD21), J Biol Chem. 2010 Apr 30; 285(18): 13550-13560.Niwa Tatsuya et al., Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins, Proc Natl Acad Sci USA 106:4201-4206.

[0006] In one aspect, the present invention aims to provide a technology for replacing egg white in foods. In one aspect, the present invention aims to provide a technology for binding food ingredients. In one aspect, the present invention aims to provide a technology for emulsifying oils and fats in food ingredients.

[0007] As a result of extensive research to solve the above problems, the present inventors have discovered that serpins derived from plants or fungi can be used to replace egg white in foods, bind food ingredients, and / or emulsify oils and fats in food ingredients, and have thus completed the present invention.

[0008] That is, the present invention can be exemplified as follows: [1] A composition containing a serpin derived from a plant or fungus, which is used for replacing egg white, binding food materials, emulsifying oils and fats in food materials, or producing foods. [2] The composition (specifically described in [1]) in which the serpin is derived from a plant of the Brassicaceae family, a plant of the Malvaceae family, a plant of the Euphorbiaceae family, a plant of the Asteraceae family, a plant of the Theaceae family, or a plant of the Apiaceae family. [3] The composition (specifically according to [1] or [2]), wherein the serpin is derived from a plant of the genus Arabidopsis, a plant of the genus Brassica, a plant of the genus Theobroma, a plant of the genus Ricinus, a plant of the genus Helianthus, a plant of the genus Camellia, or a plant of the genus Daucus. [4] The composition (specifically, the composition according to any one of [1] to [3]), wherein the serpin is derived from Arabidopsis thaliana, rapeseed (Brassica napus), cacao (Theobroma cacao), castor bean (Ricinus communis), sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), or carrot (Daucus carota subsp. sativus).[5] The composition (specifically described in any of [1] to [4]) wherein the serpin is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18; (b) a protein comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, with 1 to 10 amino acid residue substitutions, deletions, insertions, and / or additions, and having egg white replacement, binding, or emulsification functions; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having egg white replacement, binding, or emulsification functions. [6] The composition (specifically described in any of [1] to [5]) wherein the serpin has a molecular weight of 40,000 to 50,000. [7] The composition (specifically described in any of [1] to [6]) wherein the serpin has one or more properties selected from the following (1) to (4): (1) the ratio of Ser residues in the amino acid sequence is 7.5% or more; (2) the isoelectric point (pI) is 4 to 7; (3) the internal hydrophobicity is 1.5 or more; (4) the predicted scaled solubility is 0.45 or more. [8] A method for producing a food product, the method comprising the step of adding a serpin derived from a plant or fungus to a food ingredient. [9] The method (specifically described in [8]), wherein the produced food product is a food product in which egg white has been replaced, a food ingredient has been bound, and / or oils and fats in the food ingredient have been emulsified.

[10] A method for replacing egg white in a food product, the method comprising the step of adding a serpin derived from a plant or fungus to a food ingredient.

[11] A method for binding food materials, comprising the step of adding a serpin derived from a plant or fungus to the food raw materials.

[12] A method for emulsifying oils and fats in food materials, comprising the step of adding a serpin derived from a plant or fungus to the food raw materials.

[13] The method (specifically described in any of [8] to

[12] ) wherein the serpin is derived from a plant of the Brassicaceae family, a plant of the Malvaceae family, a plant of the Euphorbiaceae family, a plant of the Asteraceae family, a plant of the Theaceae family, or a plant of the Apiaceae family.

[14] The method (specifically described in any of [8] to

[13] ) wherein the serpin is derived from a plant of the genus Arabidopsis, a plant of the genus Brassica, a plant of the genus Theobroma, a plant of the genus Ricinus, a plant of the genus Helianthus, a plant of the genus Camellia, or a plant of the genus Daucus.

[15] The method (specifically according to any of [8] to

[14] ) above, wherein the serpin is derived from Arabidopsis thaliana, rapeseed (Brassica napus), cacao (Theobroma cacao), castor bean (Ricinus communis), sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), or carrot (Daucus carota subsp. sativus).

[16] The method (specifically, any of [8] to

[15] ) above, wherein the serpin is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18; (b) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having an egg white replacement function, a binding function, or an emulsification function; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having an egg white replacement function, a binding function, or an emulsification function.

[17] The method (specifically described in any of [8] to

[16] ) above, wherein the serpin has a molecular weight of 40,000 to 50,000.

[18] The method (specifically described in any of [8] to

[17] ) above, wherein the serpin has one or more properties selected from the following (1) to (4): (1) the ratio of Ser residues in the amino acid sequence is 7.5% or more; (2) the isoelectric point (pI) is 4 to 7; (3) the internal hydrophobicity is 1.5 or more; (4) the predicted scaled solubility is 0.45 or more.

[19] The method (specifically described in any of [8] to

[18] ) above, wherein the serpin is added to a concentration of 0.05 to 30% (w / w) for consumption.

[20] A food product containing a serpin derived from a plant or fungus.

[21] The food product (specifically according to

[20] ), wherein the serpin is derived from a plant of the Brassicaceae family, a plant of the Malvaceae family, a plant of the Euphorbiaceae family, a plant of the Asteraceae family, a plant of the Theaceae family, or a plant of the Apiaceae family.

[22] The food product (specifically according to

[20] or

[21] ), wherein the serpin is derived from a plant of the genus Arabidopsis, a plant of the genus Brassica, a plant of the genus Theobroma, a plant of the genus Ricinus, a plant of the genus Helianthus, a plant of the genus Camellia, or a plant of the genus Daucus.

[23] The food (specifically, the food according to any one of

[20] to

[22] ), wherein the serpin is derived from Arabidopsis thaliana, rapeseed (Brassica napus), cacao (Theobroma cacao), castor bean (Ricinus communis), sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), or carrot (Daucus carota subsp. sativus).

[24] The food (specifically according to any of

[20] to

[23] ), wherein the serpin is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18; (b) a protein comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, with 1 to 10 amino acid residue substitutions, deletions, insertions, and / or additions, and having an egg white replacement function, a binding function, or an emulsification function; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having an egg white replacement function, a binding function, or an emulsification function.

[25] The food (specifically according to any of

[20] to

[24] ), wherein the serpin has a molecular weight of 40,000 to 50,000.

[26] The food product (specifically, any one of

[20] to

[25] ) described above, wherein the serpin has one or more properties selected from the following (1) to (4): (1) the ratio of Ser residues in the amino acid sequence is 7.5% or more; (2) the isoelectric point (pI) is 4 to 7; (3) the internal hydrophobicity is 1.5 or more; (4) the predicted scaled solubility is 0.45 or more.

[27] The food product (specifically, any one of

[20] to

[26] ) described above, wherein the serpin content is 0.05 to 30% (w / w).

[0009] In one aspect, the present invention can replace egg white in foods. In one aspect, the present invention can bind food ingredients. In one aspect, the present invention can emulsify oils and fats in food ingredients.

[0010] The present invention will be described in detail below.

[0011] <1> Active ingredient In the present invention, serpin is used as the active ingredient. Serpin is also referred to as the "active ingredient."

[0012] In one aspect, by utilizing the active ingredient, it is possible to replace egg white in foods, i.e., an effect of replacing egg white in foods can be obtained. This effect is also referred to as an "egg white replacement effect." In other words, in one aspect, the active ingredient has the function of replacing egg white in foods. This function is also referred to as an "egg white replacement function." Replacement of egg white in foods is also simply referred to as "egg white replacement." Replacement of egg white may be a partial or complete replacement of egg white in foods. In other words, "replacement of egg white" may mean a reduction in the content of egg white in foods. In other words, "replacement of egg white" may mean a reduction in the amount of egg white used in the production of foods. By utilizing the active ingredient, the content of egg white in foods may be reduced to, for example, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less of the normal content of egg white in foods, or may even be reduced to zero. By utilizing the active ingredient, the amount of egg white used in the production of a food product may be reduced to, for example, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less the amount of egg white normally used in the production of a food product, or even to zero. Specifically, by utilizing the active ingredient, the function of egg white may be substituted. That is, for example, the function of egg white may be substituted by the active ingredient, thereby replacing egg white in the food product. Examples of the functions of egg white include binding ability, emulsifying ability, foaming ability, and water retention ability. By utilizing the active ingredient, for example, one or more of these egg white functions may be substituted. By utilizing the active ingredient, for example, one, two, three, or all four of these egg white functions may be substituted. Examples of egg white include egg white from birds' eggs, such as chicken eggs. The egg white replacement function (e.g., binding ability) can be measured, for example, as the gelling ability of a serpin when heated (also referred to as thermal gelling ability). The thermal gelling ability can be measured, for example, by measuring the storage elasticity when a serpin is heated to gel. The storage elasticity can be measured, for example, using a rheometer. Specifically, the thermal gelling ability can be measured, for example, by the procedure described in the Examples.

[0013] In one embodiment, the active ingredient can be used to bind food materials, i.e., to achieve the effect of binding food materials. This effect is also referred to as the "binding effect." In other words, in one embodiment, the active ingredient has the function of binding food materials. This function is also referred to as the "binding function." The binding of food materials can also be simply referred to as "binding." Examples of food materials to be bound include protein-containing ingredients such as meat and veggie meat. For example, the active ingredient may function as a binder for the food materials due to its binding function. The binding effect can be measured, for example, by measuring the texture of the food. The texture of the food can be measured, for example, by sensory evaluation by a specialist panel. The texture of the food can be measured, for example, using a texture measuring device such as a texture analyzer. Furthermore, the binding function can be measured, for example, as the gelling ability of serpins when heated (also referred to as thermal gelling ability). The thermal gelling ability can be measured, for example, by measuring the storage elasticity when serpins are heated to gel. The storage elastic force can be measured, for example, using a rheometer. The thermal gelling ability can be measured, for example, by the procedure described in the Examples.

[0014] In one embodiment, the active ingredient can be used to emulsify the fats and oils in food materials, i.e., to achieve the effect of emulsifying the fats and oils in the food materials. This effect is also referred to as the "emulsifying effect." In other words, in one embodiment, the active ingredient has the function of emulsifying the fats and oils in the food materials. This function is also referred to as the "emulsifying function." The emulsification of fats and oils in food materials is also simply referred to as "emulsification." Foods containing emulsified fats and oils include beverages, ice creams, confectioneries, bread, soups, liquid seasonings, processed meat products, processed seafood products, dairy products, health foods, medical foods, jams, preserves, syrups, processed fruits, pickles, grain flour, noodles, and liquid foods, but foods other than health foods and medical foods are preferred. The active ingredient may function as an emulsifier, for example, due to its emulsifying function. The emulsifying effect can be evaluated, for example, by calculating the creaming index (CI) of the emulsion. The creaming index (CI) is a numerical representation of the retention of oil droplets (cloudy layer) produced by emulsification. Specifically, the creaming index (CI) can be calculated, for example, using the following formula based on the height of the entire emulsion (HE) and the height of the transparent layer (HS):

[0015] Creaming Index (CI) = HS / HE

[0016] "Serpin" refers to a protein classified in the Serpin superfamily. Serpins may or may not function, for example, as serine protease inhibitors. As serpins, a single type of serpin may be used, or two or more types of serpins may be used in combination.

[0017] Serpins used as active ingredients are derived from plants or fungi. A serpin derived from organism X (where X is the name that identifies the organism) is also referred to as a "serpin derived from organism X." For example, "plant-derived serpin" refers to a serpin derived from a plant. For example, "fungal-derived serpin" refers to a serpin derived from a fungus. Serpins particularly include plant-derived serpins. The type of plant or fungus from which a serpin is derived is not particularly limited. Examples of plants include plants of the Brassicaceae family, Malvaceae family, Euphorbiaceae family, Asteraceae family, Theaceae family, and Apiaceae family. Examples of plants of the Brassicaceae family include plants of the genus Arabidopsis, Brassica, and Raphanus. Examples of plants in the Arabidopsis genus include Arabidopsis thaliana. Examples of plants in the Brassica genus include Brassica napus and Chinese cabbage (Brassica rapa subsp. pekinensis). Examples of plants in the Raphanus genus include Raphanus sativus. Examples of plants in the Malvaceae family include Theobroma cacao. Examples of plants in the Theobroma cacao genus include Theobroma cacao. Examples of plants in the Euphorbiaceae family include Ricinus communis. Examples of plants in the Ricinus genus include Ricinus communis. Plants of the Asteraceae family include plants of the genus Helianthus. Plants of the Helianthus genus include sunflowers (Helianthus annuus). Plants of the Theaceae family include plants of the genus Camellia.Examples of Camellia plants include the tea plant (Camellia sinensis var. sinensis). Examples of Apiaceae plants include the carrot (Daucus). Examples of Daucus plants include carrot (Daucus carota subsp. sativus). Examples of plants include Arabidopsis thaliana, Brassica napus, cacao (Theobroma cacao), and castor bean (Ricinus communis). Genes encoding serpins are also called "serpin genes." The nucleotide sequences of serpin genes from various organisms and the amino acid sequences of the serpins encoded by them can be obtained from public databases such as NCBI and technical literature such as patent documents. The nucleotide sequence of the Arabidopsis thaliana serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence of the Brassica napus serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequence of the Theobroma cacao serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequence of the Ricinus communis serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 7 and 8, respectively. The nucleotide sequence of the Brassica rapa subsp. pekinensis serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 9 and 10, respectively. The nucleotide sequence of the Raphanus sativus serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 11 and 12, respectively. The nucleotide sequence of the Helianthus annuus serpin gene and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 13 and 14, respectively.The nucleotide sequence of a serpin gene from Camellia sinensis var. sinensis and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 15 and 16, respectively. The nucleotide sequence of a serpin gene from Daucus carota subsp. sativus and the amino acid sequence of the serpin encoded by the gene are shown in SEQ ID NOs: 17 and 18, respectively. That is, the serpin gene may be, for example, a gene having the nucleotide sequence of one of the exemplified serpin genes (e.g., the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17). The serpin may also be, for example, a protein having the amino acid sequence of one of the exemplified serpins (e.g., the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, or 18). Unless otherwise specified, the expression "having an (amino acid or nucleotide) sequence" means "including the (amino acid or nucleotide) sequence" and also encompasses "consisting of the (amino acid or nucleotide) sequence."

[0018] The serpin gene may be a variant of the above-exemplified serpin genes (e.g., genes having the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17), so long as the original function is maintained. Similarly, the serpin may be a variant of the above-exemplified serpins (e.g., proteins having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18), so long as the original function is maintained. Such variants that maintain the original function are sometimes referred to as "conservative variants." The term "serpin gene" encompasses the above-exemplified serpin genes as well as their conservative variants. Similarly, the term "serpin" encompasses the above-exemplified serpins as well as their conservative variants. Furthermore, unless otherwise specified, the serpin genes and serpins identified in the biological species from which they are derived are not limited to those actually present in the biological species, but may also encompass conservative variants (which may or may not actually be present in the biological species). Conservative variants include, for example, the serpin genes and serpin homologs exemplified above, as well as artificially modified variants. The phrase "a serpin derived from organism X" does not necessarily mean that the serpin is a serpin found in organism X, but also encompasses cases where the serpin is an artificially modified version of a serpin found in organism X. A serpin derived from organism X may be, for example, one produced and obtained in organism X, or one obtained by heterologous expression (i.e., a recombinant protein). For example, an Arabidopsis thaliana serpin may be, for example, one produced and obtained in Arabidopsis thaliana cells, or one obtained by heterologous expression (i.e., a recombinant protein).

[0019] "Maintaining the original function" means that a gene or protein variant has a function (e.g., activity or property) corresponding to the function (e.g., activity or property) of the original gene or protein. That is, "maintaining the original function" with respect to a gene may mean that a gene variant encodes a protein in which the original function is maintained. That is, "maintaining the original function" with respect to a serpin gene may mean that a gene variant encodes a serpin. Furthermore, "maintaining the original function" with respect to a serpin may mean that a protein variant has a desired function as a serpin (e.g., egg white replacement function, binding function, or emulsification function), and may mean that these functions are maintained at 10% or more, 30% or more, 50% or more, or 80% or more compared to the function of the original protein.

[0020] Examples of conservative variants are shown below.

[0021] Serpin gene homologs or serpin homologs can be easily obtained from public databases by, for example, BLAST or FASTA searches using the nucleotide sequences of the above-listed serpin genes or the amino acid sequences of the above-listed serpins as query sequences. Serpin gene homologs can also be obtained by PCR, for example, using the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of these known serpin genes as primers.

[0022] As long as the original function is maintained, the serpin gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions in the above-mentioned amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18) have been substituted, deleted, inserted, and / or added. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. Note that the term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0023] The above-mentioned substitution, deletion, insertion, and / or addition of one or several amino acids is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted for each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted for each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted for each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted for each other when the substitution site is a basic amino acid; Asp and Glu are substituted for each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted for each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitutions of Ala with Ser or Thr, substitutions of Arg with Gln, His, or Lys, substitutions of Asn with Glu, Gln, Lys, His, or Asp, substitutions of Asp with Asn, Glu, or Gln, substitutions of Cys with Ser or Ala, substitutions of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu with Gly, Asn, Gln, Lys, or Asp, substitutions of Gly with Pro, substitutions of His with Asn, Lys, Gln, Arg, or Tyr, substitutions of Ile substitution of Lys with Leu, Met, Val, or Phe, substitution of Leu with Ile, Met, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions also include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0024] Furthermore, as long as the original function is maintained, the serpin gene may be a gene encoding a protein having an amino acid sequence that is, for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the above-mentioned entire amino acid sequence.

[0025] Furthermore, as long as the original function is maintained, the serpin gene may be a gene (e.g., DNA) that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17), such as a sequence complementary to all or part of the above-mentioned nucleotide sequence. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. One example of such conditions includes conditions under which DNAs with high identity, for example, DNAs with identity of 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, while DNAs with lower identity do not hybridize with each other; or conditions for washing once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0026] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0027] Furthermore, since codon degeneracy differs depending on the host, a serpin gene may be one in which any codon has been replaced with an equivalent codon. That is, the serpin gene may be a variant of the serpin genes exemplified above due to the degeneracy of the genetic code. For example, the expression-suppressing gene may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0028] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0029] As the serpin, for example, commercially available products may be used, or those obtained by appropriate production may be used.

[0030] The method for producing serpins is not particularly limited. Serpins can be produced, for example, by culturing serpin-producing host cells. The host cells are not particularly limited as long as they can express serpins. Examples of host cells include cells used for heterologous protein expression. Specific examples of host cells include bacteria, fungi, plant cells, animal cells, and insect cells. Examples of host cells include microorganisms such as bacteria. Examples of bacteria include Escherichia bacteria such as Escherichia coli and Corynebacterium bacteria such as Corynebacterium glutamicum. Serpin-producing host cells may naturally produce serpins or may be modified to produce serpins. Serpin-producing host cells can be obtained, for example, by introducing a serpin-encoding gene into the host cell in an expressible manner. The culture conditions for serpin-producing host cells are not particularly limited, as long as serpins are produced. Serpin-producing host cells can be cultured, for example, under normal conditions for culturing host cells.

[0031] Serpins may or may not contain components other than serpins. Serpins may contain, for example, other enzymes. That is, purified serpins may be used as serpins, or materials containing serpins may be used. Serpin-containing materials include cultures of host cells that produce serpins, culture supernatants separated from the cultures, host cells separated from the cultures, and processed products thereof. Processed products include host cell lysates. When serpins form inclusion bodies within host cells, the serpins can be solubilized as appropriate. Serpins may be purified to a desired degree.

[0032] In addition, when a material containing a serpin is used, the amount of serpin (e.g., content (concentration) or amount used) is calculated based on the amount of serpin itself in the material.

[0033] Serpins may have a molecular weight of, for example, 40,000 to 50,000. Serpins from Arabidopsis thaliana, rapeseed (Brassica napus), cacao (Theobroma cacao), castor bean (Ricinus communis), Chinese cabbage (Brassica rapa subsp. pekinensis), radish (Raphanus sativus), sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), and carrot (Daucus carota subsp. sativus) may all have a molecular weight of 40,000 to 50,000.

[0034] Serpins may have one or more properties selected from the following (1) to (4): Serpins may have one, two, three, or all four properties selected from the following (1) to (4): Serpins from Arabidopsis thaliana, rapeseed (Brassica napus), cacao (Theobroma cacao), castor bean (Ricinus communis), Chinese cabbage (Brassica rapa subsp. pekinensis), radish (Raphanus sativus), sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), and carrot (Daucus carota subsp. sativus) may all have all of the following properties (1) to (4): (1) The ratio of Ser residues in the amino acid sequence is 7.5% or more. (2) The isoelectric point (pI) is between 4 and 7. (3) The internal hydrophobicity is 1.5 or more. (4) The predicted scaled solubility is 0.45 or more.

[0035] Regarding (3) above, "internal hydrophobicity" refers to the internal hydrophobicity calculated using the AlphaFold Protein Structure Database (AlphaFold 2 DB; https: / / alphafold.ebi.ac.uk) and Biopython version 1.79 (https: / / biopython.org) according to the following procedure: The SASA value of each amino acid residue constituting the serpin was calculated using Biopython, and amino acid residues with a SASA value of less than 20 were considered to be "internal" amino acid residues. The Kyte-Doolittle scale (i.e., the hydropathic index of each amino acid listed in TABLE 2 of Kyte, J. and Doolittle, RF (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol, 157(1):105-132) was summed for all "internal" amino acid residues to determine the "internal hydrophobicity."

[0036] Regarding (4) above, "Predicted scaled solubility" is a numerical value for predicting protein solubility calculated using default parameters by Protein-Sol software (https: / / www.manchester.ac.uk). Proteins with a predicted scaled solubility of 0.45 or higher are predicted to be more soluble than the average soluble protein in E. coli. Here, the "average soluble protein in E. coli" refers to proteins in the experimental solubility dataset (Niwa et al., 2009) based on the experiment in Non-Patent Document 2.

[0037] <2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient (i.e., a serpin).

[0038] In one embodiment, the composition of the present invention can be used to replace egg white in foods, i.e., an egg white replacement effect can be obtained. In other words, the composition of the present invention has an egg white replacement function. Therefore, the composition of the present invention may be used to replace egg white. That is, the composition of the present invention may be, for example, a composition for replacing egg white.

[0039] In one embodiment, the composition of the present invention can be used to bind food materials, i.e., to provide a binding effect. In other words, the composition of the present invention has a binding function. Therefore, the composition of the present invention may be used to bind food materials. That is, the composition of the present invention may be, for example, a composition for binding food materials.

[0040] In one aspect, by using the composition of the present invention, it is possible to emulsify fats and oils in food materials, i.e., an emulsification effect is obtained. In other words, the composition of the present invention has an emulsifying function. Therefore, the composition of the present invention may be used to emulsify fats and oils in food materials. That is, the composition of the present invention may be, for example, a composition for emulsifying fats and oils in food materials.

[0041] Furthermore, by using the composition of the present invention, it is possible to produce foods that replace egg white, bind food ingredients, and / or emulsify fats and oils in food ingredients. Thus, the composition of the present invention may be used in the production of foods (specifically, the production of foods that replace egg white, bind food ingredients, and / or emulsify fats and oils in food ingredients). That is, the composition of the present invention may be, for example, a composition for use in the production of foods (specifically, the production of foods that replace egg white, bind food ingredients, and / or emulsify fats and oils in food ingredients).

[0042] The composition of the present invention may be, for example, a seasoning. Specifically, the composition of the present invention may be, for example, a seasoning for replacing egg white, a seasoning for binding food ingredients, a seasoning for emulsifying fats and oils in food ingredients, or a seasoning for food production (specifically, production of foods in which egg white is replaced, food ingredients are bound, and / or fats and oils in food ingredients are emulsified).

[0043] The composition of the present invention may be used as a substitute for egg white, to bind food ingredients, to emulsify oils and fats in food ingredients, or to produce food products in the manner described in the method of the present invention below.

[0044] The composition of the present invention may consist of the active ingredient or may contain ingredients other than the active ingredient, i.e., the active ingredient may be used as is or in combination with other ingredients in the composition of the present invention.

[0045] The ingredients other than the active ingredient are not particularly limited as long as they do not impair the desired effect (e.g., egg white replacement effect, binding effect, or emulsifying effect) (i.e., the desired effect of the active ingredient is obtained). The ingredients other than the active ingredient can be appropriately selected depending on various conditions, such as the type of food. Examples of ingredients other than the active ingredient include ingredients that are blended into foods or pharmaceuticals. Specific examples of ingredients other than the active ingredient include the food ingredients described below.

[0046] The composition of the present invention may be formulated as appropriate, for example. When formulating, additives may be used as appropriate. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, and solvents. The additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.

[0047] The form of the composition of the present invention is not particularly limited, and the composition of the present invention may be in any form, such as powder, flakes, tablets, paste, or liquid.

[0048] The content and content ratio of each component (i.e., the active ingredient and optionally other components) in the composition of the present invention are not particularly limited as long as the desired effect (e.g., egg white replacement effect, binding effect, or emulsifying effect) is obtained. The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions such as the mode of use of the composition of the present invention.

[0049] The content of the active ingredient in the composition of the present invention is more than 0% (w / w) and less than 100% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 20% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, or 70% (w / w) or more, and may be 100% (w / w). The active ingredient content in the composition of the present invention may be, for example, 0.1-1% (w / w), 1-10% (w / w), 10-30% (w / w), 30-50% (w / w), 50-70% (w / w), 70-90% (w / w), or 70-100% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 1-100% (w / w), 5-90% (w / w), or 10-70% (w / w). When the composition of the present invention contains two or more serpins, the content of those two or more serpins in the composition of the present invention may be set independently or in total within the range of the content of the active ingredient in the composition of the present invention exemplified above (provided that the total content of those two or more serpins in the composition of the present invention is 100% (w / w) or less). When the composition of the present invention contains two or more serpins, the term "content of the active ingredient in the composition of the present invention" refers to the total content of those two or more serpins in the composition of the present invention, unless otherwise specified.

[0050] The content of each component (i.e., the active ingredient and optionally other ingredients) in the composition of the present invention can be set, for example, so as to obtain the amount of each component added in the method of the present invention described below.

[0051] The components (i.e., the active ingredient and optional other ingredients) contained in the composition of the present invention may be mixed together and contained in the composition of the present invention, or may be contained separately or in any combination. For example, the composition of the present invention may be provided as a set of components each packaged separately. In such a case, the components contained in the set can be used together as appropriate when used.

[0052] <3> Method of the Present Invention The method of the present invention is a method comprising a step of utilizing an active ingredient (i.e., serpin).

[0053] In one aspect, the method of the present invention, specifically by utilizing the active ingredient, can replace egg white in food, i.e., an egg white replacement effect can be obtained. Thus, the method of the present invention may be carried out to replace egg white in food. That is, the method of the present invention may be, for example, a method for replacing egg white in food. This method is also referred to as the "egg white replacement method of the present invention."

[0054] In one aspect, the method of the present invention, specifically by utilizing an active ingredient, can bind food materials, i.e., a binding effect can be obtained. Thus, the method of the present invention may be carried out for binding food materials. That is, the method of the present invention may be, for example, a method for binding food materials. This method is also referred to as the "binding method of the present invention."

[0055] In one aspect, the method of the present invention, specifically by utilizing the active ingredient, can emulsify fats and oils in a food material, i.e., an emulsification effect can be obtained. Thus, the method of the present invention may be carried out to emulsify fats and oils in a food material. That is, the method of the present invention may be, for example, a method for emulsifying fats and oils in a food material. This method is also referred to as the "emulsification method of the present invention."

[0056] Furthermore, the method of the present invention, specifically by utilizing the active ingredient, can produce foods in which egg white has been replaced, food ingredients have been bound, and / or fats and oils in the food ingredients have been emulsified. Thus, the method of the present invention may be carried out for the production of foods (specifically, the production of foods in which egg white has been replaced, food ingredients have been bound, and / or fats and oils in the food ingredients have been emulsified). That is, the method of the present invention may be, for example, a method for producing foods (specifically, the production of foods in which egg white has been replaced, food ingredients have been bound, and / or fats and oils in the food ingredients have been emulsified). This method is also referred to as the "food production method of the present invention."

[0057] The active ingredient can be added to food ingredients during food production to replace egg white, bind food ingredients, emulsify fats and oils in food ingredients, or be used in food production. That is, an example of using the active ingredient is adding the active ingredient to food ingredients. Specifically, the method of the present invention may be, for example, a method for replacing egg white in a food product, which comprises adding the active ingredient to food ingredients. Also, the method of the present invention may be, for example, a method for binding food ingredients, which comprises adding the active ingredient to food ingredients. Also, the method of the present invention may be, for example, a method for emulsifying fats and oils in a food product, which comprises adding the active ingredient to food ingredients. Also, the method of the present invention may be, for example, a method for producing a food product (specifically, a food product in which egg white is replaced, food ingredients are bound, and / or fats and oils in a food product are emulsified), which comprises adding the active ingredient to food ingredients. "Addition" can also be referred to as "blending."

[0058] The active ingredient may be utilized in the method of the present invention, for example, in the form of a composition of the present invention. That is, "utilization of an active ingredient" also includes utilization of a composition of the present invention. For example, "addition of an active ingredient" also includes addition of a composition of the present invention.

[0059] The food obtained by the method of the present invention is also referred to as the "food of the present invention." Specifically, the food of the present invention is a food in which egg white has been replaced, food ingredients have been bound, and / or fats and oils in the food ingredients have been emulsified. In other words, the food of the present invention is a food to which an active ingredient has been added. In other words, the food of the present invention is a food containing an active ingredient.

[0060] Substitution of egg white, binding of food ingredients, emulsification of food ingredients, or production of food may be carried out in the same manner as production of ordinary foods, except for, for example, the use of an active ingredient. That is, substitution of egg white, binding of food ingredients, emulsification of food ingredients, or production of food may be carried out using the same raw materials and under the same production conditions as ordinary foods, except for, for example, the use of an active ingredient. Furthermore, any of the raw materials and production conditions of the food may be appropriately modified for use in substitution of egg white, binding of food ingredients, emulsification of food ingredients, or production of food.

[0061] The food is not particularly limited as long as it is desired to exhibit the desired effects, such as an egg white replacement effect, a binding effect, an emulsifying effect, etc. Examples of the food include foods containing egg white. Examples of the food include foods that do not contain egg white. Examples of the food include foods that may typically contain egg white, but have a reduced egg white content. Examples of the food also include beverages. Examples of the food also include seasonings. The food may be, for example, a liquid or a solid.

[0062] Specific examples of foods (e.g., foods that may typically contain egg white) include egg-containing sauces such as carbonara sauce and custard sauce, and foods using such sauces; egg-covered foods such as oyakodon (chicken and egg rice bowl), katsudon (pork cutlet rice bowl), and tenshinhan (rice with rice topped with egg and egg); tamagoyaki (Japanese omelet) such as dashimaki (rolled omelet) and omelette; scrambled egg-containing foods such as chive and egg and fried rice; marshmallows, macarons, cakes, cookies, and other sweets; chawanmushi (steamed egg custard), egg tofu, omelet rice, egg sandwiches, and puddings. An example of a food using carbonara sauce is carbonara (pasta). An example of a food using custard sauce is cream puffs.

[0063] Specific examples of foods include foods produced by processing protein-containing ingredients. Protein-containing ingredients include meat and veggie meat. "Meat" refers to meat for consumption. Meat includes animal meat. "Animal" may refer to organisms classified in the Animalia kingdom. Animals include vertebrates and aquatic organisms. Vertebrates include mammals, birds, reptiles, amphibians, and fish. Vertebrates particularly include mammals, birds, and fish. Mammals include livestock such as cows, pigs, horses, sheep, goats, and rabbits; wild animals such as boars, deer, and bears; and marine mammals such as whales, dolphins, and sea lions. Birds include chickens, turkeys, ducks, geese, guinea fowl, quails, and ostriches. Fish include horse mackerel, salmon, cod, pufferfish, whiting, conger eel, hoki, hake, eel, tuna, grouper, and sea bream. Aquatic organisms include aquatic mammals and fish, as well as crustaceans such as shrimp and crab, shellfish such as scallops and oysters, and other marine products such as squid and octopus. "Veggie meat" may refer to ingredients in which raw materials containing plant protein are processed to resemble meat, or foods produced using such ingredients. Plants from which plant proteins are derived include grains such as beans and wheat. Beans include soybeans, peas, fava beans, chickpeas, almonds, peanuts, and lupin. Wheat includes wheat, barley, and rye. Plant proteins particularly include legume proteins. Plant proteins, more particularly, include soy protein. That is, veggie meat particularly includes products produced using raw materials containing soy protein (also known as "soy meat"). Foods produced by processing protein-containing ingredients include ham, sausage, hamburger steak, meatballs, and paste products. Meatballs may include those used as fillings for stuffed noodle foods such as shumai, and those used as fillings for cabbage rolls. For example, sausages and hamburgers include sausages and hamburgers made from processed veggie meats such as soy meat, and sausages and hamburgers made from processed meats such as beef, pork, and fish. Examples of fish paste products include kamaboko, crab sticks, and fried kamaboko.In these foods, the active ingredient may function, for example, as a binder to bind protein-containing ingredients. Any of these foods may be, for example, foods that may typically contain egg white.

[0064] Specific examples of the food product include foods produced using emulsifiers.Specific examples of the food product produced using emulsifiers include beverages such as milk drinks, lactic acid bacteria drinks, carbonated drinks, vegetable drinks, fruit drinks, vegetable and fruit drinks, alcoholic drinks, coffee drinks, powdered drinks, sports drinks, supplement drinks, tea drinks, etc., frozen desserts such as ice cream, soft serve ice cream, and popsicles, confectioneries such as pudding, jelly, bavarois, gum, chocolate, candy, cookies, biscuits, cakes, and donuts, bread, soups such as potage, liquid seasonings such as separate dressing, non-oil dressing, ketchup, sauce, and so on, processed meat products such as ham and sausage, processed seafood products such as fish sausage, fish cakes, chikuwa, and kamaboko, dairy products such as yogurt, health foods and medical foods such as nutritional supplements (supplements), nutritionally functional foods, and foods for specified health uses, jams, preserves, syrups, processed fruits, pickles, grain flour, noodles, and liquid diets. In these foods, the active ingredient may function, for example, as an emulsifier for emulsifying oils and fats, an emulsifier for foaming food ingredients, etc. Any of these foods may be, for example, a food that may typically contain egg white.

[0065] The manner in which the food is provided is not particularly limited. For example, the food may be provided in a form that can be consumed as is, or in a form that requires preparation before or at the time of consumption, such as a concentrated product or a dried product. The food may also be provided in any container, such as a retort pouch, a paper pack, a plastic bottle such as a PET bottle, a metal can such as a steel can or an aluminum can, or a glass bottle. Foods are not limited to general foods, but also include so-called health foods or medical foods such as nutritional supplements (supplements), nutritionally functional foods, and foods for specified health uses. That is, for example, the foods exemplified above may be provided as general foods, health foods, or medical foods.

[0066] "Food ingredients" refers to food materials used to produce food. There are no particular limitations on the food ingredients as long as they can be used to produce food. Food ingredients can be selected appropriately depending on various conditions, such as the type of food. Examples of food ingredients include ingredients that are commonly used in food production, such as those exemplified above. Specific examples of food ingredients include grains such as rice and wheat flour; seasoning ingredients such as sugars, inorganic salts, organic acids, nucleic acids, amino acids, and protein hydrolysates; dairy products such as milk, cheese, and butter; protein-containing ingredients such as meat and veggie meat; fruits; vegetables; eggs; spices; flavorings; oils and fats; alcohol; dietary fiber; and pH buffers. Examples of eggs include avian eggs such as chicken eggs. As eggs, any fraction, such as whole eggs, egg yolks, egg whites, or a combination thereof, may be used. As eggs, a fraction containing at least egg yolk, such as whole eggs or egg yolks, may be used. As eggs, a fraction containing at least egg white, such as whole eggs or egg whites, may be used. Eggs may be used in any form, such as liquid or powder.

[0067] The active ingredient may be added to food ingredients at any stage of the food manufacturing process, as long as the desired effects, such as egg white replacement, binding, and emulsification, are achieved. In other words, the "food ingredients" to which the active ingredient is added may be those at any stage of the food manufacturing process. For example, the "food ingredients" to which the active ingredient is added may include finished foods before the active ingredient is added. The active ingredient may be added to the food ingredients either as is or after being prepared into a desired form, such as a solution. "Addition of an active ingredient" may refer collectively to the process of coexisting the active ingredient with the food ingredients. Ingredients other than the active ingredient may also be added to the food ingredients as appropriate. The description of the addition of an active ingredient also applies mutatis mutandis to the addition of ingredients other than the active ingredient. Each ingredient (i.e., the active ingredient and optionally other ingredients) may be added to the food ingredients all at the same time, separately, or in any combination. The order in which each ingredient is added to the food ingredients is not particularly limited.

[0068] The amount and ratio of each component (i.e., the active ingredient and optionally other ingredients) added in the method of the present invention are not particularly limited as long as the desired effects, such as egg white replacement effect, binding effect, emulsification effect, etc., are obtained. The amount and ratio of each component added in the method of the present invention can be appropriately set depending on various conditions, such as the type of raw material of the food and the type of food.

[0069] The active ingredient may be added to the ingredients of the food product so that the content of the active ingredient in the food product falls within a desired range (for example, the range of the content of the active ingredient described below).

[0070] The content of the active ingredient in the food product may be, for example, 0.05% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 15% (w / w) or more, or 20% (w / w) or more, or 30% (w / w) or less, 20% (w / w) or less, 15% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.5% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less, or any compatible combination thereof. The content of the active ingredient may be, for example, 0.05-0.1% (w / w), 0.1-0.2% (w / w), 0.2-0.5% (w / w), 0.5-1% (w / w), 1-2% (w / w), 2-5% (w / w), 5-10% (w / w), 10-15% (w / w), 15-20% (w / w), or 20-30% (w / w). The content of the active ingredient may be, for example, 0.05-30% (w / w), 0.1-20% (w / w), or 0.2-10% (w / w). When the food product of the present invention contains two or more serpins (e.g., when two or more serpins are added), the content of the two or more serpins may be set independently or in total within the range of the active ingredient content exemplified above. In addition, when the food of the present invention contains two or more types of serpins (for example, when two or more types of serpins are added), the ``content of active ingredient'' means the total content of those two or more types of serpins, unless otherwise specified.

[0071] The active ingredient may be added to the raw materials of the food product so that the ingestible concentration of the active ingredient falls within a desired range (for example, the range of ingestible concentration of the active ingredient described below).

[0072] The ingested concentration of the active ingredient may be, for example, 0.05% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 15% (w / w) or more, or 20% (w / w) or more, or 30% (w / w) or less, 20% (w / w) or less, 15% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.5% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less, or any compatible combination thereof. The ingested concentration of the active ingredient may be, for example, 0.05 to 0.1% (w / w), 0.1 to 0.2% (w / w), 0.2 to 0.5% (w / w), 0.5 to 1% (w / w), 1 to 2% (w / w), 2 to 5% (w / w), 5 to 10% (w / w), 10 to 15% (w / w), 15 to 20% (w / w), or 20 to 30% (w / w). The ingested concentration of the active ingredient may be, for example, 0.05 to 30% (w / w), 0.1 to 20% (w / w), or 0.2 to 10% (w / w). When the food product of the present invention contains two or more serpins (e.g., when two or more serpins are added), the ingestible concentrations of those two or more serpins, either individually or in total, may be set within the range of the ingestible active ingredient concentration exemplified above. Unless otherwise specified, when the food product of the present invention contains two or more serpins (e.g., when two or more serpins are added), the "ingestible active ingredient concentration" refers to the total ingestible concentration of those two or more serpins.

[0073] The description of the addition of an active ingredient also applies mutatis mutandis to the addition of the composition of the present invention. For example, the composition of the present invention can be added so as to obtain the amount of the active ingredient exemplified above.

[0074] In one embodiment, the food product of the present invention may be free of egg white, i.e., the food product of the present invention may be produced so as to be free of egg white.

[0075] In one embodiment, the food of the present invention may contain egg white. That is, the food of the present invention may be produced so as to contain egg white. The food containing egg white can be produced, for example, by adding egg white. That is, the method of the present invention may further comprise adding egg white to the raw materials of the food. The addition of egg white can be carried out in the same manner as the addition of the active ingredient. Furthermore, the food containing egg white can be produced, for example, using raw materials for foods containing egg white. That is, the raw materials of the food may contain egg white.

[0076] The food of the present invention is typically a food containing egg white, but may have a reduced egg white content. "Food with a reduced egg white content" refers to a food with a lower egg white content than normal (i.e., a food with a lower egg white content than normal egg white), and also includes foods that contain no egg white at all. Foods that contain a normal amount of egg white (i.e., a food with a normal egg white content) are also referred to as "normal egg white-containing foods." In other words, "food with a reduced egg white content" may specifically refer to foods of the same type as normal egg white-containing foods but with a lower egg white content than normal egg white-containing foods. More specifically, "foods with a reduced egg white content" may refer to foods of the same type as normal egg white-containing foods, but with an egg white content that is 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less of that of normal egg white-containing foods, or may also include foods that contain no egg white at all. Examples of foods with a reduced egg white content include foods that typically contain egg white, such as the foods exemplified above, but that are manufactured to have a lower egg white content than normal or to contain no egg white at all. Examples of normal egg white-containing foods include foods that contain egg white, such as the foods exemplified above, but that are manufactured to contain a normal amount of egg white. According to the present invention, for example, the reduced functionality of egg white due to the reduced egg white content may be compensated for. In other words, "egg white substitute" may also include compensating for the reduced functionality of egg white due to the reduced egg white content.

[0077] <4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the above-exemplified applications. That is, the present invention discloses, for example, use of the active ingredient as a substitute for egg white, binding food ingredients, emulsifying oils and fats in food ingredients, or for producing foods, or use of the active ingredient in producing a composition for use in a substitute for egg white, binding food ingredients, emulsifying oils and fats in food ingredients, or producing foods.

[0078] The present invention also discloses active ingredients for use in the above-exemplified applications. That is, the present invention discloses active ingredients for use, for example, as a substitute for egg white, binding food ingredients, emulsifying oils and fats in food ingredients, or in the production of foods, or active ingredients for use in the production of a composition for use in the substitution for egg white, binding food ingredients, emulsifying oils and fats in food ingredients, or in the production of foods.

[0079] The present invention will now be described more specifically with reference to the following non-limiting examples.

[0080] Example 1 Preparation of Serpins and Measurement of Elasticity (1-1) Expression and Purification of Serpins Serpins selected were Arabidopsis thaliana-derived serpin (AtSerpin), Brassica napus-derived serpin (BnSerpin), cacao Theobroma cacao-derived serpin (TcSerpin), castor bean (Ricinus communis)-derived serpin (RcSerpin), sunflower (Helianthus annuus)-derived serpin (HaSerpin), tea plant (Camellia sinensis var. sinensis)-derived serpin (CsSerpin), and carrot (Daucus carota subsp. sativus)-derived serpin (DcSerpin).

[0081] The method for preparing Serpin (AtSerpin) derived from Arabidopsis thaliana (Ath) is described below.

[0082] DNA containing the nucleotide sequence encoding AtSerpin was obtained by total synthesis and inserted into the NdeI-HindIII restriction enzyme site of pET-21b to construct the AtSerpin expression plasmid pET-AtSerpin (outsourced to Eurofins Genomics). The nucleotide sequence encoding AtSerpin and the amino acid sequence of AtSerpin are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The nucleotide sequence of pET-21b(+) is shown in SEQ ID NO: 19. The histidine tag derived from pET-21b(+) is attached to AtSerpin expressed from pET-AtSerpin.

[0083] The AtSerpin expression plasmid, pET-AtSerpin, was introduced into E. coli BL21(DE3) (Sigma-Aldrich) to obtain the BL21(DE3) / pET-AtSerpin strain. Heat shock transformation was performed as described in the protocol. This strain was inoculated into LB medium (1.0% (w / v) peptone, 0.50% (w / v) yeast extract, 1.0% (w / v) NaCl) supplemented with 100 μg / mL ampicillin and cultured overnight with shaking at 37°C. The culture was then inoculated into 100 mL of LB medium in a Sakaguchi flask to a final concentration of 1%. After 3 hours of shaking culture at 37°C, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM, and the culture was continued for an additional 2 hours.

[0084] The entire culture medium was centrifuged at 9,100 × g for 5 minutes to collect the bacterial cells, which were then washed twice with 10 mL of 10 mM Tris-HCl (pH 8.0) buffer and suspended in 50 mL of 1% NaCl solution. The resulting suspension was disrupted using an ultrasonic homogenizer (Kubota Shoji) at 170 W, 4°C, for 10 minutes, and then centrifuged at 9,100 × g for 10 minutes to obtain the precipitated fraction.

[0085] The resulting precipitated fraction was an inclusion body containing insolubilized AtSerpin within E. coli cells. 50 mL of 10 mM Tris-HCl (pH 8.0), 8 M urea, and 5 mM DTT was added as a solubilization solution, and the mixture was gently stirred at room temperature for 1 hour to obtain a solubilized solution.

[0086] 45 mL of the resulting lysate was applied to a column (Ni Sepharose Fast Flow 16 / 10, 20 mL, Cytiva) equilibrated with 10 mM Tris (pH 8.0), 160 mM urea, and 5.0 mM imidazole. The column was then washed with 9 volumes of equilibration buffer. An elution buffer containing 500 mM imidazole and 8 M urea was then applied. The fractions containing AtSerpin were collected and collected. The purified AtSerpin (36 mL) was obtained at approximately 1.0 mg / mL.

[0087] The purified AtSerpin was loaded onto 324 mL of Sephadex G-25 HiScale 26 / 20 and desalted. The Sephadex G-25 was pre-equilibrated with 1 mM Tris-HCl (pH 8.0) equilibration solution (3 times the column resin volume). After loading the purified AtSerpin, 5 times the column resin volume of equilibration solution was passed through the column. The fractions containing AtSerpin were collected, and the resulting 100 mL fraction (approximately 0.3 mg / mL) of desalted AtSerpin was used.

[0088] The resulting desalted AtSerpin was concentrated using a UF cartridge Amicon Ultra 15, 10 kDa (Millipore) to obtain approximately 10 mL of concentrated AtSerpin.

[0089] The solvent was removed from the resulting concentrated AtSerpin using a freeze dryer Genesis 25EL (SP Industries) to obtain dry AtSerpin.

[0090] Using a similar procedure, expression plasmids for BnSerpin, TcSerpin, RcSerpin, HaSerpin, DcSerpin, and CsSerpin were constructed, and heterologous expression was performed in E. coli to obtain dried BnSerpin, dried TcSerpin, dried RcSerpin, dried HaSerpin, dried CsSerpin, and dried DcSerpin. The nucleotide sequence encoding BnSerpin and the amino acid sequence of BnSerpin are shown in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequence encoding TcSerpin and the amino acid sequence of TcSerpin are shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequence encoding RcSerpin and the amino acid sequence of RcSerpin are shown in SEQ ID NOs: 7 and 8, respectively. The nucleotide sequence encoding HaSerpin and the amino acid sequence of HaSerpin are shown in SEQ ID NOs: 13 and 14, respectively. The nucleotide sequence encoding CsSerpin and the amino acid sequence of CsSerpin are shown in SEQ ID NOs: 15 and 16, respectively. The nucleotide sequence encoding DcSerpin and the amino acid sequence of DcSerpin are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0091] (1-2) Measurement of Elasticity of Serpin When Heated The elasticity of the Serpin obtained in Example 1 when heated was measured.

[0092] Each dried Serpin was dissolved in water at a concentration of 12% (wt / v) or 15% (wt / v) to prepare a Serpin solution. The solution was placed directly below a 40mm SMART-SWAP HRx0 parallel plate (TA Instruments) on the measurement platform of a Discovery HR20 rheometer (TA Instruments). The parallel plate was then lowered vertically toward the sample, and the sample was heated from 25°C to 100°C (heating rate: 5°C / min) under the following conditions: strain 1%, frequency 1 Hz, and storage modulus analysis.

[0093] The results are shown in Table 1. All Serpins gelled and exhibited elasticity.

[0094]

[0095] (Example 2) Preparation of pate-like foods and measurement of their breaking strength and emulsifying power (2-1) Preparation of pate-like foods Hereinafter, methods for preparing an additive-free pate-like food, an egg white-containing pate-like food, and a Serpin-containing pate-like food will be described.

[0096] A reconstitution solution was prepared by mixing vegetable oil and water. Next, New Fujinic (registered trademark) 80N and Apex (registered trademark) 350 (both manufactured by Fuji Oil Co., Ltd.) were reconstituted with the reconstitution solution, and then mixed in a ratio of 9:4 (New Fujinic (registered trademark) 80N:Apex (registered trademark) 350) to prepare granular soy protein. Furthermore, Apex (registered trademark) 950 (manufactured by Fuji Oil Co., Ltd.) was reconstituted with the reconstitution solution, and then crushed into flakes using a blender to prepare fibrous soy protein. The granular soy protein and fibrous soy protein were mixed to prepare a base protein, and then the base protein was mixed with methylcellulose powder (additive-free pate-like food), methylcellulose powder and egg white powder (egg white-containing pate-like food), or methylcellulose powder and each dried Serpin obtained in Example 1 (Serpin-containing pate-like food) as a binder, followed by further mixing with oils and seasonings to prepare each pate-like food. Each pate-like food was molded, baked at 180°C, flash-frozen, and placed in a vacuum pouch for frozen storage. The raw materials and their composition ratios for each pate-like food are shown in Table 2.

[0097]

[0098] (2-2) Measurement of Breaking Strength of Putty-Like Food The breaking strength of each putty-like food obtained in (2-1) was measured using a Texture Analyzer (TA) (TA-XT-plus, manufactured by Stable Microsystems). Specifically, each putty-like food was placed horizontally and broken using a wedge-shaped plunger to measure stress (g).

[0099] The measured values ​​of the breaking strength of each pate-like food are shown in Table 3. Each Serpin-containing pate-like food showed the same breaking strength as the egg white-containing pate-like food, regardless of which Serpin was used.

[0100]

[0101] (2-3) Measurement of the emulsifying effect of each Serpin: 5 g of each dried Serpin obtained in Example 1, casein (manufactured by Junsei Chemical Co., Ltd.), bovine serum albumin (manufactured by Sigma-Aldrich), or egg white albumin (manufactured by Sigma-Aldrich) was dissolved in 100 g of water as an emulsifier. 100 g of canola oil (manufactured by J-Oil Mills Co., Ltd.) was added thereto and mixed with a mixer for 1 minute to prepare an emulsion. The prepared emulsion was slowly transferred to a 500 mL graduated cylinder and allowed to stand at room temperature for 1 hour. After 1 hour, the height of the entire emulsion (HE) and the height of the clear layer (HS) were measured. The emulsion was then allowed to stand at 4°C for 24 hours, and the HE and HS were measured again. The creaming index (CI) was calculated based on the measured HE and HS values ​​using the following formula:

[0102] Creaming Index (CI) = HS / HE

[0103] The calculated creaming index (CI) for each sample is shown in Table 4. Each Serpin exhibited a creaming index (CI) equal to or greater than that of casein.

[0104]

[0105] <Sequence Listing> SEQ ID NO: 1: Nucleotide sequence encoding serpin (AtSerpin) derived from Arabidopsis thaliana 2: Amino acid sequence of serpin (AtSerpin) derived from Arabidopsis thaliana 3: Nucleotide sequence encoding serpin (BnSerpin) derived from rapeseed (Brassica napus) 4: Amino acid sequence of serpin (BnSerpin) derived from rapeseed (Brassica napus) 5: Nucleotide sequence encoding serpin (TcSerpin) derived from cacao (Theobroma cacao) 6: Amino acid sequence of serpin (TcSerpin) derived from cacao (Theobroma cacao) 7: Nucleotide sequence encoding serpin (RcSerpin) derived from castor bean (Ricinus communis) 8: Nucleotide sequence encoding serpin (RcSerpin) derived from castor bean (Ricinus communis) 9: Nucleotide sequence encoding serpin (BrSerpin) derived from Chinese cabbage (Brassica rapa subsp. pekinensis) 10: Amino acid sequence of serpin (BrSerpin) derived from Chinese cabbage (Brassica rapa subsp. pekinensis) 11: Nucleotide sequence encoding serpin (RsSerpin) derived from radish (Raphanus sativus) 12: Amino acid sequence of serpin (RsSerpin) derived from radish (Raphanus sativus) 13: Nucleotide sequence encoding serpin (HaSerpin) derived from sunflower (Helianthus annuus) 14: Amino acid sequence of serpin (HaSerpin) derived from sunflower (Helianthus annuus) 15: Nucleotide sequence encoding serpin (CsSerpin) derived from tea plant (Camellia sinensis var. sinensis) 16: Amino acid sequence of Serpin (CsSerpin) derived from tea plant (Camellia sinensis var. sinensis) 17: Amino acid sequence of Serpin (CsSerpin) derived from carrot (Daucus carota subsp.18: Amino acid sequence of serpin (DcSerpin) derived from carrot (Daucus carota subsp. sativus). 19: Nucleotide sequence of pET-21b(+).

Claims

1. A composition containing a serpin derived from a plant or fungus, the composition being used for replacing egg white, binding food ingredients, emulsifying fats and oils in food ingredients, or for producing food.

2. The composition of claim 1, wherein the serpin is derived from a plant of the Brassicaceae family, a plant of the Malvaceae family, a plant of the Euphorbiaceae family, a plant of the Asteraceae family, a plant of the Theaceae family, or a plant of the Apiaceae family.

3. The composition according to claim 1 or 2, wherein the serpin is derived from a plant of the genus Arabidopsis, a plant of the genus Brassica, a plant of the genus Theobroma, a plant of the genus Ricinus, a plant of the genus Helianthus, a plant of the genus Camellia, or a plant of the genus Daucus.

4. The composition of claim 1 or 2, wherein the serpin is derived from Arabidopsis thaliana, Brassica napus, Theobroma cacao, Ricinus communis, Helianthus annuus, Camellia sinensis var. sinensis, or Daucus carota subsp. sativus.

5. The composition according to claim 1 or 2, wherein the serpin is a protein described in (a), (b), or (c) below: (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18; (b) a protein comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, but with a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and having an egg white replacement function, a binding function, or an emulsification function; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having an egg white replacement function, a binding function, or an emulsification function.

6. The composition according to claim 1 or 2, wherein the serpin has a molecular weight of 40,000 to 50,000.

7. The composition of claim 1 or 2, wherein the serpin has one or more properties selected from the following (1) to (4): (1) the ratio of Ser residues in the amino acid sequence is 7.5% or more; (2) the isoelectric point (pI) is 4 to 7; (3) the internal hydrophobicity is 1.5 or more; (4) the predicted scaled solubility is 0.45 or more.

8. A method for producing a food product, comprising the step of adding a serpin derived from a plant or fungus to an ingredient of the food product.

9. The method of claim 8, wherein the food produced is a food in which egg white has been replaced, a food ingredient has been bound, and / or fats and oils in a food ingredient have been emulsified.

10. A method for replacing egg white in a food product, comprising the step of adding a serpin derived from a plant or fungus to an ingredient of the food product.

11. A method for binding food materials, comprising the step of adding a serpin derived from a plant or fungus to the food raw material.

12. A method for emulsifying fats and oils in a food material, comprising the step of adding a serpin derived from a plant or fungus to the food raw material.

13. The method of any one of claims 8 to 12, wherein the serpin is derived from a plant of the Brassicaceae family, a plant of the Malvaceae family, a plant of the Euphorbiaceae family, a plant of the Asteraceae family, a plant of the Theaceae family, or a plant of the Apiaceae family.

14. The method according to any one of claims 8 to 12, wherein the serpin is derived from a plant of the genus Arabidopsis, a plant of the genus Brassica, a plant of the genus Theobroma, a plant of the genus Ricinus, a plant of the genus Helianthus, a plant of the genus Camellia, or a plant of the genus Daucus.

15. The method of any one of claims 8 to 12, wherein the serpin is derived from Arabidopsis thaliana, Brassica napus, Theobroma cacao, Ricinus communis, sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), or carrot (Daucus carota subsp. sativus).

16. The method according to any one of claims 8 to 12, wherein the serpin is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18; (b) a protein comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, but with a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and having an egg white replacement function, a binding function, or an emulsification function; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having an egg white replacement function, a binding function, or an emulsification function.

17. The method according to any one of claims 8 to 12, wherein the serpin has a molecular weight of 40,000 to 50,000.

18. The method according to any one of claims 8 to 12, wherein the serpin has one or more properties selected from the following (1) to (4): (1) the ratio of Ser residues in the amino acid sequence is 7.5% or more; (2) the isoelectric point (pI) is 4 to 7; (3) the internal hydrophobicity is 1.5 or more; (4) the predicted scaled solubility is 0.45 or more.

19. The method according to any one of claims 8 to 12, wherein the serpin is added to an intake concentration of 0.05 to 30% (w / w).

20. Foods containing serpins derived from plants or fungi.

21. The food product of claim 20, wherein the serpin is derived from a plant of the Brassicaceae family, a plant of the Malvaceae family, a plant of the Euphorbiaceae family, a plant of the Asteraceae family, a plant of the Theaceae family, or a plant of the Apiaceae family.

22. The food according to claim 20 or 21, wherein the serpin is derived from a plant of the genus Arabidopsis, a plant of the genus Brassica, a plant of the genus Theobroma, a plant of the genus Ricinus, a plant of the genus Helianthus, a plant of the genus Camellia, or a plant of the genus Daucus.

23. The food product of claim 20 or 21, wherein the serpin is derived from Arabidopsis thaliana, Brassica napus, Theobroma cacao, Ricinus communis, sunflower (Helianthus annuus), tea plant (Camellia sinensis var. sinensis), or carrot (Daucus carota subsp. sativus).

24. The food according to claim 20 or 21, wherein the serpin is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18; (b) a protein comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, but with a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and having an egg white replacement function, a binding function, or an emulsification function; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 14, 16, or 18, and having an egg white replacement function, a binding function, or an emulsification function.

25. The food according to claim 20 or 21, wherein the serpin has a molecular weight of 40,000 to 50,000.

26. The food of claim 20 or 21, wherein the serpin has one or more properties selected from the following (1) to (4): (1) the ratio of Ser residues in the amino acid sequence is 7.5% or more; (2) the isoelectric point (pI) is 4 to 7; (3) the internal hydrophobicity is 1.5 or more; (4) the predicted scaled solubility is 0.45 or more.

27. The food according to claim 20 or 21, wherein the content of the serpin is 0.05 to 30% (w / w).

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