JP5829844B2 - Method for producing water-soluble elastin peptide - Google Patents

Description

  The present invention relates to a method for producing a water-soluble elastin peptide derived from elastin.

  Conventionally, peptides are produced by enzymatic treatment of arterial spheres such as tuna. For example, Patent Document 1 describes that after elastin-containing meat is shredded and subjected to alkali decomposition, insoluble matter is recovered by filtration, and thermolysin and papain are added to the insoluble matter to decompose elastin. ing. Patent Document 2 describes that a soluble peptide containing high elastin is obtained by using a fish arterial sphere as a raw material and then removing blood, lipids, soluble proteins, and collagen, followed by solubilization treatment. Patent Document 3 describes that the arterial sphere pieces of fish after washing are solubilized with a proteolytic enzyme as it is.

JP 2010-239919 A JP 2007-151453 A JP 2010-241708 A

  However, in any of the above methods, since an alkaline aqueous solution such as an aqueous sodium hydroxide solution is used in the washing step before the enzyme treatment, there is a risk in the washing step. In addition, there is a problem that a cleaning process and a neutralization process for removing alkali are required before obtaining a final product, which complicates the process. Furthermore, there is a possibility that an alkaline substance may remain in the final product due to insufficient washing or neutralization, and there is a problem that safety is low.

  The present invention has been made in view of the above points, and an object of the present invention is to provide a highly safe method for producing a water-soluble elastin peptide by a simple process.

The method for producing a water-soluble elastin peptide according to the present invention comprises removing an elastin-containing product using an alkaline protease, washing it, then inactivating the alkaline protease, and then further using the new alkaline protease. It is characterized by decomposing elastin in the elastin-containing material after washing into water-soluble elastin peptide.

The elastin-containing material is preferably a fish arterial sphere.
Moreover, in the said washing | cleaning, it is preferable to make the said alkaline protease into the mixture ratio of 0.1-0.5 weight part with respect to 100 weight part of said elastin containing materials.

  By washing the elastin-containing material with an alkaline protease, the elastin-containing material can be washed without using an alkaline aqueous solution or an organic solvent, and safety can be improved. Further, since no alkaline aqueous solution or organic solvent is used, washing for neutralizing or removing them is not necessary, and the process can be simplified.

It is a flowchart which shows the process of an Example. It is a flowchart which shows the process of a comparative example. It is a graph which shows the molecular weight distribution of an Example.

  Hereinafter, modes for carrying out the present invention will be described.

  In the present invention, elastin in an elastin-containing product is decomposed into a water-soluble elastin peptide. In this case, all of the elastin in the elastin-containing material may be decomposed into peptides, or part of the elastin in the elastin-containing material may be decomposed into peptides, and the remainder may be decomposed into amino acids. Any elastin-containing material may be used as long as it contains elastin, but it is preferable to use a fish portion rich in elastin. Use of fish sites can reduce the chances of being infected with viruses and bacteria like mammalian tissues. Arterial spheres can be exemplified as fish sites rich in elastin, which makes it easier to obtain specific amino acids contained in elastin such as desmosine and isodesmosine. In addition, among fish, it is preferable to use fish belonging to the genus Tuna genus, Sabaceae, which contains abundant elastic fibers in the arterial sphere. Specifically, arterial spheres such as bluefin tuna, southern bluefin tuna, bigeye, yellowfin, albacore, cocinaga and Atlantic tuna can be used.

  The present invention includes a washing step, a solubilization step, and a solid-liquid separation step. Moreover, you may perform a drying process after a solid-liquid separation process as needed.

[Washing process]
The washing step is a step of removing contaminants such as blood, lipids and collagen adhering to and remaining on the elastin-containing material. In the present invention, in this washing step, after washing with water, washing with an alkaline protease (endopeptidase) is performed, so that compared with washing with hot water alone, the water-soluble elastin peptide of the final product is not brominated. Can be improved. In addition, when alkaline protease is used, there are fewer problems with corrosion of manufacturing equipment and safety for workers compared to washing with alkaline chemicals and organic solvents. There is no need for desalination work and large ultrafiltration equipment.In addition, due to lack of washing when washing alkaline chemicals or organic solvents with water, elution of off-flavors and poor taste in the water-soluble elastin peptide of the final product You don't have to worry about the problem. In addition, when alkaline protease is used, the removal rate of contaminants is less likely to be lower than when acidic protease or neutral protease is used, and the elastin body is hardly degraded.
As the alkaline protease, an enzyme having an optimum pH of 6 or more is preferable. , Penicillium citrinum, Rhizomucor miehei, Rhizopus chinensis, Rhizopus delemar, Rhizopus niveus, basidiomycete (Pycnopporrus coccineus), radiophile (Streptomyces), bacteria (Bacillus amyloliquefaciens, Bacillus coaglans, lenti , Bacillus subtilis, Bacillus thermoproteolyticus, Pseudomonas paucimobilis) or yeast (Saccharomyces) or the like. The washing can be performed by immersing the elastin-containing material in water and then adding an alkaline protease. In this case, it is preferable to use 200 to 800 parts by weight of water and 0.1 to 0.5 parts by weight of alkaline protease for 100 parts by weight of the elastin-containing material. Within this range, the amount of water and alkaline protease is not too much or too little with respect to the elastin-containing material, washing can be performed sufficiently and the alkaline protease can be made difficult to be wasted. Moreover, it is preferable that the alkaline protease aqueous solution in the case of washing | cleaning shall be pH 6.0-7.5. Within this range, the activity of alkaline protease is unlikely to be impaired, and washing can be performed sufficiently. Moreover, it is preferable that the temperature of alkaline protease aqueous solution in the case of washing | cleaning is 50-70 degreeC. Within this range, the activity of alkaline protease is unlikely to be impaired, and washing can be performed sufficiently. The washing time (immersion time) of the elastin-containing material is preferably 5 to 10 minutes. Within this range, the alkaline protease can sufficiently act on the elastin-containing material, so that the washing can be sufficiently performed and the washing process can be prevented from being prolonged.

  The washing step is terminated after separating the alkaline protease aqueous solution and the elastin-containing material after deactivating the alkaline protease.

[Solubilization process]
The solubilization process involves hydrolyzing part or all of the elastin in the elastin-containing product after the washing process into peptides and amino acids, solubilizing (water-solubilizing) elastin, which is essentially an insoluble protein, and a commercially available product It is easy to process as.

  In the present invention, an alkaline protease (alkaline endopeptidase) is used in this solubilization step. Thus, compared to the case of using an acidic protease or a neutral protease, a filter paper or a filter is used in the solid-liquid separation step. The clogging of cloth is less likely to occur, the filterability and the workability of filtration are improved, the odor of elastin-containing materials such as fish odor is almost eliminated, and the yield of water-soluble elastin peptide is also improved by about 20-25% Can be made.

  Here, the alkaline protease used in the solubilization step may be the same as or different from the alkaline protease used in the washing step. pilosus, Mucor circinelloides, Mucor javanicus, Mucor miehei, Mucor rouxii, Penicillium citrinum, Rhizomucor miehei, Rhizopus chinensis, Rhizopus delemar, Rhizopus niveus), basidiomycete (Pycnopporrus coccineus), ces , J4, Bacillus lentus, Bacilluslicheniformis, Bacillus polymixa, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thermoproteolyticus, Pseudomonas paucimobilis) or yeast (Saccharomyces), etc. Can be used.

  The solubilization step can be performed by immersing the elastin-containing material in water and then adding an alkaline protease. In this case, it is preferable to pulverize (mince) the elastin-containing material in order to increase the reactivity. In the solubilization step, it is preferable that the mixing ratio is 200 to 800 parts by weight of water and 0.1 to 0.7 parts by weight of alkaline protease with respect to 100 parts by weight of the elastin-containing material. Within this range, water and alkaline protease are not too much or too little of the elastin-containing material, and elastin can be sufficiently reduced in molecular weight by enzymatic treatment (hydrolysis), making it water-soluble. The yield of elastin peptide can be improved. Moreover, it is preferable that pH of the alkaline protease aqueous solution in the solubilization step is 6.0 to 7.5. Within this range, the activity of alkaline protease is unlikely to be impaired, and the enzyme treatment can be sufficiently performed. Moreover, it is preferable that the temperature of alkaline protease aqueous solution in the case of a solubilization process is 50-70 degreeC. Within this range, the activity of alkaline protease is unlikely to be impaired, and the enzyme treatment can be sufficiently performed. The enzyme treatment time (immersion time) of the elastin-containing material is preferably 1 to 24 hours. Within this range, the alkaline protease can sufficiently act on the elastin-containing material, the enzyme treatment can be sufficiently performed, and the solubilization process can be prevented from being prolonged.

  Then, by performing enzyme inactivation of alkaline protease after the enzyme treatment, the solubilization step is completed, and an aqueous solution containing an elastin-derived peptide or amino acid can be obtained.

[Solid-liquid separation process]
The solid-liquid separation step is a step in which the aqueous solution containing the elastin-derived peptide or amino acid after the solubilization step is treated with activated carbon to remove impurities by adsorption, and then separated into a liquid and a solid. The liquid contains an elastin-derived peptide and, in some cases, an elastin-derived amino acid. The solid is a component (residue) that has not been decomposed in the solubilization process. The solid-liquid separation step can be performed by a perforated wall centrifuge, a non-porous wall centrifuge, a pressure filter, a filter cloth, a filter paper, or the like.

  In the present invention, an elastin peptide dissolved in water can be obtained by a washing step, a solubilizing step and a solid-liquid separation step. Moreover, the water-soluble elastin peptide powder derived from a powdery elastin containing material can be obtained by concentrating and drying this elastin peptide containing aqueous solution. The water-soluble elastin peptide powder may contain an amino acid powder.

  As described above, the water-soluble elastin peptide obtained in a dissolved or powdered form in an aqueous solution can be used as a raw material for foods and cosmetics. Elastin-derived elastin peptide has an effect of promoting cell growth and collagen production, and oral ingestion is expected to improve skin elasticity and flexibility and improve skin texture and wrinkles. In particular, skin quality is expected to be improved by ingesting a water-soluble elastin peptide derived from elastin together with collagen, hyaluronic acid, and chondroitin sulfate.

  In the present invention, since alkaline protease is used in the washing step, the yield of the water-soluble elastin peptide can be increased as compared with the case of washing with an alkaline aqueous solution or an organic solvent. This is because (1) a long treatment is required when washing with an alkaline aqueous solution or the like, and elastin peptide may be eluted at the same time when washing the alkali with water, 2) When an enzyme treatment is carried out using acidic or neutral protease, especially in the case of fish, the filtration rate becomes extremely slow, and undegraded products remain on the filter paper or filter cloth, which causes clogging. The reason is that a degradation product (peptide or amino acid) cannot be efficiently obtained in the filtrate.

  Hereinafter, the present invention will be specifically described by way of examples.

(Example)
A water-soluble elastin peptide powder was produced by the process shown in FIG.

[Washing process]
200 g of tuna (south tuna) arterial spheres were prepared and immersed in 1000 g of water five times the weight of the arterial spheres and held for 0.5 hours. In this way, washing with water was performed, and blood, lipids and the like adhering to and remaining on the arterial sphere were roughly washed. Thereafter, the washing solution (water used for washing) and the arterial sphere are separated by filtration. The biuret reaction of this washing solution was negative (no 365 nm blue fluorescence).

  Next, the arterial sphere after washing with water is mixed with 1000 g of hot water, and further, an alkaline protease (Orientase 22BF (HBI Enzymes Inc.)) is added at 0.1% based on the weight of the arterial sphere. Washing with an aqueous solution was performed. At this time, the temperature of the aqueous alkaline protease solution was 60 ° C., the immersion time was 10 minutes, and the pH was 6.2. Next, the alkaline protease was inactivated by maintaining the temperature of the aqueous alkaline protease solution at 90 ° C. for 10 minutes. After inactivation, the aqueous alkaline protease solution had a Brix of 3.6% and a pH of 6.2.

  Next, the alkaline protease aqueous solution after enzyme deactivation was filtered to separate it into a washing solution and an arterial sphere. The biuret reaction of this washing solution was negative (no 365 nm blue fluorescence).

[Solubilization process]
After pulverizing the arterial sphere after the washing step (to mince), 400 g of water is added thereto, and 0.5% of an alkaline protease (Orientase 22BF (HBI Enzymes Inc.)) is added to the enzyme. Treatment (hydrolysis treatment) was performed. The temperature of the alkaline protease aqueous solution at this time was 60 ° C., the reaction time was 60 minutes, and the pH was 6.2. Next, the alkaline protease was inactivated by maintaining the temperature of the aqueous alkaline protease solution at 90 ° C. for 10 minutes. After the inactivation, the aqueous alkaline protease solution had a Brix of 3.2% and a pH of 6.2. The aqueous alkaline protease solution after inactivation is an elastin peptide-containing aqueous solution containing an elastin-derived peptide or amino acid.

[Solid-liquid separation process]
The aqueous solution containing the elastin peptide obtained in the solubilization step was brought into contact with activated carbon to perform activated carbon treatment. The aqueous solution containing the elastin peptide treated with activated carbon had a Brix of 3.0% and a pH of 5.9.

  Next, the elastin peptide-containing aqueous solution after the activated carbon treatment was filtered using a pressure filter to separate the residue and the filtrate. The filtrate had a Brix of 3.0% and a pH of 5.8. Next, the filtrate was concentrated to a liquid volume of 68.3 g, Brix of 34%, and pH of 5.9. Next, the concentrated solution was spray-dried to obtain a powdery elastin peptide. 19.1 g of this powder was obtained, and water-soluble elastin peptide powder was obtained from 200 g of arterial sphere with a yield of 9.5%. This powder was positive for the biuret reaction (having 365 nm blue fluorescence).

(Comparative Example 1)
A water-soluble elastin peptide powder was produced by the process shown in FIG.

[Washing process]
200 g of a tuna arterial sphere similar to the example was prepared and immersed in 1000 g of water five times the weight of the arterial sphere and held for 0.5 hour. In this way, washing with water was performed, and blood, lipids and the like adhering to and remaining on the arterial sphere were roughly washed. Thereafter, the washing solution (water used for washing) and the arterial sphere were separated by filtration. The biuret reaction of this washing solution was negative (no 365 nm blue fluorescence).

  Next, washing was performed by mixing the arterial sphere after washing with 1000 g of hot water. The temperature of the hot water at this time was 60 ° C., and the immersion time was 2 hours. Next, it was separated into a washing solution (hot water) and an arterial sphere by filtration. The biuret reaction of this washing solution was negative (no 365 nm blue fluorescence).

[Solubilization process]
After pulverizing the arterial sphere after the washing step (to mince), 400 g of water was added thereto, and then 0.5% of neutral protease (Orientase 90N (HBI Enzymes Inc.)) was added. Enzymatic treatment (hydrolysis treatment) was performed. At this time, the temperature of the neutral protease aqueous solution was 60 ° C., the immersion time was 60 minutes, and the pH was 6.2. Next, the neutral protease was inactivated by maintaining the temperature of the neutral protease aqueous solution at 90 ° C. for 10 minutes. The neutral protease aqueous solution after inactivation is an aqueous solution containing an elastin-derived peptide or amino acid.

[Solid-liquid separation process]
The water-soluble elastin peptide-containing aqueous solution obtained in the solubilization step was brought into contact with activated carbon to perform activated carbon treatment. Next, the water-soluble elastin peptide-containing aqueous solution after the activated carbon treatment was filtered to separate the residue and the filtrate. Next, the filtrate was concentrated, the liquid volume was 60.0 g, and Brix was 26%. Next, the concentrated solution was spray-dried to obtain a powdery water-soluble elastin peptide. 15.5 g of this powder was obtained, and water-soluble elastin peptide powder was obtained from 200 g of arterial sphere with a yield of 7.8%. In addition, this powder had a positive biuret reaction (with 365 nm blue fluorescence).

(Comparative Example 2)
In Comparative Example 1, an alkaline aqueous solution was used instead of hot water in [Washing Step]. Sodium hydroxide was used as the alkali and the concentration was 0.1%. Other configurations were the same as those in Comparative Example 1. In this case, it took 3 to 4 days to wash the arterial spheres by removing alkali, and since the washing solution also showed a positive biuret reaction, it was judged to be impractical and the subsequent treatment was stopped.

  The amino acid composition of the water-soluble elastin peptide powder obtained from the examples was measured. The results are shown in Table 1. The amino acid composition was measured as follows.

(Preparation of standard solution)
An amino acid standard solution (amino acid mixed standard solution, type H) and elastin, desmosine, tryptophan, and hydroxyproline were made up to a constant volume with 0.01 M hydrochloric acid to obtain a standard solution.

(Preparation of sample solution)
An appropriate amount of the elastin peptide powder obtained in the examples was taken, 6M hydrochloric acid was added, hydrolyzed at 110 ° C. for 24 hours, neutralized, and then constant volume with 0.01M hydrochloric acid to obtain a sample solution. Cystine was subjected to hydrolysis with performic acid in advance and then hydrolyzed. Moreover, the sample which hydrolyzed using the alkali was used for the measurement of tryptophan.

(Quantitative)
The content of each amino acid, desmosine, and isodesmosine was measured for the standard solution and the sample solution by a liquid chromatography method or the like. The content of tryptophan was measured by reverse phase HPLC.

  Moreover, about the Example, the nutrient component value of water-soluble elastin peptide powder was measured. The results are shown in Table 2.

  Moreover, about the Example, molecular weight distribution of the water-soluble elastin peptide powder was measured. The results are shown in Tables 3 and 4 and FIG. The molecular weight distribution was measured as follows.

(Preparation of standard solution)
As molecular weight markers, (1) Cytochrome C (MW = 12,500), (2) Aprotinin (MW = 6512), (3) Bacitracin (MW = 1450), (4) Angiotensin II (MW) = 1046), (5) Gly-Gly-Tyr-Arg (MW = 451), (6) Gly-Gly-Gly (MW = 189), taking appropriate amounts, based on elution time and molecular weight A calibration curve was created.

(Preparation of sample solution)
An appropriate amount of the water-soluble elastin peptide powder obtained in the examples was taken and dissolved by adding a mobile phase to obtain a sample solution.

(Measurement)
Based on the calibration curve prepared using each standard solution, the molecular weight at each peak in the sample solution was examined under the following conditions.
[HPLC conditions]
Column; TSKgel G2500PW 7.8mm x 300mm
Column temperature: 40 ° C
Mobile phase; water / acetonitrile / T. F. A. Mixed liquid (55/45 / 0.1)
Measurement wavelength: 220 nm

Claims (3)

After removing impurities and washing the elastin-containing material using an alkaline protease, the alkaline protease is deactivated, and then the elastin in the washed elastin-containing material is further dissolved in water-soluble elastin using a new alkaline protease. A method for producing a water-soluble elastin peptide, which comprises decomposing the peptide into peptides. The method for producing a water-soluble elastin peptide according to claim 1, wherein the elastin-containing substance is a fish arterial sphere. 3. The water-soluble elastin peptide according to claim 1, wherein in the washing, the alkaline protease is mixed in an amount of 0.1 to 0.5 parts by weight with respect to 100 parts by weight of the elastin-containing material. Manufacturing method.

Images