Saltiness-enhancing oligopeptide

Abstract

The purpose of the present invention is to provide a component capable of enhancing saltiness. Provided is an oligopeptide that is a degradation product of γ-glutamyl bonds and peptide bonds of a pea protein and has a molecular weight of 900-1000 Da, specifically an oligopeptide that is obtained by causing glutaminases and proteases to act on a pea protein, that has a molecular weight of 900-1000 Da, and that can enhance saltiness.

Description

Salt-enhancing oligopeptide

[0001] The present invention relates to salt-enhancing oligopeptides.

[0002] In recent years, in the food market, there has been a demand for replacing animal-derived foods with plant-derived foods, not only due to the inclinations of some consumers such as vegetarians or vegans, but also due to growing awareness of health, dieting, environmental issues, animal welfare, etc. Furthermore, with the rise in health and diet awareness, there is also a growing need to reduce salt content.

[0003] On the other hand, even for health foods, various attempts have been made to process them to improve their sensory characteristics such as taste, flavor, aroma, etc. For example, Patent Document 1 describes that the body, umami, and savory flavor are improved by allowing glutaminase derived from bacteria of the genus Bacillus, protease derived from filamentous fungi, and protease derived from the genus Bacillus or Geobacillus to act on a protein raw material.

[0004] International Publication No. 2022 / 191303

[0005] Glutamyl peptide hydrolases and proteases can produce glutamic acid, a savory component, from proteins, thereby altering taste, such as enhancing umami. However, it is not known whether protein processing can produce components that can enhance saltiness.

[0006] Therefore, an object of the present invention is to provide a component that can enhance the salty taste.

[0007] As a result of extensive research, the present inventors have found that oligopeptides of a specific molecular weight fraction, which are obtained by treating pea protein with glutaminase and protease, have the property of enhancing salty taste. The present invention was completed based on this finding and through further research.

[0008] That is, the present invention provides the following aspects. Item 1. An oligopeptide having a molecular weight of 900 to 1000 Da, which is a degradation product of pea protein at γ-glutamyl bonds and peptide bonds. Item 2. An oligopeptide having a molecular weight of 900 to 1000 Da, which is obtainable by allowing at least one glutaminase and a protease to act on pea protein. Item 3. The oligopeptide according to Item 2, wherein the glutaminase is glutaminase (EC 3.5.1.2). Item 4. The oligopeptide according to Item 2 or 3, wherein the protease includes a bacterial protease. Item 5. The oligopeptide according to any one of Items 2 to 4, wherein the protease is a filamentous fungal protease and a bacterial protease. Item 6. Item 4 or 5, wherein the bacterial protease is a protease derived from a bacterium of the genus Bacillus or Geobacillus. Item 7. The oligopeptide according to Item 5 or 6, wherein the amount of the filamentous fungal protease used per 1 U of the glutaminases is 1,000 U or more. Item 8. The oligopeptide according to any of Items 4 to 7, wherein the amount of the bacterial protease used per 1 U of the glutaminases is 500 U or more. Item 9. The oligopeptide according to any of Items 5 to 8, wherein the amount of the bacterial protease used per 1 U of the filamentous fungal protease is 0.01 to 2 U. Item 10. A method for producing an oligopeptide, comprising an enzyme treatment step of obtaining an enzyme-treated product of pea protein with glutaminases and proteases, and a fractionation step of obtaining a fraction containing an oligopeptide having a molecular weight of 900 to 1,000 Da. Item 11. A salty taste enhancer comprising the oligopeptide according to any of Items 1 to 9. Item 12. A structured vegetable protein material comprising the oligopeptide according to any one of Items 1 to 9.

[0009] According to the present invention, there is provided an oligopeptide that can enhance salty taste.

[0010] 1. Saltiness-Enhancing Oligopeptides The oligopeptides of the present invention are oligopeptides having a molecular weight of 900 to 1,000 Da that are degradation products of pea protein at γ-glutamyl bonds and peptide bonds. Specifically, the oligopeptides of the present invention are oligopeptides having a molecular weight of 900 to 1,000 Da that are obtainable by treating pea protein with glutaminase and protease. More specifically, the oligopeptides of the present invention are obtainable by a production method that includes an enzyme treatment step of obtaining a product of pea protein treated with glutaminase and protease, and a fractionation step of obtaining oligopeptides having a molecular weight of 900 to 1,000 Da. That is, the oligopeptides of the present invention are oligopeptides having a molecular weight of 900 to 1,000 Da that are separated from a product of pea protein treated with glutaminase and protease. This oligopeptide has the property of enhancing the saltiness.

[0011] 1-1. Enzyme Treatment Step In the enzyme treatment step, a product of enzyme treatment of pea protein with at least one type of glutaminase and a protease is obtained.

[0012] 1-1-1. Specific Embodiments Specific embodiments of the enzyme treatment step include the following: [A] An embodiment in which glutaminase and protease are allowed to act on a pea protein-containing composition (a) to obtain an enzyme-treated product; [B] An embodiment in which at least glutaminase is allowed to act on a pea protein-containing composition (b) that has been treated with a protease to obtain an enzyme-treated product; and [C] An embodiment in which protease is allowed to act on a pea protein-containing composition (c) that has been treated with glutaminase to obtain an enzyme-treated product.

[0013] The above-mentioned embodiment [B] further includes the following embodiments: [B1] An embodiment in which an enzyme-treated product is obtained by allowing glutaminase or similar compounds to act on a pea protein-containing composition (b1) that has been treated with a protease derived from a filamentous fungus and a protease derived from a bacteria; [B2] An embodiment in which an enzyme-treated product is obtained by allowing glutaminase or similar compounds and a protease derived from a bacteria to act on a pea protein-containing composition (b2) that has been treated with a protease derived from a filamentous fungus; and [B3] An embodiment in which an enzyme-treated product is obtained by allowing glutaminase or similar compounds and a protease derived from a filamentous fungus to act on a pea protein-containing composition (b3) that has been treated with a protease derived from a bacteria.

[0014] Hereinafter, these pea protein-containing compositions (a), (b) (including (b1) to (b3); the same applies below), and (c) will also be collectively referred to as "pea protein-containing compositions". Furthermore, the proteases used in the preparation of the pea protein-containing composition (b) and the treatment therewith, and the glutaminases used in the preparation of the pea protein-containing composition (c) and the treatment therewith will also be referred to as "pre-treatment enzymes" and "pre-enzyme treatments", respectively.

[0015] 1-1-2. Pea Protein-Containing Composition Specific properties of the pea protein-containing composition that is the raw material for the oligopeptide of the present invention include liquid, slurry, paste, etc. (hereinafter, these properties will be collectively referred to as "liquid, etc.").

[0016] Specific examples of pea protein-containing compositions include the following: (i) a liquid in which crushed pea protein is dispersed in water; (ii) a liquid obtained by prior enzymatic treatment of the liquid described in (i); (iii) a liquid obtained by removing at least one component other than pea protein (which also includes pea protein hydrolysates in the case of the liquid described in (ii)) from the liquid described in (i) or (ii) above, thereby increasing the proportion of the pea protein content (which also includes the content of pea protein hydrolysates; the same applies hereinafter) per dry weight equivalent of the liquid; (iv) a liquid obtained by prior enzymatic treatment of the liquid described in (iii) above; (v) a liquid obtained by mixing water with a dry powder prepared by removing water from the liquids described in (i) to (iv) above; (vi) a liquid obtained by prior enzymatic treatment of the liquid described in (v) above. The content of pea protein in (iii) above is 60% by weight or more, preferably 70% by weight or more, and more preferably 75% by weight or more, based on the dry weight of the liquid or the like.

[0017] The content of pea protein in the pea protein-containing composition is not particularly limited and is set appropriately depending on the properties, and is therefore widely acceptable in the range of, for example, 0.01% by weight or more and 98% by weight or less. For example, the pea protein content may be 0.01% by weight or more, preferably 0.02% by weight or more, more preferably 0.1% by weight or more or 0.5% by weight or more, even more preferably 1% by weight or more or 5% by weight or more, and even more preferably 8% by weight or more, and may be, for example, 40% by weight or less, 30% by weight or less, 20% by weight or less, or 12% by weight or less. Specific ranges for the content of pea protein in the pea protein-containing composition include 0.01 to 98% by weight, preferably 0.02 to 98% by weight, more preferably 0.1 to 98% by weight, or 0.5 to 98% by weight, even more preferably 1 to 98% by weight, or 5 to 98% by weight, and even more preferably 8 to 98% by weight, 8 to 40% by weight, 8 to 30% by weight, 8 to 20% by weight, or 8 to 12% by weight.

[0018] The pea protein-containing composition may or may not contain any other components in addition to the pea protein and water. These other components include pea-derived components (e.g., lipids, carbohydrates, etc.) and food additives. Examples of food additives include thickeners, binders, seasonings, pH adjusters, buffers, colorants, flavors, etc.

[0019] Details of the enzymes used in the enzyme treatment step, specifically the enzymes used in the treatment of the pea protein-containing composition (a), as well as the pre-treatment enzymes used in the preparation of the pea protein-containing compositions (b) and (c) and the enzymes used in the treatment of the pea protein-containing compositions (b) and (c), are as follows:

[0020] 1-1-3-1. Glutaminases Glutaminase is a general term for enzymes that have the activity of hydrolyzing γ-glutamyl bonds, and is not particularly limited as long as it has this activity. Therefore, glutaminases may be either those that use free glutamine as a substrate or those that use glutamine residues as a substrate, and may be either those that do not have transferase activity or those that have transferase activity. Examples of glutaminases that can be used in the present invention include glutaminase (EC 3.5.1.2; glutaminase; an enzyme that hydrolyzes free glutamine to glutamic acid), protein glutaminase (EC 3.5.1.44; proteininglutaminase; an enzyme that deamidates glutamine residues in proteins or peptides), and transglutaminase (EC 2.3.2.13; transglutaminase; an enzyme that transfers glutamine residues in proteins or peptides to other amino acid residues). In the present invention, one of these enzymes may be used alone as the glutaminase, or multiple types may be used in combination. Among these glutaminases, glutaminase (EC 3.5.1.2; glutaminase; an enzyme that hydrolyzes free glutamine to glutamic acid) is preferred from the viewpoint of further enhancing the salty taste enhancing effect.

[0021] Glutaminase (EC 3.5.1.2; glutaminase; an enzyme that hydrolyzes free glutamine to glutamic acid) is not particularly limited in terms of its origin, but from the viewpoint of further enhancing the salty taste enhancing effect, glutaminase derived from a microorganism is preferred, glutaminase derived from the genus Bacillus or Geobacillus is more preferred, and glutaminase derived from Bacillus amyloliquefaciens is even more preferred.

[0022] When using the above-mentioned microbial glutaminase (EC 3.5.1.2), a culture solution, a disruption solution, or an extract of a microorganism from which the glutaminase is derived or a heterologous host into which a gene encoding the above-mentioned microbial glutaminase has been introduced may be used, or a purified product obtained by purifying these to an arbitrary level to increase the concentration of glutaminase may be used. Furthermore, when using glutaminase, a commercially available enzyme preparation may be used.

[0023] The amount of glutaminase used is not particularly limited, but may be, for example, 0.001 U or more per 1 g of pea protein. From the viewpoint of further enhancing the salty taste enhancing effect, the amount of glutaminase used per 1 g of pea protein is preferably 0.005 U or more, more preferably 0.01 U or more, 0.05 U or more, 0.1 U or more, 0.3 U or more, 0.4 U or more, 0.5 U or more, or 0.6 U or more, even more preferably 0.7 U or more, even more preferably 0.8 U or more, and still more preferably 0.9 U or more. The upper limit of the amount of glutaminase used is not particularly limited, but may be, for example, 100 U or less, or 50 U or less, preferably 10 U or less, more preferably 8 U or less, 6 U or less, 4 U or less, 3 U or less, 2 U or less, or 1.5 U or less per 1 g of pea protein. Specific ranges of the amount of glutaminases to be used per gram of pea protein include 0.001 to 100 U, preferably 0.005 to 100 U, more preferably 0.01 to 50 U, 0.05 to 50 U, 0.1 to 50 U, 0.3 to 10 U, 0.4 to 8 U, 0.5 to 6 U, or 0.6 to 4 U, even more preferably 0.7 to 3 U, even more preferably 0.8 to 2 U, and even more preferably 0.9 to 1.5 U.

[0024] For glutaminase (EC 3.5.1.2) activity, 1 unit (1 U) is the amount of enzyme that produces 1 μmol of L-glutamic acid per minute using L-glutamine as a substrate. For protein glutaminase (EC 3.5.1.44) activity, 1 unit (1 U) is the amount of enzyme that liberates 1 μmol of ammonia per minute using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as a substrate. For transglutaminase (EC 2.3.2.13) activity, 1 unit (U) is the enzyme activity that produces 1 μmol of hydroxamic acid per minute using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates.

[0025] 1-1-3-2. The protease used in the present invention is an endopeptidase. The protease is not particularly limited, but preferred examples include proteases derived from filamentous fungi and proteases derived from bacteria. These proteases may be used alone or in combination of two or more.

[0026] 1-1-3-2 (1) Proteases derived from filamentous fungi Proteases derived from filamentous fungi are endopeptidases originating from filamentous fungi. Specific examples of proteases derived from filamentous fungi include proteases derived from the genera Aspergillus, Rhizopus, Mucor, Neurospora, Penicillium, Rhizomucor, and Sclerotinia. Examples of proteases derived from the genus Aspergillus include proteases derived from Aspergillus oryzae and proteases derived from Aspergillus melleus. Furthermore, proteases derived from filamentous fungi may be acidic proteases or neutral proteases. More specific examples of proteases derived from filamentous fungi include acidic proteases derived from Aspergillus oryzae, neutral proteases derived from Aspergillus oryzae, and neutral proteases derived from Aspergillus melleus.

[0027] These filamentous fungal proteases may be used singly or in combination of two or more. Among these filamentous fungal proteases, from the viewpoint of further enhancing the salty taste enhancing effect, proteases derived from the genus Aspergillus are preferred, proteases derived from Aspergillus oryzae are more preferred, and acidic proteases derived from Aspergillus oryzae are even more preferred.

[0028] When using a protease derived from a filamentous fungus, a culture solution, a lysate, or an extract of the above-mentioned filamentous fungus or a heterologous host into which a gene encoding the above-mentioned protease derived from a filamentous fungus has been introduced may be used, or a purified product obtained by purifying these to an arbitrary level to increase the concentration of the protease may be used. Furthermore, when using a protease derived from a filamentous fungus, a commercially available enzyme preparation may be used.

[0029] The amount of filamentous fungal protease used is not particularly limited, but can be 1000 U or more of filamentous fungal protease per 1 U of the glutaminases. From the viewpoint of further enhancing the salty taste enhancing effect, the amount is preferably 1500 U or more, more preferably 2000 U or more, and even more preferably 2500 U or more. The upper limit of the amount of filamentous fungal protease used per 1 U of the glutaminases is also not particularly limited, but examples include ratios of 4000 U or less, preferably 3500 U or less, more preferably 3000 U or less, and even more preferably 2800 U or less. Specific ranges of the amount of filamentous fungal protease used per 1 U of glutaminases include 1000 to 4000 U, preferably 1500 to 3500 U, more preferably 2000 to 3000 U, and even more preferably 2500 to 2800 U.

[0030] Specific examples of the amount of the filamentous fungus-derived protease used per 1 g of pea protein include, for example, at least 10 U or at least 50 U, preferably at least 100 U or at least 500 U, more preferably at least 1000 U or at least 2000 U, and even more preferably at least 2500 U. There are no particular limitations on the upper limit of the amount of the filamentous fungus-derived protease used per 1 g of pea protein, and examples include 50,000 U or less, 20,000 U or less, 10,000 U or less, 8,000 U or less, 5,000 U or less, 4,000 U or less, or 3,500 U or less. Specific ranges of the amount of the filamentous fungus-derived protease to be used per gram of pea protein include 10 to 50,000 U or 50 to 20,000 U, preferably 100 to 10,000 U or 500 to 8,000 U, more preferably 1,000 to 5,000 U or more or 2,000 to 4,000 U, and even more preferably 2,500 to 3,500 U or more.

[0031] Regarding the activity of the fungal protease, one unit (1 U) is defined as the amount of enzyme that causes an increase in the Folin test solution color substance equivalent to 1 μg of tyrosine per minute using casein as a substrate.

[0032] 1-1-3-3 (2) Bacterial proteases Bacterial proteases are endopeptidases originating from bacteria. Specific examples of bacterial proteases include proteases derived from the genus Bacillus and Geobacillus, and more specifically, proteases derived from Bacillus amyloliquefaciens, Bacillus cereus, Bacillus clausii, Bacillus intermedius, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus spp. Examples of proteases that can be used include proteases derived from Geobacillus sp., Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thermoproteolyticus, and proteases derived from these Geobacillus sp.

[0033] These proteases derived from the genus Bacillus and Geobacillus may be used singly or in combination of two or more. Among these proteases derived from the genus Bacillus and Geobacillus, Bacillus stearothermophilus and Geobacillus stearothermophilus are preferred from the viewpoint of further enhancing the salty taste enhancing effect.

[0034] When using bacterial proteases, the culture medium, lysate, or extract of the above-mentioned bacteria or a heterologous host into which a gene encoding the above-mentioned bacterial protease has been introduced may be used, or a purified product obtained by purifying these to an arbitrary level to increase the concentration of the protease may be used. Furthermore, when using bacterial proteases, commercially available enzyme preparations may be used.

[0035] The amount of bacterial protease used is not particularly limited, but can be 500 U or more per 1 U of the glutaminases. From the viewpoint of further enhancing the salty taste enhancing effect, the bacterial protease can be used at a ratio of preferably 700 U or more, more preferably 900 U or more, and even more preferably 1000 U or more. The upper limit of the range of the amount of bacterial protease used per 1 U of the glutaminases is also not particularly limited, but examples include ratios of 2000 U or less, preferably 1700 U or less, more preferably 1400 U or less, and even more preferably 1200 U or less. Specific ranges of the amount of bacterial protease used per 1 U of glutaminases include 500 to 2000 U, preferably 700 to 1700 U, more preferably 900 to 1400 U, and even more preferably 1000 to 1200 U.

[0036] A specific amount of bacterial protease to be used per 1 g of pea protein is, for example, 10 U or more, preferably 50 U or more, more preferably 100 U or more, even more preferably 500 U or more, and even more preferably 900 U or more. The upper limit of the amount of bacterial protease to be used per 1 g of pea protein is not particularly limited, and examples include 30,000 U or less, 20,000 U or less, 10,000 U or less, 8,000 U or less, 5,000 U or less, 3,000 U or less, 2,000 U or less, 1,500 U or less, or 1,200 U or less. Specific ranges of the amount of bacterial protease to be used per gram of pea protein include 10 to 30,000 U, preferably 50 to 30,000 U, more preferably 100 to 30,000 U, even more preferably 500 to 30,000 U, and even more preferably 900 to 30,000 U, 900 to 20,000 U, 900 to 10,000 U, 900 to 8,000 U, 900 to 5,000 U, 900 to 3,000 U, 900 to 2,000 U, 900 to 1,500 U, or 900 to 1,200 U.

[0037] The amount of bacterial protease used per 1 U of filamentous fungal protease is, for example, 0.01 to 2 U, preferably 0.05 to 1 U, more preferably 0.1 to 0.7 U, even more preferably 0.2 to 0.6 U, and even more preferably 0.3 to 0.5 U.

[0038] Regarding the activity of bacterial protease, one unit (1 U) is defined as the amount of enzyme that causes an increase in the color substance of Folin's test solution equivalent to 1 μg of tyrosine per minute using casein as a substrate.

[0039] 1-1-4. Enzyme Treatment Reaction The enzyme treatment reaction can usually be carried out by subjecting a reaction mixture containing the protein-containing composition and an enzyme to the reaction conditions of the enzyme. The combination of the protein-containing composition and the enzyme is determined depending on the above-mentioned aspects [A] to [C], and in the case of aspect [B], depending on the aspects [B1] to [B3]. This allows the reaction to proceed, producing the salty taste-enhancing oligopeptide.

[0040] When two or more enzymes are used in the treatment of the protein-containing composition, the enzyme treatments with the two or more enzymes may be carried out simultaneously or sequentially. In a preferred embodiment, the enzyme treatment reaction involves simultaneously acting a glutaminase and a protease on the pea protein-containing composition (a) to obtain an enzyme-treated product.

[0041] The reaction conditions (temperature and pH) can be appropriately set depending on the thermal and pH characteristics of the enzyme used. Specific examples of the reaction temperature include, for example, 10 to 90°C, preferably 20 to 80°C, more preferably 25 to 70°C, even more preferably 30 to 65°C, even more preferably 35 to 60°C, and even more preferably 40 to 55°C or 45 to 55°C. Specific examples of the pH (25°C) of the mixture of the protein-containing composition and the enzyme include, for example, 3 to 9, preferably 4 to 8, more preferably 4.5 to 7.5, even more preferably 5 to 7, and even more preferably 5.5 to 6.5. The reaction time can be appropriately set through preliminary experiments depending on the reaction scale and / or the desired yield of the salty taste-enhancing oligopeptide, and can be, for example, 30 seconds to 48 hours, preferably 1 minute to 24 hours, more preferably 5 minutes to 12 hours, even more preferably 10 minutes to 6 hours, even more preferably 15 minutes to 2 hours, and even more preferably 20 minutes to 1 hour.

[0042] 1-2. Separation Step In the separation step, a fraction containing an oligopeptide having a molecular weight of 900 to 1000 Da (salty-enhancing oligopeptide) is obtained from the enzyme-treated product obtained in the enzyme treatment step. In the separation step, at least one of proteins, peptides, oligopeptides, and amino acids other than the salty-enhancing oligopeptide is removed from the enzyme-treated product, so that the content of the salty-enhancing oligopeptide per dry weight of the fraction can be increased to be higher than the content of the salty-enhancing oligopeptide per dry weight of the enzyme-treated product.

[0043] Fractions containing salty taste-enhancing oligopeptides can be obtained using any molecular weight fractionation method, such as gel filtration, ultrafiltration, or size exclusion chromatography. These molecular weight fractionation methods can be used alone or in combination.

[0044] 1-3. Other Steps The production method of the present invention may or may not include other steps in addition to the enzyme treatment step and the separation step described above. Examples of other steps include a step of preparing a protein-containing composition, a step of deactivating an enzyme, a purification step, a drying treatment, etc. These other steps may be performed singly or in combination of two or more steps.

[0045] 1-3-1. Protein-Containing Composition Preparation Step The protein-containing composition preparation step is carried out before the enzyme treatment step described above in "1-1. Enzyme Treatment Step." The protein-containing composition may be prepared in accordance with aspects [A] to [C] described above in "1-1-1. Specific Embodiments" and specific examples (i) to (vi) described above in "1-1-2. Pea Protein-Containing Composition." The type, amount used, and treatment conditions of the pretreatment enzymes used in the preparation of the pea protein-containing compositions (b) and (c) are as described above in "1-1-3. Enzymes." Furthermore, enzyme deactivation may be carried out as appropriate in the same manner as described below in "1-3-2. Enzyme Deactivation Step."

[0046] 1-3-2. Enzyme inactivation step The enzyme inactivation step can be carried out after the enzyme treatment step described above in "1-1. Enzyme treatment step" and before the above "1-2. Fractionation step". In the enzyme inactivation step, the enzyme-treated product obtained in the enzyme treatment step described above in "1-1. Enzyme treatment step" is subjected to conditions that inactivate the enzyme. The inactivation conditions may be appropriately selected so as to denature the enzyme, and are typically heat inactivation. Specific temperature conditions for heat inactivation may be set depending on the thermal properties of the enzyme actually used, and include, for example, 70°C or higher, preferably 80°C or higher, and more preferably 85°C or higher. The time for heat inactivation is not particularly limited, and may be, for example, 5 to 20 minutes, preferably 8 to 15 minutes.

[0047] 1-3-3. Purification Step In the purification step, components other than proteins, peptides, oligopeptides, amino acids, and water are removed from the fraction containing the salty taste enhancing oligopeptide obtained in the above "1-2. Separation Step" to purify the salty taste enhancing oligopeptide. Methods used in the purification step include, for example, desalting and precipitation.

[0048] 1-3-4. Drying Step In the drying step, water is removed from the fraction containing the salty taste enhancing oligopeptide obtained in the above "1-2. Separation Step" or a purified product thereof. In the drying step, any drying method can be used, such as freeze drying or spray drying.

[0049] 2. Saltiness Enhancer 2-1. Active Ingredient The saltiness enhancer of the present invention contains, as an active ingredient, an oligopeptide having a molecular weight of 900 to 1000 Da, which is a degradation product of pea protein at the γ-glutamyl bond and peptide bond, specifically, an oligopeptide having a molecular weight of 900 to 1000 Da obtained by allowing glutaminase and protease to act on pea protein. Details of the oligopeptide are as described above in "1. Saltiness-Enhancing Oligopeptide."

[0050] 2-2. Other Components The salty taste enhancer of the present invention may or may not contain any other component in addition to the active ingredient described above, as long as it exhibits the salty taste enhancing effect.

[0051] Examples of other components include components that are inevitably contained in the process of obtaining oligopeptides having a molecular weight of 900 to 1000 Da in the production method described above in "1. Salty Taste-Enhancing Oligopeptides," such as pea-derived components (proteins, peptides, oligopeptides, amino acids, lipids, carbohydrates, etc.), food additives, etc.

[0052] Examples of other components include additives and / or bases acceptable for the formulation of enzyme preparations. Such additives and bases include excipients, buffers, antioxidants, UV inhibitors, preservatives, antiseptics, pH adjusters, dispersants, emulsifiers, solubilizers, carriers, solvents (water, etc.), etc. These additives and bases may be used alone or in combination of two or more. The content of these additives and bases may be appropriately determined depending on the type of the components and / or the formulation form, etc.

[0053] 2-3. Properties There are no particular limitations on the properties of the salty taste enhancer of the present invention, and examples include dry preparations in the form of powder, fine granules, or granules, and liquid preparations.

[0054] 2-4. Uses The salty taste enhancer of the present invention is used for the purpose of enhancing saltiness. More specifically, the salty taste enhancer of the present invention is added to a food or beverage composition together with sodium chloride and / or potassium chloride, and is used for the purpose of enhancing the saltiness of the food or beverage beyond the saltiness corresponding to the content of the sodium chloride and / or potassium chloride. Furthermore, since the salty taste enhancer of the present invention enhances the saltiness of a food or beverage beyond the saltiness corresponding to the content of the sodium chloride and / or potassium chloride, it can be used for the purpose of reducing salt in a food or beverage.

[0055] The food and drink composition is not particularly limited as long as it is provided as a food and drink or a food and drink additive by adding sodium chloride and / or potassium chloride. Such food and drink or food and drink additives include any food and drink as long as they have a salty taste. Examples of foods and beverages or food additives include foods with high salt concentrations such as pickles, tsukudani (simmered foods), and shiokara (shiokara), processed meat products such as ham, sausage, bacon, dry sausage, and beef jerky, seafood paste products such as fish ham, fish sausage, kamaboko (fish paste), chikuwa (fish cake), hampen (hampen), and tempura, foods using textured vegetable protein materials (also referred to as meat substitutes or pseudo-meat), various soups or sauces such as ramen, udon, soba, potage, consommé, bouillon, miso soup, clear soup, dashi stock, stew, curry roux, and pasta sauce (all of which include any form such as liquid, paste, block, or powder), noodles such as ramen, udon, soba, yakisoba, and pasta, breads, condiments such as soy sauce, powdered soy sauce, miso, dried miso, dressing, salad seasoning, sauce, powdered sauce, ketchup, mayonnaise, and grilled meat sauce, and seasoning powders for snacks.

[0056] When the food or beverage is a food using a textured vegetable protein material (meat substitute, artificial meat), the salty taste enhancer of the present invention may be added during cooking of the textured vegetable protein material, or may be kneaded into the textured vegetable protein material. A textured vegetable protein material kneaded with the salty taste enhancer of the present invention can be prepared by kneading the salty taste enhancer of the present invention into a raw material mixture containing vegetable protein and water, extruding the mixture using an extruder or the like, and drying or freezing it to create a meat-like texture. The "meat" that the textured vegetable protein material imitates refers to the muscle of an animal that is used for food, and the term "meat" is used to encompass not only the muscle of mammals and birds, but also the flesh of seafood. The shape of the textured protein-containing composition can be granular or fibrous. Granular shapes include block shapes of various sizes such as small (minced), large, and block types (sizes increase in the order of small, large, and block types); and flat shapes of various sizes such as flake, fillet, and slice types (sizes increase in the order of flake, fillet, and slice types).

[0057] The specific amount of the salty taste enhancer of the present invention to be used can be determined appropriately depending on the desired salty taste enhancement effect, and may be, for example, an amount such that the salty taste enhancing oligopeptide is 0.02 parts by weight or more per 1 part by weight of the total amount of sodium chloride and potassium chloride. From the viewpoint of enhancing the salty taste enhancement effect, a preferred amount of the salty taste enhancer of the present invention to be used is, in terms of the amount of salty taste enhancing oligopeptide per 1 part by weight of the total amount of sodium chloride and potassium chloride, 0.05 parts by weight or more, more preferably 0.1 parts by weight or more, and even more preferably 0.15 parts by weight or more. The specific amount of the salty taste enhancer of the present invention to be used is not particularly limited in its upper limit, and may be, for example, 1 part by weight or less per 1 part by weight of the total amount of sodium chloride and potassium chloride. Because the salty taste enhancer of the present invention has an excellent salty taste enhancement effect, an effective salty taste enhancement effect can be obtained even when the specific amount of the salty taste enhancer of the present invention to be used is, in terms of the amount of salty taste enhancing oligopeptide per 1 part by weight of the total amount of sodium chloride and potassium chloride, 0.5 parts by weight or less, 0.3 parts by weight or less, or 0.25 parts by weight or less.

[0058] The total amount of sodium chloride and potassium chloride in a food or beverage or food or beverage additive may vary depending on the type of food or beverage or food or beverage additive, but examples of the content in the food or beverage at the time of consumption include 0.2 to 1.9 wt %, 0.3 to 1.2 wt %, 0.35 to 1 wt %, 0.4 to 0.80 wt %, 0.45 to 0.6 wt %, and 0.45 to 0.55 wt %.

[0059] The present invention will be specifically described below with reference to examples, but the present invention should not be construed as being limited to the following examples.

[0060] [Enzyme used]

[0061] (Glutaminase Activity Measurement Method) 1 mL of the enzyme solution was weighed into a test tube and left in a 37°C thermostatic water bath for 5 minutes. 1 mL of 2% (w / v) L-glutamine solution (0.1 mol / L acetate buffer (pH 6.0)) preheated to 37°C was added, mixed, and left for exactly 10 minutes. After leaving, 1 mL of 5% (v / v) perchloric acid test solution was added, mixed, and immediately placed in ice water. After leaving for at least 1 minute, 1 mL of sodium hydroxide test solution (0.75 mol / L) was added and mixed to form a reaction solution. L-glutamic acid in the reaction solution was quantified using the L-glutamic acid measurement kit "Yamasa" NEO (Yamasa Shoyu Co., Ltd.). Under these conditions, the amount of enzyme that produces 1 μmol of L-glutamic acid per minute was defined as 1 unit.

[0062] (Protease Activity Measurement Method) 5 mL of 0.6% (v / w) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0 [for bacterial proteases] or 0.7% (v / w) lactic acid, pH 3.0 [for fungal proteases]) was heated at 37°C for 10 minutes, and then 1 mL of a sample solution containing protease was added and immediately shaken. This solution was then left at 37°C for 10 minutes, after which 5 mL of trichloroacetic acid test solution (containing 1.8% trichloroacetic acid, 1.8% sodium acetate, and 0.33 mol / L acetic acid [for bacterial proteases] or 0.44 mol / L trichloroacetic acid [for fungal proteases]) was added and shaken, and the solution was again left at 37°C for 30 minutes and filtered. The first 3 mL of filtrate was discarded, and the next 2 mL of filtrate was measured, to which 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added, shaken well, and allowed to stand for 30 minutes at 37° C. The absorbance AT of this solution (enzyme reaction solution) at a wavelength of 660 nm was measured, using water as a control.

[0063] Separately, 1 mL of the protease-containing sample solution was measured, and 5 mL of trichloroacetic acid test solution (containing 1.8% trichloroacetic acid, 1.8% sodium acetate, and 0.33 mol / L acetic acid [for bacterial proteases] or 0.44 mol / L trichloroacetic acid [for fungal proteases]) was added and shaken. 5 mL of 0.6% (v / w) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0 [for bacterial proteases] or 0.7% (v / w) lactic acid, pH 3.0 [for fungal proteases]) was then added, immediately shaken, and left at 37°C for 30 minutes. The absorbance (AB) of this blank solution was measured in the same manner as the enzyme reaction solution described above. One unit (1 U) was defined as the amount of enzyme that increases the Folin test solution color product equivalent to 1 μg of tyrosine per minute.

[0064] 1 mL, 2 mL, 3 mL, and 4 mL of a 1 mg / mL tyrosine standard stock solution (0.2 mol / L hydrochloric acid) were measured, and 0.2 mol / L hydrochloric acid test solution was added to each to make a 100 mL solution. 2 mL of each solution was measured, and 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added. The mixture was immediately shaken and left at 37°C for 30 minutes. For each of these solutions, 2 mL of 0.2 mol / L hydrochloric acid test solution was measured and the resulting solution was used as a control. Absorbances A1, A2, A3, and A4 at a wavelength of 660 nm were measured. A calibration curve was created, plotting absorbances A1, A2, A3, and A4 on the vertical axis and the amount of tyrosine (μg) in 2 mL of each solution on the horizontal axis, and the amount of tyrosine (μg) per 2 mL of each solution was calculated.

[0065]

[0066] Test Example 1 (1) Preparation of Salty Taste Enhancing Oligopeptide (1-1) Preparation of Pea Protein-Containing Composition A pea protein material (PURIS Pea 870 (manufactured by PURIS, protein content 80% by weight)) was suspended in water so that the pea protein content was 10% by weight, to obtain a pea protein-containing composition.

[0067] (1-2) Enzyme Treatment The obtained pea protein-containing composition was heated to 50°C with stirring, and glutaminase (1 U / g-pea protein), Aspergillus oryzae-derived acid protease (3,000 U / g-pea protein), and Geobacillus stearothermophilus-derived protease (1,000 U / g-pea protein) were added, followed by a reaction at 50°C for 1 hour. The temperature was then raised to 85°C and maintained for 10 minutes to inactivate the enzymes. The obtained enzyme-treated solution was powdered using a spray dryer (model: Henningsen Pilot Plant Tower Spray Dryer Model T-20, inlet temperature: 140-150°C, outlet temperature: 90-100°C), to obtain a powdered enzyme-treated product.

[0068] (1-3) The powdered enzyme-treated product was suspended in purified water to a final concentration of 20 wt%. The pH was adjusted to 4, and the mixture was centrifuged at 9,000 g for 10 minutes. The supernatant was collected, and the low-molecular-weight peptide fraction was fractionated using a 3 kDa cutoff ultrafiltration column. The resulting fraction was subjected to lyophilization and powderization. The resulting powder was dissolved in purified water to a concentration of 100 mg / mL and filtered through a 0.45 μm filter to obtain a peptide solution. 2 mL of the resulting peptide solution was fractionated using a TSK-gel G2000SWXL mobile phase containing 45 v / v% acetonitrile containing 0.1 v / v% trifluoroacetic acid at a flow rate of 0.5 mL / min (25°C). The detection peak was measured at 220 nm UV. The four fractions shown in Table 2 were collected, and each fraction was lyophilized to obtain a powder.

[0069]

[0070] (2) Sensory Test: Test samples with the compositions shown in Tables 3 and 4 were prepared using the powders of each fraction obtained in (1) above. The peptide concentrations in the test samples were measured by the BCA method. Three sensory panelists scored the saltiness intensity based on the following criteria, and the average value was calculated as the saltiness score. The results are shown in Tables 3 and 4.

[0071] -1: The saltiness intensity was lower than that of Comparative Example 1. 0: The saltiness intensity was the same as that of Comparative Example 1. +1: The saltiness intensity was slightly stronger than that of Comparative Example 1. +2: The saltiness intensity was stronger than that of Comparative Example 1. +3: The saltiness intensity was significantly stronger than that of Comparative Example 1.

[0072]

[0073]

[0074] As shown in Table 3, none of the peptides in any fractions exhibited a salty taste by themselves. However, as shown in Table 4, when combined with sodium chloride, only the peptide from Fraction 3 of Example 1 (saltiness-enhancing oligopeptide) enhanced the saltiness of sodium chloride.

[0075] Test Example 2 Water was added to a mixture of 15 parts by weight of the powder of fraction 3 peptide (salty taste enhancing oligopeptide) obtained in Test Example 1, 15 parts by weight of dextrin (Pinex #2, Matsutani Chemical Industry Co., Ltd.), and 70 parts by weight of a pea protein material (PURIS Pea 870, manufactured by PURIS), and the mixture was kneaded and textured in an extruder (Wenger Manufacturing TX-57 model) (pressure: 150-175 psi, stage 1 temperature: 60°C, stage 2 temperature: 80°C, stage 3 temperature: 90°C, stage 4 temperature: 100°C) to give a granular textured vegetable protein material.

[0076] The resulting granular textured vegetable protein material was pulverized using a mortar and pestle. The pulverized material was passed through an 80-mesh sieve and powdered. The resulting powder was fractionated in the same manner as in "(1-3) Fractionation" of Test Example 1, yielding the same four fractions (fractions 1 to 4) as those listed in Table 2. Each of the resulting fractions was lyophilized and powdered, and a test similar to "(2) Sensory Test" of Test Example 1 was conducted. Results similar to those in Tables 3 and 4 were obtained. This confirms that only the Fraction 3 peptide enhanced the saltiness of sodium chloride. These results confirm that the Fraction 3 peptide (saltiness-enhancing oligopeptide) of Example 1 maintains its properties even when kneaded into a textured vegetable protein material.

Claims

1. An oligopeptide with a molecular weight of 900-1000 Da, which is a degradation product of pea protein at the γ-glutamyl bond and peptide bond.

2. An oligopeptide with a molecular weight of 900 to 1000 Da obtained by acting on pea protein with at least one type of glutaminase and protease.

3. The oligopeptide according to claim 2, wherein the glutaminase is glutaminase (EC 3.5.1.2).

4. The oligopeptide of claim 2, wherein the protease comprises a bacterial protease.

5. The oligopeptide according to claim 2, wherein the protease is a fungal protease or a bacterial protease.

6. The oligopeptide according to claim 4, wherein the bacterial protease is a protease derived from a bacterium of the genus Bacillus or Geobacillus.

7. The oligopeptide according to claim 5, wherein the amount of the filamentous fungal protease used per 1 U of the glutaminase is 1,000 U or more.

8. The oligopeptide according to claim 4, wherein the amount of the bacterial protease used per 1 U of the glutaminase is 500 U or more.

9. The oligopeptide according to claim 5, wherein the amount of the bacterial protease used is 0.01 to 2 U per 1 U of the filamentous fungal protease.

10. A method for producing an oligopeptide, comprising an enzyme treatment step of obtaining an enzyme-treated product of pea protein with glutaminase and protease, and a fractionation step of obtaining a fraction containing oligopeptides having a molecular weight of 900 to 1000 Da.

11. A salty taste enhancer comprising the oligopeptide according to claim 1 or 2.

12. A structured vegetable protein material comprising the oligopeptide of claim 1 or 2.

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