Production of peptide compositions by alkaline hydrolysis

Abstract

This invention provides a method for suppressing gas generation during the production of peptide compositions by alkaline hydrolysis of solid proteins, and a method for producing novel peptide compositions that are safe and yield-efficient. [Solution] A method for suppressing gas generation in the production of a peptide composition by alkaline hydrolysis of a solid protein, comprising alkaline hydrolysis of a solid protein in a solution containing hydrogen peroxide, wherein the solid protein is avian eggshell membrane, or cartilage of a mammal, bird, or fish, and the gas comprises hydrogen sulfide gas.

Description

Technical Field

[0001] The present invention relates to a method for producing a peptide composition from a solid protein by alkaline hydrolysis and a method for solubilizing a solid protein.

Background Art

[0002] The eggshell membrane of birds has antibacterial and antimicrobial activities and acts as a protective membrane until the embryo hatches. In addition, the main component of the eggshell membrane of birds is protein, and it has been used for the treatment of burns and wounds in folk medicine since ancient times. In recent years, as research and development on the skin care use of the eggshell membrane of birds has become active, various functions of the eggshell membrane and its decomposition products have become clear.

[0003] Since the eggshell membrane is water-insoluble, it needs to be solubilized for application to cosmetics, pharmaceuticals, foods, etc. To solubilize the eggshell membrane, decomposition methods such as enzymatic treatment (Patent Documents 1 and 2) and acid / alkali hydrolysis (Patent Documents 3 and 6) have been studied. However, hydrolysis under acid / alkali conditions, for example, alkaline hydrolysis using NaBH4 (sodium borohydride) or KOH (potassium hydroxide), can generate toxic gases such as hydrogen sulfide, which can cause health hazards (Patent Document 1). In addition, in general acid hydrolysis using hydrochloric acid, active ingredients with physiological activity may be lost (Non-Patent Document 1).

[0004] It is necessary to remove the salts generated in the process after hydrolysis of the eggshell membrane, and ion exchange resins and gel filtration are used (Patent Documents 4 and 5). However, ion exchange resins cannot be used for food raw materials, and gel filtration has a problem that useful components are also excluded together with salts, resulting in a decrease in yield.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] [Non-Patent Document 1] Pharmacology and Clinical Practice, Vol. 27, No. 4, pp. 187-119 (2017) [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a method for suppressing gas generation in the production of peptide compositions by alkaline hydrolysis of solid proteins. A further object of the present invention is to provide a safe and high-yield method for producing novel peptide compositions. Specifically, the object of the present invention is to provide a safe and high-yield method for producing novel eggshell membrane hydrolysates. A further object of the present invention is to provide a safe and novel method for solubilizing solid proteins. Specifically, the object of the present invention is to provide a safe and novel method for solubilizing eggshell membranes. [Means for solving the problem]

[0008] As a result of diligent research, the inventors discovered that gas generation can be suppressed by performing alkaline hydrolysis of eggshell membrane in a solution containing hydrogen peroxide. Based on this finding, the inventors completed the present invention.

[0009] [1-1] A method for suppressing gas generation in the production of a peptide composition by alkaline hydrolysis of a solid protein, This includes alkaline hydrolysis of a solid protein in a solution containing hydrogen peroxide. The solid protein is the eggshell membrane of birds, or the cartilage of mammals, birds, or fish. A method in which the gas contains hydrogen sulfide gas.

[0010] [1-2] The method according to [1-1], wherein the solvent of the solution containing hydrogen peroxide is water. [1-3] The method according to [1-1] or [1-2], wherein the alkali used in alkaline hydrolysis is sodium hydroxide, potassium hydroxide, or sodium boro hydroxide.

[0011] [1-4] The method according to any of [1-1] to [1-3], wherein the solution before the start of alkaline hydrolysis contains alkali at a concentration of 5-50 mg / ml, 10-40 mg / ml, or 15-30 mg / ml.

[0012] [1-5] The method according to any of [1-1] to [1-4], wherein hydrogen peroxide is present in the solution before the start of alkaline hydrolysis at a concentration of 5-50 mg / ml, 5-30 mg / ml, or 10-20 mg / ml.

[0013] [1-6] The method according to any of [1-1] to [1-5], wherein the amount of solid protein subjected to the reaction is 10 to 5000 g, 50 to 300 g, or 100 to 300 g. [1-7] The method according to any of [1-1] to [1-6], wherein the alkaline hydrolysis is carried out at a temperature of 10°C to 80°C, 30°C to 60°C, or 30°C to 50°C.

[0014] [1-8] The method according to any of [1-1] to [1-7], wherein the reaction time for alkaline hydrolysis is 1 to 24 hours, 3 to 20 hours, or 15 to 18 hours. [1-9] The method according to any one of [1-1] to [1-8], wherein the birds are selected from one or more of the group consisting of chickens, quail, turkeys, ducks and geese.

[0015] [1-10] The method according to any one of [1-1] to [1-9], wherein the solid protein is chicken or quail eggshell membrane. [1-11] The method according to any one of [1-1] to [1-10], wherein the amount of gas generated during the alkali hydrolysis process is 5.0 ppm or less, 3.0 ppm or less, or 2.0 ppm or less.

[0016] [1-12] The method according to any one of [1-1] to [1-11], wherein the amount of gas generated during the alkali hydrolysis process is 50% or less, 30% or less, or 20% or less compared to the case where the solid protein is alkali hydrolyzed under the same conditions except that it does not contain hydrogen peroxide solution.

[0017] [1-13] The method according to any one of [1-1] to [1-12], wherein the total amount of gas generated throughout the entire process of manufacturing the peptide composition is 10.0 ppm or less, 5.0 ppm or less, or 3.0 ppm or less.

[0018] [1-14] The method according to any one of [1-1] to [1-13], wherein the amount of gas generated throughout the entire process of manufacturing the peptide composition is 50% or less, 30% or less, or 20% or less compared to the case where the solid protein is alkali hydrolyzed under the same conditions except that it does not contain hydrogen peroxide solution.

[0019] [1-15] The method according to any one of [1-1] to [1-14], wherein the amount of residual hydrogen peroxide in the peptide composition is 250 ppm or less, 150 ppm or less, or 50 ppm or less. [1-16] The method according to any one of [1-1] to [1-15], wherein the peptide composition is obtained in a yield of 40% or more, or 50% or more.

[0020] [2-1] Suppressing the generation of gas by alkali hydrolyzing a solid protein in a solution containing hydrogen peroxide A method for producing a peptide composition comprising: The solid protein is an eggshell membrane of a bird or cartilage of a mammal, bird, or fish, The method, wherein the gas contains hydrogen sulfide gas.

[0021] [2-2] The method according to [2-1], further comprising performing pH adjustment. [2-3] The method according to [2-1] or [2-2], further comprising desalting.

[0022] [2-4] The method described in [2-3], which uses ultrafiltration with an exclusion limit of 6,000 molecular weight for desalting. [2-5] The method according to any one of [2-1] to [2-4], further comprising sterilization.

[0023] [2-6] The method according to any of [2-1] to [2-5], wherein the solvent of the solution containing hydrogen peroxide is water. [2-7] The method according to any of [2-1] to [2-6], wherein the alkali used for alkaline hydrolysis is sodium hydroxide, potassium hydroxide, or sodium boro hydroxide.

[0024] [2-8] The method according to any of [2-1] to [2-7], wherein the solution before the start of alkaline hydrolysis contains alkali at a concentration of 5-50 mg / ml, 10-40 mg / ml, or 15-30 mg / ml.

[0025] [2-9] The method according to any of [2-1] to [2-8], wherein hydrogen peroxide is present in the solution before the start of alkaline hydrolysis at a concentration of 5-50 mg / ml, 5-30 mg / ml, or 10-20 mg / ml.

[0026] [2-10] The method according to any of [2-1] to [2-9], wherein the amount of solid protein subjected to the reaction is 10 to 5000 g, 50 to 300 g, or 100 to 300 g. [2-11] The method according to any of [2-1] to [2-10], wherein the alkaline hydrolysis is carried out at a temperature of 10°C to 80°C, 30°C to 60°C, or 30°C to 50°C.

[0027] [2-12] The method according to any of [2-1] to [2-11], wherein the reaction time for alkaline hydrolysis is 1 to 24 hours, 3 to 20 hours, or 15 to 18 hours. [2-13] The method according to any of [2-1] to [2-12], wherein the birds are selected from one or more of the group consisting of chickens, quail, turkeys, ducks and geese.

[0028] [2-14] The method according to any of [2-1] to [2-13], wherein the solid protein is chicken or quail eggshell membrane. [2-15] The method according to any one of [2-1] to [2-14], wherein the amount of gas generated during the alkaline hydrolysis process is 5.0 ppm or less, 3.0 ppm or less, or 2.0 ppm or less.

[0029] [2-16] The method according to any one of [2-1] to [2-15], wherein the amount of gas generated during the alkaline hydrolysis process is 50% or less, 30% or less, or 20% or less compared to when a solid protein is alkaline hydrolyzed under identical conditions except that hydrogen peroxide solution is not included.

[0030] [2-17] The method according to any one of [2-1] to [2-16], wherein the total amount of gas generated throughout the entire process of manufacturing the peptide composition is 10.0 ppm or less, 5.0 ppm or less, or 3.0 ppm or less.

[0031] [2-18] The method according to any one of [2-1] to [2-17], wherein the amount of gas generated throughout the entire process of manufacturing the peptide composition is 50% or less, 30% or less, or 20% or less compared to when a solid protein is alkaline hydrolyzed under identical conditions except that hydrogen peroxide solution is not included.

[0032] [2-19] The method according to any one of [2-1] to [2-18], wherein the amount of residual hydrogen peroxide in the peptide composition is 250 ppm or less, 150 ppm or less, or 50 ppm or less. [2-20] The method according to any of [2-1] to [2-19], wherein the peptide composition is obtained in a yield of 40% or more, or 50% or more.

[0033] A peptide composition obtained by any of the methods described in [2-21], [2-1], to [2-20]. [3-1] Suppressing gas generation by alkaline hydrolysis of solid proteins in a solution containing hydrogen peroxide. A method for solubilizing a solid protein containing, The solid protein is the eggshell membrane of birds, or the cartilage of mammals, birds, or fish. A method in which the gas contains hydrogen sulfide gas.

[0034] [3-2] The method according to [3-1], further comprising performing pH adjustment. [3-3] The method according to [3-1] or [3-2], further comprising desalting.

[0035] [3-4] The method described in [3-3], which uses ultrafiltration with an exclusion limit of 6,000 molecular weight to perform desalting. [3-5] The method according to any one of [3-1] to [3-4], further comprising sterilization.

[0036] [3-6] The method according to any of [3-1] to [3-5], wherein the solvent of the solution containing hydrogen peroxide is water. [3-7] The method according to any of [3-1] to [3-6], wherein the alkali used for alkaline hydrolysis is sodium hydroxide, potassium hydroxide, or sodium boro hydroxide.

[0037] [3-8] The method according to any of [3-1] to [3-7], wherein the solution before the start of alkaline hydrolysis contains alkali at a concentration of 5-50 mg / ml, 10-40 mg / ml, or 15-30 mg / ml.

[0038] [3-9] The method according to any of [3-1] to [3-8], wherein hydrogen peroxide is present in the solution before the start of alkaline hydrolysis at a concentration of 5-50 mg / ml, 5-30 mg / ml, or 10-20 mg / ml.

[0039] [3-10] The method according to any of [3-1] to [3-9], wherein the amount of solid protein subjected to the reaction is 10 to 5000 g, 50 to 300 g, or 100 to 300 g. [3-11] The method according to any of [3-1] to [3-10], wherein the alkaline hydrolysis is carried out at a temperature of 10°C to 80°C, 30°C to 60°C, or 30°C to 50°C.

[0040] [3-12] The method according to any of [3-1] to [3-11], wherein the reaction time for alkaline hydrolysis is 1 to 24 hours, 3 to 20 hours, or 15 to 18 hours. [3-13] The method according to any of [3-1] to [3-12], wherein the birds are selected from one or more of the group consisting of chickens, quail, turkeys, ducks and geese.

[0041] [3-14] The method according to any of [3-1] to [3-13], wherein the solid protein is chicken or quail eggshell membrane. [3-15] The method according to any of [3-1] to [3-14], wherein the amount of gas generated during the alkaline hydrolysis process is 5.0 ppm or less, 3.0 ppm or less, or 2.0 ppm or less.

[0042] [3-16] The method according to any of [3-1] to [3-15], wherein the amount of gas generated during the alkaline hydrolysis process is 50% or less, 30% or less, or 20% or less compared to when a solid protein is alkaline hydrolyzed under identical conditions except that hydrogen peroxide solution is not included.

[0043] [3-17] The method according to any one of [3-1] to [3-16], wherein the total amount of gas generated throughout the entire process of manufacturing the peptide composition is 10.0 ppm or less, 5.0 ppm or less, or 3.0 ppm or less.

[0044] [3-18] The method according to any one of [3-1] to [3-17], wherein the amount of gas generated throughout the entire process of manufacturing the peptide composition is 50% or less, 30% or less, or 20% or less compared to when a solid protein is alkaline hydrolyzed under identical conditions except that hydrogen peroxide is not included. [Effects of the Invention]

[0045] According to the present invention, gas generation during alkaline hydrolysis can be suppressed, and peptide compositions can be produced safely and in high yield. Furthermore, solid proteins can be safely solubilized (peptidized). [Modes for carrying out the invention]

[0046] One aspect of the present invention is a method for suppressing gas generation in the production of a peptide composition by alkaline hydrolysis of a solid protein, comprising alkaline hydrolysis of the solid protein in a solution containing hydrogen peroxide. In one aspect of the present invention, the solid protein is the eggshell membrane of a bird, or the cartilage of a mammal, bird, or fish, and the gas is hydrogen sulfide gas.

[0047] Alkaline hydrolysis In one embodiment of the present invention, the alkaline solution mediating alkaline hydrolysis comprises an organic solvent selected from methanol, ethanol, acetone, n-propanol, and isopropanol, or water, or a combination thereof, as the solvent. In one embodiment of the present invention, the solvent in the alkaline solution mediating alkaline hydrolysis is water. In one embodiment of the present invention, the alkaline solution comprises sodium hydroxide, potassium hydroxide, or sodium borohydrate as the alkali. The concentration of alkali in the alkaline solution before the start of alkaline hydrolysis is 5-50 mg / ml, 10-40 mg / ml, or 15-30 mg / ml.

[0048] In one embodiment of the present invention, the alkaline solution before the initiation of alkaline hydrolysis contains hydrogen peroxide at concentrations of 5-50 mg / ml, 5-30 mg / ml, or 10-20 mg / ml. If the amount of hydrogen peroxide exceeds 50 mg / ml, residual hydrogen peroxide may remain in the final product.

[0049] In one embodiment of the present invention, alkaline hydrolysis is carried out at temperatures of 10°C to 80°C, 30°C to 60°C, or 30°C to 50°C. In one embodiment of the present invention, the reaction time for alkaline hydrolysis is 1 to 24 hours, 3 to 20 hours, or 15 to 18 hours.

[0050] In one embodiment of the present invention, the gas generated by alkaline hydrolysis includes hydrogen sulfide. In one embodiment of the present invention, the amount of gas generated during the alkaline hydrolysis process is 5.0 ppm or less, 3.0 ppm or less, or 2.0 ppm or less. In one embodiment of the present invention, the amount of gas generated during the alkaline hydrolysis process is 50% or less, 30% or less, or 20% or less compared to when solid protein is alkaline hydrolyzed under the same conditions except that hydrogen peroxide solution is not included.

[0051] In one aspect of the present invention, the total amount of gas generated throughout the entire process of producing the peptide composition is 10.0 ppm or less, 5.0 ppm or less, or 3.0 ppm or less. Furthermore, the amount of gas generated throughout the entire process of producing the peptide composition is 50% or less, 30% or less, or 20% or less compared to when solid protein is alkaline hydrolyzed under identical conditions except for the absence of hydrogen peroxide.

[0052] Solid proteins In one embodiment of the present invention, the solid protein is a tissue of a vertebrate, such as a mammal, bird, or fish, such as eggshell membrane or cartilage. For example, it may be a bird's eggshell membrane, or cartilage from a mammal, bird, or fish.

[0053] Peptide composition In one embodiment of the present invention, the peptide composition may include avian eggshell membrane, or a degradation product of cartilage from mammals, birds, or fish.

[0054] In one embodiment of the present invention, the molecular weight range of the peptide in the peptide composition is 10,000 to 75,000 and 15,000 to 25,000. In one embodiment of the present invention, in the production of a peptide composition, the solid protein may be subjected to alkaline hydrolysis followed by a heating step, a pH adjustment step, a desalting step, and a sterilization step.

[0055] In one embodiment of the present invention, the peptide composition is heated to a temperature of 70-200°C, 70-100°C, or 70-90°C during the heating step. The heating step is sustained for 10-120 minutes, 10-60 minutes, or 10-40 minutes.

[0056] In one embodiment of the present invention, the peptide composition is adjusted to a pH of 8-9, for example, 8.1-8.9, during the pH adjustment step. In one embodiment of the present invention, the pH of the obtained peptide composition is 6.0-8.5. In one embodiment of the present invention, hydrochloric acid, acetic acid, or sulfuric acid may be used as the pH adjuster.

[0057] In one embodiment of the present invention, desalting is performed in the desalting step using ultrafiltration, gel filtration, or electrodialysis. In one embodiment of the present invention, the exclusion limit for ultrafiltration is a molecular weight of 6,000.

[0058] In one embodiment of the present invention, the yield of the peptide composition obtained from the solid protein is 40% or more, or 50% or more. In one aspect of the present invention, the amount of salt contained in the obtained peptide composition is 15.0% or less, 10.0% or less, or 7.0% or less. In addition, the amount of salt equivalent to sodium chloride is 37.5% or less, 25.0% or less, or 17.5% or less.

[0059] In one embodiment of the present invention, the obtained peptide composition may be in powder form. In one aspect of the present invention, the amount of residual hydrogen peroxide in the peptide composition is 250 ppm or less, 150 ppm or less, or 50 ppm or less.

[0060] Purpose In one embodiment of the present invention, the peptide composition can be used in cosmetics, pharmaceuticals, quasi-drugs, or food products. [Examples]

[0061] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. <Example 1> Preparation of eggshell membrane hydrolysate by the method of the present invention 40 g of KOH was dissolved in 2000 ml of water, and then 40 ml of 30% hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code: 081-04215) was added to prepare an alkaline aqueous solution. 150 g of powdered quail eggshell membrane (manufactured by Tengu Canned Foods Co., Ltd.) was added to the alkaline aqueous solution, and after heating to 40°C, it was stirred for 16 hours to hydrolyze it and obtain an eggshell membrane hydrolysate (extraction step). Next, the alkaline aqueous solution containing the eggshell membrane hydrolysate was heated to 85 ± 5°C and maintained at the same temperature for 30 minutes while stirring (heating step). Hydrochloric acid was added to the aqueous solution to adjust the pH to 8.5 and it was rapidly cooled to 60°C (pH adjustment step). The pH was measured using a YOKOGAWA Personal pH / ORP Meter (model PH72, electrode pH72SN). The aqueous solution was clarified by diatomaceous earth filtration, and then desalted by ultrafiltration with an exclusion limit of 6,000 Da (desalting step). Next, hydrochloric acid was added again to adjust the pH to 6.5, and then the mixture was heated to 90°C for 30 minutes for sterilization (sterilization step) before being spray-dried. The yield of the obtained peptide composition was 50-60% by weight relative to the raw material quail eggshell membrane. At this time, the potassium ion concentration measured with a HORIBA compact potassium ion meter LAQUA twin K-11 was 4.4-8.6%.

[0062] <Comparative Example 1> Preparation of eggshell membrane hydrolysate by conventional method Eggshell membrane hydrolysates were prepared in the same manner as in Example 1, except that hydrogen peroxide was not added, and subjected to the same heating, pH adjustment, desalting, and sterilization processes as in Example 1.

[0063] For the samples obtained in Example 1 and Comparative Example 1, the generation of hydrogen sulfide at each of the above steps was measured. Hydrogen sulfide was measured by aspirating gas from 1-2 cm above the liquid surface using a gas detection tube (Gastec, product name: hydrogen sulfide, part number: 4LB). In addition, residual hydrogen peroxide was measured using a hydrogen peroxide 0-400 test strip (Serim Research, part number: 5202).

[0064] Table 1 shows the hydrogen sulfide concentration at each stage, and Table 2 shows the residual hydrogen peroxide concentration.

[0065] [Table 1]

[0066] [Table 2]

[0067] Table 1 shows that the amount of hydrogen sulfide generated during the extraction process in Example 1 was 1.0 ppm, which is lower than that in Comparative Example 1 (6.0 ppm or higher). Furthermore, Table 1 shows that in Example 1, the generation of hydrogen sulfide was completely suppressed to below the detection limit even in the processes after heating.

[0068] Table 2 shows that in Example 1, residual hydrogen peroxide is below the detection limit after the heating step. From these results, it is understood that the method of the present invention using hydrogen peroxide is a very safe method that allows for the acquisition of only the target peptide, and that the method of the present invention is a manufacturing method that can replace conventional methods.

[0069] This example illustrates a process for obtaining eggshell membrane peptides, but it is also possible to obtain high-quality products in a safe working environment without generating harmful gases such as hydrogen sulfide, using cartilage from other animals or fish, etc.

Claims

1. A method for suppressing gas generation in the production of peptide compositions by alkaline hydrolysis of solid proteins, This includes alkaline hydrolysis of a solid protein in a solution containing hydrogen peroxide. The solid protein is the eggshell membrane of birds, or the cartilage of mammals, birds, or fish. A method in which the gas contains hydrogen sulfide gas.

2. The method according to claim 1, wherein the solvent of the solution containing hydrogen peroxide is water.

3. The method according to claim 1 or 2, wherein the alkali used in alkaline hydrolysis is sodium hydroxide, potassium hydroxide, or sodium boro hydroxide.

4. The method according to any one of claims 1 to 3, wherein the solution before the start of alkaline hydrolysis contains alkali at a concentration of 5 to 50 mg / ml, 10 to 40 mg / ml, or 15 to 30 mg / ml.

5. The method according to any one of claims 1 to 4, wherein hydrogen peroxide is contained in the solution before the commencement of alkaline hydrolysis at a concentration of 5 to 50 mg / ml, 5 to 30 mg / ml, or 10 to 20 mg / ml.

6. The method according to any one of claims 1 to 5, wherein the amount of solid protein subjected to the reaction is 10 to 5000 g, 50 to 300 g, or 100 to 300 g.

7. The method according to any one of claims 1 to 6, wherein the alkaline hydrolysis is carried out at a temperature of 10°C to 80°C, 30°C to 60°C, or 30°C to 50°C.

8. The method according to any one of claims 1 to 7, wherein the reaction time for alkaline hydrolysis is 1 to 24 hours, 3 to 20 hours, or 15 to 18 hours.

9. The method according to any one of claims 1 to 8, wherein the birds are one or more selected from the group consisting of chickens, quail, turkeys, ducks and geese.

10. The method according to any one of claims 1 to 9, wherein the solid protein is chicken or quail eggshell membrane.

11. The method according to any one of claims 1 to 10, wherein the amount of gas generated during the alkaline hydrolysis process is 5.0 ppm or less, 3.0 ppm or less, or 2.0 ppm or less.

12. The method according to any one of claims 1 to 11, wherein the amount of gas generated during the alkaline hydrolysis process is 50% or less, 30% or less, or 20% or less compared to when a solid protein is alkaline hydrolyzed under identical conditions except that hydrogen peroxide solution is not included.

13. The method according to any one of claims 1 to 12, wherein the amount of residual hydrogen peroxide in the peptide composition is 250 ppm or less, 150 ppm or less, or 50 ppm or less.

14. The method according to any one of claims 1 to 13, wherein a peptide composition is obtained in a yield of 40% or more, or 50% or more.

15. The process of suppressing gas generation by alkaline hydrolysis of solid proteins in a hydrogen peroxide-containing solution. A method for producing a peptide composition containing, The solid protein is the eggshell membrane of birds, or the cartilage of mammals, birds, or fish. A method in which the gas contains hydrogen sulfide gas.

16. The process of suppressing gas generation by alkaline hydrolysis of solid proteins in a hydrogen peroxide-containing solution. A method for solubilizing a solid protein containing, The solid protein is the eggshell membrane of birds, or the cartilage of mammals, birds, or fish. A method in which the gas contains hydrogen sulfide gas.

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