Method for producing protein hydrolysates, and enzyme preparations

Abstract

The present invention addresses the problem of providing a practical means for generating cysteine from protein, said means also being suited for use in the field of foodstuffs (foodstuff applications). According to the present invention, provided is a cysteine-containing protein degradation product production method including a step for causing γ-glutamyl peptide hydrolase, filamentous bacteria-derived protease, and bacterial protease to act on a protein material.

Description

Technical Field

[0001] The present invention relates to a method for producing cysteine from a protein, an enzyme agent for producing a cysteine-containing protein hydrolyzate, and uses thereof.

Background Art

[0002] Proteins in foods are important as nutrients, and from the viewpoint of palatability, mainly animal proteins such as livestock meat and seafood have been preferred. In recent years, due to the perspective of environmental protection and the increasing awareness of health, the demand for plant proteins as an alternative to animal proteins has been increasing. However, there are many problems in terms of palatability, and various studies have been conducted for the purpose of improving physical properties and taste properties. For example, Patent Document 1 discloses that in a method for producing soy protein using transglutaminase and protease, the protease is used for the purpose of reducing the viscosity increased by transglutaminase. On the other hand, amino acids and peptides are not only important as nutrients but also important elements in the taste and flavor of foods. For example, glutamic acid and aspartic acid exhibit umami and sourness, glycine, alanine, threonine, etc. exhibit sweetness, and tryptophan, isoleucine, valine, etc. exhibit bitterness. Therefore, research has been conducted on producing amino acids and peptides from proteins. Patent Document 2 discloses a method for producing low molecular weight peptides by allowing two or more enzymes having only endoprotease activity to act on soy protein. In addition, methods for producing amino acids as seasonings to impart taste properties have also been studied. For example, Patent Document 3 discloses a method for obtaining an amino acid seasoning rich in glutamic acid content by hydrolyzing a protein raw material with a heat-resistant protease and a heat-resistant glutaminase.

[0003] Incidentally, cysteine ​​is known to impart flavor to meat by forming Maillard reaction products. Cysteine ​​is mainly extracted from animal-derived raw materials (hair and feathers), and its addition to food is not preferred. Patent document 4 discloses a method for producing cysteine ​​by reacting glutathione with acid protease and glutaminase, but the glutathione content varies depending on the food, so its applications are limited.

[0004] Furthermore, Patent Document 5 discloses that treating soy protein with glutamate-specific endoprotease increases free aspartic acid and free glutamate. Patent Document 6 also discloses that when soy protein is treated with Bacillus licheniformis-derived subtilisin protease (ALCALASE) or Nocardiopsis prasina-derived serine protease (SP1), the SP1 hydrolysate is less bitter than the ALCALASE hydrolysate. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2006-141231 [Patent Document 2] Japanese Patent Application Publication No. 5-252979 [Patent Document 3] Japanese Unexamined Patent Publication No. 48-82068 [Patent Document 4] WO2021 / 002195 issue [Patent Document 5] Special Publication No. 2008-526261 [Patent Document 6] Special Publication No. 2011-530274 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of this invention is to provide a practical means that can improve the richness or umami flavor of food products. [Means for solving the problem]

[0007] Under the above-mentioned challenges, the inventors conducted extensive research aimed at improving the richness or umami of food using enzymes. As a result, they discovered that food with improved richness or umami can be produced by using glutaminase, a type of γ-glutamyl peptide hydrolase, in combination with filamentous fungal protease and bacterial protease. The present invention was completed by further research based on this finding. That is, the present invention provides the invention in the following embodiments.

[0008] [1] A method for producing a protein hydrolysate, comprising the step of reacting a protein material with γ-glutamyl peptide hydrolase, a protease derived from filamentous fungi, and a bacterial protease. [2] The γ-glutamyl peptide hydrolase is glutaminase, γ- Glutamil The method according to [1], wherein the transferase is a γ-glutamylcyclotransferase. [3] The method according to [1] or [2], wherein the γ-glutamyl peptide hydrolase is a glutaminase derived from a microorganism of the genus Bacillus. [4] The method according to any one of [1] to [3], wherein the γ-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloricephaciens. [5] The method according to any one of [1] to [4], wherein the filamentous fungal protease is an acidic protease derived from a microorganism of the genus Aspergillus or a neutral protease derived from a microorganism of the genus Aspergillus. [6] The method according to any one of [1] to [5], wherein the filamentous fungal protease is an acidic protease derived from Aspergillus oryzae, a neutral protease derived from Aspergillus oryzae, or a neutral protease derived from Aspergillus meleus. [7] The method according to any one of [1] to [6], wherein the bacterial protease is a protease derived from a microorganism of the genus Bacillus or Diobacillus. [8] The method according to any one of [1] to [7], wherein the bacterial protease is a protease derived from Geobacillus stearothermophilus. [9] The method according to any one of [1] to [8], wherein the protein material is an animal protein material, a plant protein material, or a microbial protein material.

[10] The method according to any one of [1] to [9], wherein the protein material is a plant protein material.

[11] The method according to any one of [1] to [9], wherein the protein material is a protein material derived from peas, soybeans, broad beans, chickpeas, barley, wheat, oats, rice, buckwheat, millet, foxtail millet, hemp, algae, almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, or coconut. Foods containing protein hydrolysates produced by any one of the methods described in

[12] [1] to

[11] .

[13] Enzymatic preparations for the production of protein hydrolysates, comprising γ-glutamyl peptide hydrolase, fungal proteases, and bacterial proteases.

[14] The γ-glutamyl peptide hydrolase is glutaminase, γ- Glutamil The enzyme preparation described in

[13] , which is a transferase or γ-glutamylcyclotransferase.

[15] The enzyme preparation according to

[13] or

[14] , wherein the γ-glutamyl peptide hydrolase is a glutaminase derived from a microorganism of the genus Bacillus.

[16] The enzyme preparation according to any one of

[13] to

[15] , wherein the γ-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloricephaciens.

[17] The enzyme preparation according to any one of

[13] to

[16] , wherein the filamentous fungal protease is an acidic protease derived from a microorganism of the genus Aspergillus or a neutral protease derived from a microorganism of the genus Aspergillus.

[18] The enzyme agent according to any one of

[13] to

[17] , wherein the filamentous fungus protease is an acidic protease derived from Aspergillus oryzae, a neutral protease derived from Aspergillus oryzae, or a neutral protease derived from Aspergillus melleus.

[19] The enzyme agent according to any one of

[13] to

[18] , wherein the bacterial protease is a protease derived from a microorganism of the genus Bacillus or Geobacillus.

[20] The enzyme agent according to any one of

[13] to

[19] , wherein the bacterial protease is a protease derived from Geobacillus stearothermophilus.

Advantages of the Invention

[0009] According to the present invention, it is possible to impart kokumi taste or umami taste to foods and the like, or to enhance the kokumi taste or umami taste of foods.

Brief Description of the Drawings

[0010] [Figure 1] FIG. 1 shows the results of the evaluation of firmness in Example 2. [Figure 2] FIG. 2 shows the results of the free amino acid analysis in Example 2. [Figure 3] FIG. 3 shows the results of the free amino acid analysis in Example 3. [Figure 4] FIG. 4 shows the results of the free amino acid analysis in Example 3. [Figure 5] FIG. 5 shows the results of the free amino acid analysis in Example 3. [Figure 6] FIG. 6 shows the results of the free amino acid analysis in Example 3.

Modes for Carrying Out the Invention

[0011] According to the present invention, there is provided a method for producing a proteolysate, which includes a step of allowing a γ-glutamylpeptide hydrolase, a protease derived from a filamentous fungus, and a bacterial protease to act on a protein material. The present invention further provides an enzyme preparation for the production of protein degradation products, comprising γ-glutamyl peptide hydrolase, a filamentous fungal protease, and a bacterial protease.

[0012] The step of treating the protein material with γ-glutamyl peptide hydrolase, filamentous fungal protease, and bacterial protease may be carried out in a single step (i.e., a step of treating the protein material with the three enzymes simultaneously), or in a two-step or three-step step (i.e., a step of treating the protein material with one or two of the three enzymes, and then a step of treating it with another of the three enzymes).

[0013] Examples of γ-glutamyl peptide hydrolases include glutaminase and γ- Glutamil Examples include transferases or γ-glutamylcyclotransferases. As γ-glutamyl peptide hydrolases, γ-glutamyl peptide degrading enzymes derived from microorganisms can be used. Examples include glutaminase, γ-glutamyltransferase, and γ-glutamylcyclotransferase derived from Bacillus microorganisms. The γ-glutamyl peptide hydrolase is preferably a glutaminase derived from a microorganism of the genus Bacillus, and more preferably a glutaminase derived from Bacillus amyloliquefaciens (for example, glutaminase SD-C100S provided by Amano Enzyme Co., Ltd.).

[0014] The microbial-derived γ-glutamyl peptide hydrolase does not have to be a purified product; for example, a culture medium, lysate / extract, or partially purified product of a microorganism that produces γ-glutamyl peptide hydrolase may be used. Two or more microbial-derived γ-glutamyl peptide hydrolases may also be used in combination. Several microbial-derived γ-glutamyl peptide hydrolases are commercially available (for example, the above-mentioned glutaminase SD-C100S) and are readily available and usable.

[0015] Preferred examples of filamentous fungal proteases include acidic proteases derived from Aspergillus microorganisms and neutral proteases derived from Aspergillus microorganisms.

[0016] An example of an acid protease derived from an Aspergillus microorganism is the acid protease derived from Aspergillus oryzae (for example, Protease M "Amano" SD and Protease HF "Amano" 150SD provided by Amano Enzyme Co., Ltd.).

[0017] Examples of neutral proteases derived from Aspergillus microorganisms include neutral proteases derived from Aspergillus oryzae and neutral proteases derived from Aspergillus melleus, such as those provided by Amano Enzyme Co., Ltd.: Aspergillus oryzae-derived neutral protease (PR-AX, product name: ProteaX), Aspergillus oryzae-derived neutral protease (PR-ASD, product name: Protease A "Amano" SD), Aspergillus melleus-derived neutral protease (PR-P6SD, product name: Protease P "Amano" 6SD), and Aspergillus oryzae-derived neutral protease (Aspergillus It is a neutral protease (PR-AN100SD) derived from *Oryzae*.

[0018] The proteases derived from filamentous fungi do not necessarily have to be purified products; for example, culture media, lysates / extracts, or partially purified products thereof of microorganisms that produce proteases derived from filamentous fungi may be used. Two or more types of proteases derived from filamentous fungi may also be used in combination. Several proteases derived from filamentous fungi are commercially available (for example, the above-mentioned Protease M "Amano" SD, Protease HF "Amano" 150S, as well as ProteaX, Protease A "Amano" SD, Protease P "Amano" 6SD, PR-AN100SD), and are readily available and usable.

[0019] As the bacterial protease, a metalloprotease is preferred. The bacterial protease can preferably be derived from a microorganism of the genus Bacillus or Diobacillus, and more preferably from Diobacillus stearothermophilus. An example of a bacterial protease is samoase PC10F provided by Amano Enzyme Co., Ltd.

[0020] Bacterial proteases do not necessarily have to be purified products; for example, culture media of microorganisms that produce bacterial proteases, lysates / extracts, or partially purified products thereof may be used. Two or more types of bacterial proteases may also be used in combination. Several bacterial proteases are commercially available (for example, the samoase PC10F mentioned above) and are readily available and usable.

[0021] The enzyme preparation of the present invention, which contains γ-glutamyl peptide hydrolase, filamentous fungal protease, and bacterial protease, may also contain excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc., in addition to the active ingredients (the three enzymes mentioned above). Excipients that can be used include lactose, sorbitol, D-mannitol, maltodextrin, sucrose, etc. Buffers that can be used include phosphates, citrates, acetates, etc. Stabilizers that can be used include propylene glycol, ascorbic acid, etc. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc.

[0022] The amount of active ingredients (the three enzymes mentioned above) in this enzyme preparation is not particularly limited and can be set as appropriate. The enzyme preparation of the present invention is usually provided in solid form (for example, granules, powders, immobilized enzymes in which the enzyme is immobilized on a material capable of fixing the enzyme on or inside the surface of silica or porous polymers) or in liquid form.

[0023] The conditions for reacting the protein material with γ-glutamyl peptide hydrolase, filamentous fungal protease, and bacterial protease are, for example, a reaction temperature of 15°C to 70°C, preferably 30°C to 65°C, and more preferably 40°C to 60°C. The reaction time and enzyme amount are not particularly limited as long as the expected effect is achieved. Examples of reaction times include 5 minutes to 48 hours, preferably 10 minutes to 12 hours, and more preferably 15 minutes to 6 hours. The enzyme amount can be such that the concentration of each of the three enzymes in the reaction solution is, for example, 0.001% (W / W) to 10% (W / W), preferably 0.01% (W / W) to 2% (W / W).

[0024] The protein material is preferably an animal protein material, a plant protein material, or a microbial protein material, and more preferably a plant protein material. Specific examples of protein materials include peas, soybeans, broad beans, chickpeas, barley, wheat, oats, rice, buckwheat, millet, foxtail millet, hemp, algae, almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, and coconut-derived protein materials.

[0025] According to the present invention, it is possible to produce a protein degradation product (cysteine-containing protein degradation product) that contains a large amount of cysteine ​​capable of forming a Maillard reaction. The cysteine ​​capable of forming a Maillard reaction includes not only cysteine ​​in the form of a free amino acid, but also peptides having cysteine ​​residues in a state capable of forming a Maillard reaction. Preferably, cysteine ​​in the form of a free amino acid is mentioned. The present invention further provides a food product comprising a cysteine-containing protein hydrolysate produced by the method for producing cysteine-containing protein hydrolysates according to the present invention.

[0026] The present invention will be specifically described by the following examples, but the present invention is not limited to these examples. [Examples]

[0027] <Example 1> The following experiments were conducted with the aim of establishing an efficient method for producing cysteine ​​from proteins.

[0028] 1. Examination of cysteine ​​production (1) Method A protein solution (15% (W / W), pH 5) was prepared by suspending 12 g of various protein materials in water. An enzyme solution was prepared by dissolving 1.4 g of filamentous fungal protease (Protease HF "Amano" 150SD, Amano Enzyme Co., Ltd.), 0.7 g of glutaminase (Glutaminase SD-C100S, Amano Enzyme Co., Ltd.), and 0.7 g of bacterial protease (Samoase PC10F, Amano Enzyme Co., Ltd.) in 20 mL of water. 1 mL of the enzyme solution was added to 30 mL of the protein solution at 50°C, mixed, and treated at 50°C for 2 hours. The treated solution was centrifuged, and the supernatant was collected. The thiol groups of cysteine ​​contained in the supernatant were quantified using DTNB reagent. To 2.6 mL of 0.1 M Tris buffer (1 mM EDTA, pH 7.05), 0.2 mL of 25 mM DTNB solution (50 mM ammonium acetate, 1 mM EDTA, pH 5.0) and 0.2 mL of supernatant diluted to an appropriate concentration were added and mixed. After mixing, the reaction was allowed to proceed for 10-15 minutes. Immediately after the reaction was complete, the absorbance at 412 nm was measured. The amount of cysteine ​​residues in the supernatant was calculated from a calibration curve created using a 10 μM-50 μM cysteine ​​solution (1 mM EDTA) instead of the supernatant. Furthermore, to confirm the change in taste, 2 mL of the supernatant neutralized to pH 7 with hydrochloric acid was taken, 1 mL of 20% glucose was added, and the Maillard reaction was carried out by boiling for 30 minutes. The richness of the taste was then checked. Specifically, the change in taste was confirmed by sensory evaluation by panelists.

[0029] (2) Results Enzymatic treatment confirmed the release of cysteine ​​in all protein materials. Furthermore, in all but hemp protein, an improvement in umami, a key element of meat flavor, was observed in the Maillard reaction products. However, with hemp protein, the strong, characteristic hemp flavor prevented any observation of a change in umami.

[0030] [Table 1]

[0031] <Example 2> 70g of soy meat (Soy TVP (Textured Vegetable Protein) (Daizu Lab Soy Meat Minced Type, Marukome Co., Ltd.)) was mixed with 105g (1.5 times the amount) of water and left to stand for 10 minutes. 8.7g of Metrolose, 48.5g of sunflower oil, and 26.7g of Sun Rubber 10 (soy protein powder) were then mixed uniformly into the mixture. 25g of this mixture was mixed with 1% of the enzyme solution shown in Table 2 (relative to the soy TVP) and reacted at 50°C for 1 hour. After the reaction, 6mL of water was added, the mixture was shaped, and then baked (in an oven at 110°C for 10 minutes). The resulting baked product was subjected to a sensory evaluation (5-point scale for umami and aroma). The results of the sensory evaluation are shown in Table 3. Using the no-enzyme product as a baseline, a higher number indicates improvement, with 1 indicating no change and 4 indicating significant improvement.

[0032] [Table 2]

[0033] [Table 3]

[0034] The hardness of the resulting fired product was evaluated under the following conditions. Equipment used: Rheometer (COMPAC-100II) Pushing speed: 60 mm / min Platform travel distance: 7mm (Measure the firmness at a point 7mm after touching the meat.) The results of the hardness evaluation are shown in Figure 1. The unit on the vertical axis of Figure 1 is N (Newtons).

[0035] The free amino acids in the obtained calcined product were analyzed as follows. 1 mL of distilled water was added to 1 g of the calcined sample, mixed, and centrifuged. The supernatant and ethanol were mixed in a 1:1 ratio (removal of protein). The mixture was centrifuged, the supernatant was collected, diluted 12.5 times with water, filtered through a microfiltration membrane (MF), and HPLC analysis was performed. The HPLC analysis conditions were as follows. The results of the free amino acid analysis are shown in Figure 2.

[0036] Agilent HPLC (1260 InfinityII) A buffer: 20mM Na2HPO4·H3PO4pH8.2 B buffer:methanol:acetonitrile:water = 45:45:10 Column: HPH-C18 2.7 μm 3.0 × 100 mm (Poroshell) Flow rate: 0.65 mL / min 0-0.35 min: A: 96%, B: 4% 0.35-13.4 min: A: 43%, B: 57% 13.4-13.5 min: A: 0%, B: 100% 13.5-15.7 min: A: 0%, B: 100% 15.7-15.8 min: A: 96%, B: 4% 15.8-18 min: A: 96%, B: 4%

[0037] <Example 3> To a 10% (w / v) plant protein solution (pea: pea protein (Usuki Pharmaceutical), soybean: Sun Rubber 10 (Fuji Oil Co., Ltd.)), 4% enzyme was added relative to the weight of the plant protein (2% filamentous fungal protease + 1% bacterial protease + 1% glutaminase). The mixture was treated at 50°C for 2 hours at 400 rpm to allow it to react. The reaction product was boiled for 10 minutes, centrifuged, and the supernatant was collected. Sensory evaluation and free amino acid analysis were performed on the obtained sample supernatant.

[0038] (Sensory evaluation) Four milliliters of the sample supernatant were collected, and two milliliters of 20% glucose were added. The resulting mixture was placed in boiling water and treated for 30 minutes, followed by a sensory evaluation. The results are shown in Table 4. The higher the number, the greater the improvement compared to the baseline (no enzyme). 1 indicates no change, and 5 indicates a significant improvement.

[0039] [Table 4]

[0040] (Free amino acid analysis) The sample supernatant and ethanol were mixed in a 1:1 ratio (protein removal). The mixture was centrifuged, the supernatant was collected, diluted 12.5 times with water, filtered through a microfiltration membrane (MF), and HPLC analysis (amino acid analysis) was performed under the same conditions as in Example 2. The results of the free amino acid analysis are shown in Figures 3 to 6.

Claims

1. The process includes reacting a protein material with γ-glutamyl peptide hydrolase, a protease derived from filamentous fungi, and a bacterial protease. A method for producing a cysteine-containing protein degradation product, wherein the filamentous fungus-derived protease is a neutral protease derived from Aspergillus oryzae or a neutral protease derived from Aspergillus meleus.

2. The method according to claim 1, wherein the γ-glutamyl peptide hydrolase is glutaminase, γ-glutamyltransferase, or γ-glutamylcyclotransferase.

3. The method according to claim 1 or 2, wherein the γ-glutamyl peptide hydrolase is glutaminase derived from a microorganism of the genus Bacillus.

4. The method according to any one of claims 1 to 3, wherein the γ-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloricephaciens.

5. The method according to any one of claims 1 to 4, wherein the protein material is an animal protein material, a plant protein material, or a microbial protein material.

6. The method according to any one of claims 1 to 5, wherein the protein material is a plant-based protein material.

7. The method according to any one of claims 1 to 6, wherein the protein material is a protein material derived from peas, soybeans, broad beans, chickpeas, barley, wheat, oats, rice, buckwheat, millet, foxtail millet, hemp, algae, almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, or coconut.

8. It contains γ-glutamyl peptide hydrolase, filamentous fungal protease and bacterial protease, An enzyme preparation for producing cysteine-containing protein degradation products, wherein the filamentous fungus-derived protease is a neutral protease derived from Aspergillus oryzae or a neutral protease derived from Aspergillus meleus.

9. The enzyme preparation according to claim 8, wherein the γ-glutamyl peptide hydrolase is glutaminase, γ-glutamyltransferase, or γ-glutamylcyclotransferase.

10. The enzyme preparation according to claim 8 or 9, wherein the γ-glutamyl peptide hydrolase is glutaminase derived from a microorganism of the genus Bacillus.

11. The enzyme preparation according to any one of claims 8 to 10, wherein the γ-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloricephaciens.

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