JP2000053696A - Hydrolyzed poriferan protein and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively utilize dried poriferan sponge which is a scrap of sponge used for body-cleaning or make-up and hitherto discarded as a waste. SOLUTION: The objective hydrolyzed poriferan protein is produced by dissolving a porifer and neutralizing the supernatant of the solution or further treating the obtained poriferan protein solution with a proteinase. A functional low-molecular peptide and/or amino acid produced by treating the hydrolyzed poriferan protein with an ultrafiltration membrane, precision filtration membrane, gel-filtration membrane or ion-exchange resin are used as a food raw material, a raw material for pet food, cosmetics, pharmaceuticals or an agent having proliferation promoting action on the cells originated from mammalian.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a food material, a pet food material, a cosmetic or pharmaceutical, and a cell derived from a mammal obtained by dissolving a dried sponge and then neutralizing the supernatant. The present invention relates to a sponge protein solution for use in applications having a growth promoting action. In addition, the present invention provides a raw material for food and a raw material for pet food obtained by dissolving a dried sponge and then neutralizing the supernatant to obtain a protein solution of the sponge, which is then reacted with a protein hydrolase. The present invention relates to a sponge protein hydrolyzate for use in cosmetics, pharmaceuticals, and applications having a growth-promoting effect on mammalian cells, and a method for producing the same. In addition, the present invention provides a method for ultrafiltration of a sponge protein hydrolyzate obtained by allowing a protein hydrolase to act on a sponge protein solution obtained by dissolving a dried sponge and then neutralizing the supernatant. Membrane, microfiltration membrane, gel filtration or purification by using ion exchange resin, for use in foods, pet foods, cosmetics or pharmaceuticals, and in applications that have a growth promoting effect on mammalian cells The present invention relates to a functional low-molecular peptide and / or amino acid to be provided and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, useful proteins are separated from those normally discarded, such as blood coming out at the time of slaughter for animal meat, or stick water, fish water-exposed waste liquid concentrate, and broth produced during canning for fish and shellfish. -Attempts to purify have been made. However, it is a fibrous sponge,
No information on the effective use of proteins from sponges is known. Sponge animals have conventionally been used as sponges for washing and makeup of the body, and are still favored as high-grade sponges. The reason is that sponges are rich in water absorption and have a very good skin feel compared to other natural and synthetic sponges, but they are almost effectively used in fields other than cleaning and cosmetics. It is not at present. Further, for the portion used as the above-mentioned sponge, a useful use was not found, and it had to be discarded. When analyzing the components of these fibrous substances used as sponge sponges, most of them are proteins except for water, and amino acid analysis of the proteins reveals the usefulness of glycine, aspartic acid, glutamic acid, proline, arginine, etc. It has been found to contain a lot of unique amino acids, but no useful use has yet been found.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies on the effective use of sponges, for example, a method of producing a protein hydrolyzate having high physiological activity and taste. It has been found that a protein hydrolyzate having high physiological activity and high taste can be produced by dissolving the protein contained in E. coli with an alkaline solution, an acidic solution or a protein hydrolase. That is, in the present invention, after dissolving a dried sponge, the sponge protein solution itself obtained by neutralizing the supernatant and the sponge protein solution obtained by neutralizing the supernatant are proteolytically hydrolyzed. The sponge protein hydrolyzate obtained by the action of an enzyme has extremely high physiological activity and taste, and is a raw material for food,
It has been discovered that it can be used for pet food materials, cosmetics or pharmaceuticals, and for applications having a growth promoting action on mammalian cells. Further, the protein hydrolyzate thus obtained is additionally subjected to an ultrafiltration membrane,
Functional low molecular peptides and amino acids obtained by purification and separation by microfiltration membrane, gel filtration, or ion-exchange resin are also used as raw materials for foods, raw materials for pet food, cosmetics or pharmaceuticals and mammals as well as protein hydrolysates. It has also been found that the cells have a growth promoting effect on cells.

[0004]

According to the present invention, there is provided a method for producing a lysate of sponge protein, comprising the steps of adding sodium hydroxide, potassium hydroxide, calcium hydroxide, water Alkali diluted solution such as magnesium oxide, preferably 0.01 to 50% by weight
And stir until well dissolved. Or
A highly acidic solution such as hydrochloric acid, sulfuric acid, nitric acid, formic acid or acetic acid, preferably 0.01 to 50% by weight, is added to the dry raw material, and the mixture is stirred until it is sufficiently dissolved. It is efficient to heat both the alkali dissolution and the acid dissolution to promote dissolution. Next, after dissolving the fibrous sponge, the fibrous sponge is neutralized, and the insoluble matter is removed by centrifugation or a filter cloth. Subsequently, the filtrate is treated with activated carbon to remove off-flavors and brown colors. At this stage, a clear viscous sponge protein lysate is obtained. Further, in the case of purifying the obtained protein lysate, salting out precipitation such as ammonium sulfate, precipitation using an organic solvent such as ethyl alcohol, methyl alcohol or acetone, gel filtration treatment, ion exchange resin treatment and the like are performed. It was found that the sponge-derived protein solution thus obtained can be used in a wide range of fields such as raw materials for foods, raw materials for pet foods, and raw materials for cosmetics and pharmaceuticals.

A method for producing a sponge hydrolyzate is as follows. Since a sponge protein solution is a polymer, it becomes very viscous at a high concentration and becomes difficult to handle. It is appropriate to do so. Degradation by this protein hydrolase not only solves the problem of handling by using the above-mentioned sponge protein lysate as a substrate for the protein hydrolase, but also reduces cell activation and anti-cancer effects by reducing the molecular weight. A certain physiologically active peptide or amino acid can be obtained. Further, the concentration of the sponge protein lysate as a substrate is not particularly limited, but is usually 0.1%.
A range of １５15% by weight is preferred. 1 protein concentration
When the amount exceeds 5% by weight, when the reaction product generated in the hydrolysis reaction is continuously separated by an ultrafiltration membrane or a microfiltration membrane as described later, clogging of the membrane is promoted to promote protein degradation. Reduces productivity. On the other hand, when the concentration is lower than 0.1% by weight, the concentration of the reaction product becomes excessively low, so that a large amount of energy and time are required for concentration of the proteolysate.

[0006] The protein hydrolase used in the method of the present invention is not particularly limited, and generally, a protein hydrolase derived from animals, plants, or microorganisms is preferably used. Depending on the purpose, endo- or exo-type protein hydrolases can be used alone or in a mixture at an appropriate ratio. Further, those in which the protein hydrolase is in a free state, one in which the protein hydrolase is cross-linked, one in which the protein hydrolase is included, or one in which the protein hydrolase is immobilized on an insoluble carrier. It is also possible to use

Further, the protein hydrolase used in the method of the present invention can be immobilized on an insoluble carrier by a method such as covalent bond, ionic bond, or physical adsorption. As an insoluble carrier, carboxymethyl cellulose,
Ethylene-maleic acid copolymer, carboxychloride resin, carbodiimide resin, acrylamide-methacrylic acid copolymer, cyanogen bromide-activated polysaccharide, DEAE-cellulose, DEAE-Sephadex, Amberli
Tes, activated carbon, porous glass, acid clay, silica gel, alumina, bentonite, polysulfone, polyamide, polyimide, polyethersulfone, cellulose acetate, polyacrylonitrile, and the like.

[0008] Further, those obtained by crosslinking protein hydrolases can be used. Glutaraldehyde, isocyanate derivatives, bisdiazobenzidine, N, N'-polymethylenebisiodoacetamide, N, N'-ethylenebismaleimide, etc. are used as the crosslinking agent. Furthermore, entrapping and immobilizing protein hydrolase in a matrix of polymer gel by encapsulating it in a state where it cannot be detached (lattice type) or coating the enzyme with a translucent polymer film ( (Microcapsule type). Examples of the polymer used as the lattice type include polyacrylamide gel, polyvinyl alcohol gel, silicon resin, starch matrix, konjac powder and the like. Examples of the microcapsule type polymer include nylon, polyurethane, ethyl cellulose, polystyrene, collodion, cellulose nitrate, cellulose butyl acetate, polyurea, and polyamide.

[0009] The sponge protein solution itself obtained by dissolving the dried sponge obtained in the present invention and neutralizing the supernatant with an alkaline solution or an acidic solution is used as a raw material for food, pet food. It has a growth promoting effect on raw materials, cosmetics or pharmaceuticals, and mammalian cells. Further, a protein hydrolyzate obtained by allowing a protein hydrolase to act on the obtained sponge protein solution can also be used for the same uses as the sponge protein solution. The protein hydrolase used at this time is not particularly limited, and may be in a free state, a cross-linked state between protein hydrolases, a protein hydrolase included, or immobilized on an insoluble carrier. What has been done can be used. As the protein hydrolase, endo-type or exo-type protein hydrolase can be used alone or in a mixture of both at an appropriate ratio.

As described above, the sponge protein hydrolyzate can be applied to foods, cosmetics, medicines and the like as it is, but can be concentrated by an appropriate concentration means as needed. As a concentration means, for example, a general means such as a heating evaporation method can be adopted, but the concentration can be easily and efficiently performed by a reverse osmosis method. Further, after the hydrolyzate is appropriately concentrated as described above, it can be pulverized by means such as spray drying. Furthermore, by obtaining a specific hydrolyzate fraction by means such as gel filtration, ion exchange resin, microfiltration membrane or ultrafiltration membrane, it is possible to obtain foods, cosmetics, pharmaceutical products or mammal-derived products with higher added value. Can be used for applications having a growth promoting effect on cells.

[0011]

The present invention will be described below with reference to examples.
The present invention is not limited by these examples.

Example 1 A dried fibrous sponge was cut into small pieces, and 900 g of a 20% by weight aqueous sodium hydroxide solution was added to 100 g of the small pieces, and the mixture was heated to 50 ° C. and stirred for 1 hour. After confirming dissolution, the mixture was neutralized with 30% by weight of hydrochloric acid, and the insoluble residue was removed with a filter cloth to obtain a supernatant. The color and odor of the supernatant were removed by pouring the supernatant into a Buchner funnel on which activated carbon had been previously laminated. This supernatant liquid could be used directly or as a raw material for fortified and functional foods and a raw material for pet foods by pulverizing with a spray dryer.

[0012]

Example 2 A dried fibrous sponge was cut into small pieces, and 100 g of the pieces were added with 900 g of a 30% by weight aqueous solution of potassium hydroxide, heated to 50 ° C., and stirred for 1 hour. After confirming dissolution, the mixture was neutralized with 30% by weight of hydrochloric acid, and the insoluble residue was removed by centrifugation to obtain a supernatant. An activated carbon-acid clay mixture (50% -5%) was added to 500 g of the supernatant.
(0%) was added, and the mixture was stirred for 30 minutes and then filtered using filter paper. Subsequently, for protein purification, the filtrate was sieved by gel filtration to collect a protein fraction having a molecular weight of about 50,000 to 1,000,000, and then concentrated with a reverse osmosis membrane. This concentrated solution was thinly coated on a PET film with a smoother or the like, and then dried to obtain a sponge protein thin film. The obtained thin film was usable as artificial skin for medical use.

[0013]

Example 3 After drying a dried fibrous sponge into small pieces, 9 kg of a 20% by weight aqueous sodium hydroxide solution was added to 1 kg of the small pieces, heated to 50 ° C., and stirred for 1 hour. After confirming dissolution, the mixture was neutralized with 30% by weight of hydrochloric acid, and the insoluble residue was removed by centrifugation to obtain a supernatant. Approximately 10 kg of the supernatant was added with 1 kg of activated carbon, stirred for 30 minutes, and filtered using filter paper. In order to subsequently hydrolyze the sponge protein solution, endo- and exo-type proteolytic enzymes derived from microorganisms were dried at 1 g dry weight.
Each 500 U was added per (the dry weight of the protein solution was measured in advance) and reacted at 50 ° C. for 8 hours. In addition, the reaction status of the hydrolysis was periodically confirmed by gel filtration. After completion of the reaction, the solution was desalted with a reverse osmosis membrane, and the reaction product was pulverized with a spray dryer. The obtained powder could be used as a seasoning powder containing low molecular weight peptides and amino acids.

[0014]

Example 4 An enzyme-free membrane bioreactor was constructed to hydrolyze the sponge protein solution obtained in Example 3. That is, 1 kg of a sponge protein solution and 1 U of a microbial endoprotease are added to a substrate tank per 1 g of dry weight (the dry weight of the protein solution is measured in advance), and the flow rate is set to 0.10 m / p by a pump. The mixture was adjusted to S and circulated until the temperature of the substrate-enzyme mixture in the substrate tank reached 50 ° C. After the temperature rise, the membrane pressure is increased to 0.5 kg / cm
^The mixture was adjusted to ² , and the reaction product was continuously collected as a filtrate by an ultrafiltration membrane (average molecular weight cut-off: 20,000). In addition, the sponge animal protein solution is supplied from the reservoir tank to the substrate tank by the amount obtained as the filtrate, and 500 U per 1 g of dry weight of the protease is measured once every 2 hours (the dry weight of the protein solution is measured in advance). Was added. After completion of the reaction, the solution was desalted with a reverse osmosis membrane, and the reaction product was pulverized with a spray dryer. The results of use are shown in Table 1.

[0015]

Example 5 In order to hydrolyze the sponge protein solution obtained in Example 3, instead of the ultrafiltration membrane of the enzyme-free membrane bioreactor of Example 4, a protease was previously introduced into the membrane. An ultrafiltration membrane (amount of enzyme immobilized: 50 U / cm ² -membrane area) immobilized on the sponge layer of Example 1 by glutaraldehyde by a crosslinking method was used. That is, after adding 1 kg of the sponge protein solution to the substrate tank, the flow rate was adjusted to 0.11 m / S by a pump, and the solution was circulated until the temperature of the substrate solution in the substrate tank reached 50 ° C. After the temperature rises, the membrane pressure is increased to 0.5 kg / cm ²
The reaction product was continuously collected as a filtrate using an enzyme-immobilized ultrafiltration membrane (average molecular weight cut off: 10,000). The sponge protein solution was supplied from the reservoir tank to the substrate tank in an amount obtained as the filtrate. After the reaction,
The filtrate as a reaction product was desalted with a reverse osmosis membrane and powdered with a spray dryer. The results of use are shown in Table 1.

[0016]

Example 6 An enzyme-immobilized column bioreactor was constructed to hydrolyze the plum wool protein solution obtained in Example 3. That is, endo-type and exo-type proteolytic enzymes derived from microorganisms were previously immobilized on the ion-exchange resin packed in the column by an ion binding method (immobilization amount: 20 U / cm ² -end-exchange resin surface area of the end-type). 10 exo type 35 U / cm ² -ion exchange resin surface area). In the reaction, after adjusting the column temperature to 45 ° C. with a heater, the substrate solution was supplied to the column at a flow rate of 10 ml / min from the substrate tank containing the sponge protein solution. The sponge protein solution was continuously supplied from the reservoir tank to the substrate tank in an amount obtained as a reaction product. After completion of the reaction, the reaction product was desalted with a reverse osmosis membrane and powdered with a spray dryer. The results of use are shown in Table 1.

[0017]

Example 7 After the dried fibrous sponge was cut into pieces,
A 20.0% by weight aqueous solution of hydrochloric acid 9 was added to 1.0 kg of the strips.
Then, the mixture was heated to 85 ° C. and stirred for 5 hours. After confirming dissolution, the mixture was neutralized with 20.0% by weight of sodium hydroxide, and the insoluble residue was removed by centrifugation to obtain a supernatant. 1 kg of activated carbon was added to 10 kg of the supernatant, stirred for 30 minutes, and then filtered using a filter paper. Subsequently, the filtrate was separated by gel filtration for protein purification, and the molecular weight was about 5%.
After collecting 000 to 500,000 protein fractions, they were concentrated with a reverse osmosis membrane. This concentrate was pulverized with a spray dryer. The sponge protein hydrolyzate powders obtained in Examples 4 to 7 were respectively added to a liquid for lotion. As controls, no sponge protein hydrolyzate powder was added (the weight loss was replaced with ion-exchanged water), collagen addition, silk protein (sericin) addition, and glycine addition were also prepared at the same time to improve the feeling of use on the skin. Examined. The results of use are shown in Table 1.

[0018]

[Table 1]

According to the results shown in Table 1, Examples 4 to 4 of the present invention are shown.
The sponge protein hydrolyzate obtained in 7 is conventionally
A feeling of use comparable to the formulations (control formulations 1 to 4) using proteins and amino acids used as cosmetic raw materials was obtained. It is clear that a great effect is exhibited in consideration of the economical cost of the material in the present invention.

[0020]

Example 8 Human-derived epidermis using the sponge protein hydrolyzed powder obtained in Examples 4 to 7 and the control as sponge protein and hydrolyzed collagen before activated carbon treatment in Examples 4 to 7 The growth promoting effect on keratinocytes was examined. That is, each protein concentration was calculated from the amount of nitrogen obtained by the Geldar method.
Adjusted to 0%. 2.0 wells of F-12 medium (hydrocortisone / adenine / fetal calf serum added) were added to a 24-well culture plate, and the number of keratinocytes was 2 × 10 5
⁶ /, the final concentration of sponge protein was 0.003125-
The cells were cultured under conditions of 0.1%, 30 ° C. and 5.0% CO ₂ for 7 days. After the culture, the number of viable cells
TT (3- [4,5-dimethylethiazol-2-yl] -2,5-diphenyltet
razoliumbromide) (Optical Density: 570n
m-650 nm). The results are shown in Fig. 1 (Example-4: OD value without activated carbon treatment was calculated as 100%). In the case of the collagen-added group, there was no promoting effect on the proliferation of epidermal keratinocytes, but the sponge protein showed the promoting effect on cell proliferation at a concentration of 0.00625% or more.

[0021]

According to the present invention, a sponge sponge which has been conventionally used only as a sponge for body washing or cosmetics, and the shavings portion has been discarded, is used as a raw material to produce a high value-added food, Obtaining a sponge protein hydrolyzate that is a raw material for pet foods and cosmetics / medicines is a just fit for social needs. Furthermore, by purifying the sponge protein hydrolyzate obtained in the present invention, it can be used as a more economical product material, and the social utility of the present invention is extremely high.

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[Procedure amendment]

[Submission date] April 8, 1999 (1999.4.8)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1. Effect of sea surface protein and hydrolyzed collagen on the proliferation of human-derived epidermal keratinocytes

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C07K 1/34 C07K 1/34 14/435 14/435 // A23J 1/04 A23J 1/04 3/04 3/04 3/34 3/34 A61K 7/00 A61K 7/00 K 38/00 A61L 27/00 C A61L 27/00 V A61K 37/18 (72) Inventor Koji Nakamura 6-3 Nishiawaji, Higashiyodogawa-ku, Osaka-shi, Osaka No. 41 Nakamura Bussan Co., Ltd. Awaji Plant (72) Inventor Takeshi Okada 6-3-3 Nishi Awaji, Higashiyodogawa-ku, Osaka City, Osaka Prefecture Nakamura Bussan Co., Ltd. Awaji Plant (72) Inventor Tadao Miyake Higashiyodogawa, Osaka City, Osaka Prefecture 6-41 Nishi-Awaji-ku Nakamura Bussan Awaji Plant

Claims (19)

[Claims] 1. A sponge protein solution obtained by dissolving a dried sponge and neutralizing the supernatant. 2. The sponge protein solution according to claim 1, wherein the concentration is in the range of 0.1 to 15% by weight. 3. The composition according to claim 1, wherein the composition is used as a raw material for foods, a raw material for pet foods, cosmetics or pharmaceuticals.
Sponge protein solution. 4. The method according to claim 1, wherein the cell is used for an application having a growth promoting effect on mammalian cells.
Sponge protein solution. 5. A sponge protein hydrolyzate obtained by dissolving a dried sponge, neutralizing the supernatant, and allowing a protein hydrolase to act on a sponge protein solution obtained. 6. A protein hydrolase in a free state, a protein hydrolase cross-linked, a protein hydrolase inclusive, or a protein hydrolase immobilized on an insoluble carrier. The sponge protein hydrolyzate according to claim 5, characterized in that: 7. The protein hydrolase according to claim 5, wherein the endo- or exo-type protein hydrolase is used alone or in a mixture of both at an appropriate ratio. A sponge protein hydrolyzate according to any one of the above. 8. The composition according to claim 5, which is used as a raw material for foods, a raw material for pet foods, cosmetics or pharmaceuticals.
A sponge protein hydrolyzate according to any one of the above. 9. The method according to claim 5, wherein the cell is used for an application having a growth promoting effect on mammalian cells.
A sponge protein hydrolyzate according to any one of the above. 10. The sponge protein hydrolyzate according to claim 5, comprising an ultrafiltration membrane, a microfiltration membrane,
Functional low molecular weight peptides and / or amino acids obtained using gel filtration or ion exchange resins. 11. The functional low-molecular-weight peptide and / or amino acid according to claim 10, wherein the functional low-molecular-weight peptide and / or amino acid is used as a raw material for food, a raw material for pet food, a cosmetic or a pharmaceutical. 12. The functional low-molecular-weight peptide and / or amino acid according to claim 10, wherein the functional low-molecular-weight peptide and / or amino acid is used for an application having a growth promoting effect on mammalian cells. 13. A method for producing a sponge protein hydrolyzate, comprising dissolving a dried sponge animal and neutralizing a supernatant thereof, and allowing a protein hydrolase to act on a sponge protein solution obtained. 14. The method according to claim 13, wherein an alkaline solution or an acidic solution is used as a solution for the dried sponge.
A method for producing a sponge protein hydrolyzate. 15. The method for producing a sponge protein hydrolyzate according to claim 13, wherein the concentration of the alkaline solution or the acidic solution of the dried sponge is 0.01 to 15% by weight. 16. Use of a protein hydrolase in a free state, a protein hydrolase cross-linked, a protein hydrolase inclusive, or a protein hydrolase immobilized on an insoluble carrier. The method for producing a sponge protein hydrolyzate according to any one of claims 13 to 15, wherein: 17. The protein hydrolase according to claim 13, wherein an endo-type or exo-type protein hydrolase is used alone or in a mixture of both at an appropriate ratio. The method for producing a sponge protein hydrolyzate according to item 1. An ultrafiltration membrane, a microfiltration membrane, or a sponge protein hydrolyzate according to any one of claims 13 to 17,
A method for producing a functional low-molecular peptide and / or amino acid, comprising separating a functional low-molecular peptide and / or amino acid using gel filtration or an ion exchange resin. 19. The cut material of a dried sponge to be discarded is used as a raw material.
19. The method for producing a sponge protein according to any one of 18.