Description

METHOD FOR PREPARING POL Y-GAMMA-GLUT AMIC

ACID HYDROGEL

Technical Field

[1] The present invention relates to a method for preparing poly(gamma-glutamic acid) hydrogel, and more particularly, to a method for preparing poly(gamma-glutamic acid) hydrogel which is nontoxic and has high water absorption rate, the method comprising dissolving poly-gamma-glutamic acid in a salt solution and subjecting the resulting solution to cross-linking through irradiation of gamma rays.

[2]

Background Art

[3] Hydrogel is a three-dimensional, hydrophilic, polymeric network capable of containing large amounts of water, which is made up of a polymer or a copolymer, and form a stable three-dimensional network structure in which structural anistropy is hardly induced by an external force. Since such a structure is formed by forces including covalent bonding, hydrogen bonding, van der Waals interaction or physical interaction etc., hydrogel is thermodynamically stable and does not change its degree of swelling in an aqueous solution, and thus, has mechanical and physicochemical properties corresponding to those of an intermediary form between liquid and solid.

[4]

[5] Swelling behavior of hydrogel can be adjusted according to a chemical structure and hydrophilicity of a polymer and crosslinking rate between polymeric chains, so that hydrogels with various properties can be produced in various shapes according to components and preparation methods thereof. Applications of hydrogels in the field of biomaterials have been drawing increasing attention since the first hydrogels based on poly(hydroxyethyl methacrylate)(PHEMA) were developed in 1960s by wichterle et al., and, after calcium alginate hydrogel was developed in 1980s, various hydrogels have been developed as a biomaterial using natural polymers or synthetic polymers as a result of mainly studies on physicochemical properties of hydrogel.

[6]

[7] Starting with sanitary products based on high absorption capacity, hydrogels have been widely used in the cosmetic industry, and currently, are being usefully used from medical applications to industrial applications, including a drug delivery system, embolization, scaffolds, chemical valves, separation, concentration and stabilization of proteins, immune assay, bioreactors, sensors, chromatography, cosmetic filler, etc., by introducing various additional functions thereto.

[8]

[9] When hydrogels are produced from natural polymers, they may not have sufficient mechanical properties, and may contain pathogens or evoke immune/inflammatory responses but they do offer several advantageous properties such as biocompatibility, biodegradability and may contain biologically recognizable residues capable of facilitating smooth cell activities, whereas when hydrogels are produced from synthetic polymers, they don't have original physiological activities, but may provide suitably structured matrices whose degradability and functionality were controlled. Thus, combining natural polymers with synthetic polymers has been attempted in such a way that disadvantages of the respective natural polymers and synthetic polymers are complemented and advantages thereof are strengthened.

[10]

[11] In the present invention, it is intended to prepare hydrogel using poly- gamma-glutamic acid. Poly gamma-glutamic acid is a viscose polymer consisting of D-L-glutamic acid polymerized through {gamma} -glutamyl bonds, which is a natural amino acid polymer produced from the genus Bacillus strain. The inventive poly(gamma-glutamic acid) hydrogel is produced using poly-gamma-glutamic acid having ultra high molecular weight, and thus it has both advantages of natural polymers, i.e, biocompatibility and biodegradability, and advantageous physical properties of synthetic polymers, compared with the existing poly(gamma-glutamic acid) hydrogels.

[12]

[13] The present inventors obtained a patent relating to poly-gamma- glutamate with high molecular weight and a method for using the same (Korean Patent Registration No. 10-0399,091), and a patent relating to a method for producing γ-PGA using a halophilic Bacillus subtilis var. chungkookjang that produces γ-PGA with high molecular weight (Korean Patent Registration No. 10-0500,796). Also, the present inventors obtained patents relating to an anticancer composition, an immune adjuvant and an immune enhancing agent, which contain γ-PGA (Korean Patent Registration Nos. 10-0496,606; 10-0517,114; and 10-0475,406). In addition, the present inventors have identified the effects of γ-PGA by continuously developing applications for γ- PGA, including studies on the use of γ-PGA in medical applications, such as development of hyaluronidase inhibitor containing poly-gamma-glutamic acid(Korean Patent Registration No. 10-0582,120) and the discovery of anticancer effects of poly- gamma-glutamic acid through enhanced immunity (Poo, H.R. et al., Journal of Immunology, 178:775, 2007).

[14]

[15] However, the molecular weight of traditional poly-gamma-glutamic acid is

50,000- 1,000,000Da, so that it is a limiting factor in application thereof, especially in industrial production.

[16]

[17] Poly(gamma-glutamic acid) hydrogel is known to be an eco-friendly material characterized by absorption, biodegradability and plasticity, which is synthesized by inter- molecular cross-linking of a biopolymer used as a raw material, poly-gamma-glutamic acid produced from Bacillus subtilis var. chungkookjang through microbial fermentation.

[18]

[19] In the production of poly(gamma-glutamic acid) hydrogel, cross-linking methods mainly include chemical treatments using cross-linking agents such as epoxy resins or gamma rays or e-beam irradiation, but the chemical treatment methods have a safety problem due to toxicity of chemical materials and problems involving a complicated process due to a recovery process, and radiation crosslinking methods have a disadvantage in that they produce low yields of hydrogel due to cross-linking inefficiency.

[20]

[21] Thus, the present inventors have made extensive efforts to overcome the problems occurring in the prior art, and, as a result, confirmed that, when poly(gamma-glutamic acid) hydrogel is produced by dissolving poly-gamma-glutamic acid in a salt solution and then cross-linking the resulting solution by irradiating gamma rays, the produced poly(gamma-glutamic acid) hydrogel is nontoxic, has superior absorption property and moisturizing property, and the yield thereof increases, thereby completing the present invention.

[22]

[23] SUMMARY OF INVENTION

[24] It is an object of the present invention to provide a method for preparing poly(gamma-glutamic acid) hydrogel which is nontoxic, and has superior absorption property and moisturizing property, using poly-gamma-glutamic acid.

[25]

[26] To achieve the above object, the present invention provides a method for preparing poly(gamma-glutamic acid) hydrogel, which comprises dissolving poly- gamma-glutamic acid in a salt solution and then irradiating gamma rays thereto.

[27]

[28] Other features and embodiments of the present invention will be more apparent from the following detailed description and the appended claims.

[29]

Brief Description of Drawings

[30] Fig. 1 is a graph showing the results obtained by measuring water absorption rates of the inventive poly(gamma-glutamic acid) hydrogel and conventional hydrogels.

[31]

[32] Fig. 2 is a graph showing the results obtained by measuring water absorption rates of the inventive poly(gamma-glutamic acid) hydrogel, according to the molecular weight thereof.

[33]

[34] Fig. 3 is a graph showing the results obtained by measuring water absorption rates of the inventive poly(gamma-glutamic acid) hydrogel at various salt concentrations.

[35]

[36] Fig. 4 is a graph showing the results obtained by measuring safety of the inventive poly(gamma-glutamic acid) hydrogel, according to the molecular weight thereof.

[37]

[38] DETAILED DESCRIPTION OF THE INVENTION. AND PREFERRED EM-

BODIMENTS

[39] The present invention relates to a method for preparing poly(gamma-glutamic acid) hydrogel, which comprises dissolving poly-gamma-glutamic acid in a salt solution and then irradiating gamma rays thereto.

[40]

[41] Specifically, the present invention is intended to provide a method for preparing poly(gamma-glutamic acid) hydrogel having high water absorption rate in high yield, which solves safety problems such as toxicity of chemical substances, occurring by conventional chemical cross-linkage, and doesn't require a treatment process such as a recovery process, etc. by preparing poly-gamma-glutamic acid using microorganisms, adding salt to the prepared poly-gamma-glutamic acid, and cross-linking the resulting solution through irradiation of gamma rays.

[42]

[43] In the present invention, the poly-gamma-glutamic acid is produced by a microorganism. The microorganism is Bacillus subtilis var. chungkookjang (KCTC 0697BP), and a process for the isolation and identification thereof, and its physiological properties and the like thereof are described in detail in Korean Patent Registration 10-2001-1481 and WO 02/055671.

[44]

[45] The morphological and physiological properties of the strain are as follows. When cultured in an LB agar plate, the strain forms milky white colonies, and the strain is characterized in that the growth thereof since it is gram positive bacteria growing

actively at a culture temperature higher than 37 ⁰ C under aerobic conditions, is reduced at a culture temperature higher than 55 ⁰ C. The inventive strain is halotolerant bacteria capable of growing at a salt(NaCl) concentration of 9.0%, which can also be grown at sodium chloride (NaCl), which is higher than the salt concentration at which a general Bacillus subtilis strain can grow, and it is a typical Bacillus strain forming endospores upon liquid culture or solid culture in an LB liquid medium for more than 70hrs. As a result of comparatively analyzing the 16S rDNA sequence of the inventive strain and the 16S rDNA sequence of the prior Bacillus sp. strain, the sequence of the inventive strain shows high homology (99.0%) with that of Bacillus subtilis.

[46]

[47] In the present invention, the average molecular weight of poly-gamma-glutamic acid, which is produced by Bacillus subtilis chungkookjang (KCTC 0697BP), is 13,00OkDa, and more than 95% of the γ-PGA has molecular weight ranging from 3,000 to 15,00OkDa.

[48]

[49] Therefore, according to application thereof, if necessary, the prepared poly- gamma-glutamic acid may be cut with a suitable method to have a given molecular weight for use weight to use, or may be separated with a suitable method to recover according to molecular weight for use.

[50]

[51] The molecular weight of the poly-gamma-glutamic acid, which is used to prepare the inventive poly(gamma-glutamic acid) hydrogel having ultra high molecular weight, is preferably 2,00OkDa- 15,00OkDa, and more preferably 5,00OkDa- 15,00OkDa, and when the molecular weight thereof is less than 2,00OkDa, there is a problem in that hydrogel properties deteriorate to cause low yield, and when the molecular weight thereof is more than 15,00OkDa, there is a problem in that it is hard to produce hydrogel in a general process.

[52]

[53] In the present invention, the salt is used to produce poly(gamma-glutamic acid) hydrogel in high yield by promoting cross-linking of poly-gamma-glutamic acid with gamma rays, and may be selected from the group consisting of H ₂ PO ₄ , K ₂ HPO ₄ , NaH ₂ PO ₄ , Na ₂ HPO ₄ , (NH ₄ ) ₂ SO ₄ , (NH ₄ ) ₂ S ₂ O ₈ , MgSO ₄ , NaCl, CaCl ₂ and a mixture thereof and preferably K ₂ HPO ₄ . In addition, the amount of salt added is 0.001-2 parts by weight based on 100 parts by weight of the poly-gamma-glutamic acid and preferably 0.01-lpart by weight. When the salt is added at a concentration of less than 0.001 parts by weight to poly-gamma-glutamic acid, there is a problem in that hydrogel properties deteriorate to cause low yield, and, when the salt is added at a concentration of more than 2 parts by weight to poly-gamma-glutamic acid, product safety problems may

occur due to high salt concentration.

[54]

[55] The inventive cross-linking method using irradiation of gamma-ray has advantages in that it involves no safety problems such as toxicity and the like as well as does not require treatment process including recovery process to simplify production process, compared with the prior chemical cross-linking method.

[56]

[57] In the present invention, poly-gamma-glutamic acid may be irradiated with gamma rays at a dose range of 1 to 10OkGy, and preferably, 5 to 5OkGy. If poly- gamma-glutamic acid is cross-linked by irradiation with a dose lower than IkGy, there may be a problem in that hydrogel properties deteriorate to cause low yield, and if poly-gamma-glutamic acid is cross-linked by irradiation with a dose higher than 10OkGy, there may be a problem of discoloration and shape change.

[58]

[59] The inventive poly(gamma-glutamic acid) hydrogel has a high water absorption rate due to the use of poly-gamma-glutamic acid with high molecular weight as well as a very high level of safety against toxicity and the like by a cross-linkage using gamma- ray irradiation of poly-gamma-glutamic acid added with salt, not by chemical cross linkage.

[60]

[61] The inventive poly(gamma-glutamic acid) hydrogel may be added to a cosmetic composition. For the cosmetic composition according to the present invention, the poly(gamma-glutamic acid) hydrogel may be added thereto at a concentration of 0.001-5 parts by weight per 100 parts by weight of the cosmetic composition based on dry weight, and preferably 0.01-3 parts by weight based on the dry weight thereof. If it is added thereto at a concentration of less than 0.001 parts by weight per 100 parts by weight of the cosmetic composition based on dry weight of the cosmetic composition, the effect of poly(gamma-glutamic acid) hydrogel cannot be expected, and if it is added thereto at a concentration of more than 5 parts by weight, the effect thereof is not significantly different, and there may be a problem in that viscosity excessively increases.

[62]

[63] The cosmetic composition according to the present invention can be formulated into toner, nutrient lotion, nutrient cream, massage cream, essence, pack and the like, and according to the type of formulation, the purpose of use, etc, the composition may additionally contain additives such as colorants, preservatives, surfactants, oil, water, moisturizing agents, thickeners, chelating agents, fragrances, and the like. The cosmetic composition according to the present invention may be used according to the

conventional method, and frequency of use thereof may be varied according to user's skin condition or preference.

[64]

[65] Examples

[66] Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

[67]

[68] Example 1: Production of poly-gamma-glutamic acid

[69]

[70] 3L of a basal medium for poly-gamma-glutamic acid production (a medium containing 5% L-glutamic acid: 5% glucose, 1% (NH ₄ ) ₂ SO ₄ , 0.27% KH ₂ PO ₄ , 0.42% Na ₂ HPO ₄ 12H ₂ O, 0.9% NaCl, 0.3% MgSO ₄ 7H ₂ O, 5m/L Vitamin solution, pH 6.8) in a 5L fermentor was inoculated with 2% culture broth of Bacillus subtilis var chungkookjang (KCTC 0697BP) and then cultured at a stirring speed of 150 rpm, an air injection rate of 1.2 vvm and a temperature of 37 ⁰ C for 60 hours. After completion of the culture, bacterial cells were removed using a filter press to obtain a γ- PGA-containing sample solution.

[71]

[72] 2N sulfuric acid solution was added to the γ-PGA-containing sample solution and left to stand at 8 ⁰ C for 18 hours to collect a γ-PGA precipitate. The collected precipitate was washed with a sufficient amount of distilled water to obtain poly- gamma-glutamic acid using a Nutsche filter. Molecular weight of the obtained poly- gamma-glutamic acid was measured using GPC (gel permeation column), and as a result, it was confirmed that poly-gamma-glutamic acids having molecular weights of 1-15,00OkDa was produced, and it were separated according to molecular weight through a heating process poly-gamma-glutamic acids having average molecular weights of 5OkDa, 50OkDa, 2,00OkDa, 5,00OkDa and 7,00OkDa, respectively, thus producing poly-gamma-glutamic acid.

[73]

[74] Example 2: Production of poly(gamma-glutamic acid) hydrogel

[75]

[76] KH2PO4 was added to poly-gamma-glutamic acid prepared according to molecular weight in Example 1, at a concentration of 0.01%, 0.05% and 1.0%, and then irradiated with 2OkGy of gamma ray, thus preparing gel type hydrogel. The gel type hydrogel thus prepared was lyophilized, and then pulverized, thus preparing poly(gamma-glutamic acid) hydrogel powders.

[78] Experimental example 1: Measurement of water absorption rate of poly(gamma-glutamic acid) hydrogel

[79]

[80] In order to measure water absorption rate of poly(gamma-glutamic acid) hydrogel,

O.lg of the poly (gamma- glutamic acid) hydrogel having an average molecular weight of 7,00OkDa prepared in Example 2 was added to 200ml of distilled water to gelatinize and then allowed to stand at room temperature for 48 hrs, followed by filtering the resulting solution with a sieve to remove unabsorbed water. The weight of gelatinized material was measured to calculate water absorption rate of the added hydrogel. Moreover, in order to compare its water absorption rate to that of conventional products, water absorption rate of poly(gamma-glutamic acid) hydrogel from Vedan Co. was measured by the same method as described above.

[81]

[82] As a result, as shown in FIG.1, the poly(gamma- glutamic acid) hydrogel of the present invention absorbed water 3500 times its own weight while poly- gamma-glutamic acid from Vedan Co. absorbed water 300 times its own weight, thus confirming that the inventive poly(gamma-glutamic acid) hydrogel is an excellent material having high water absorption rate compared with other company's product.

[83]

[84] Experimental example 2: Measurement of water absorption rate of hydrogel according to molecular weight of poly-gamma-glutamic acid

[85]

[86] Hydrogels containing poly(gamma-glutamic acid) having respective molecular weights prepared in Example 2, was compared and analyzed for water absorption rate using the same method as described in Experimental example 1.

[87]

[88] As a result, as shown in FIG. 2, the use of poly-gamma-glutamic acid of 5OkDa did not produce hydrogel, and it was shown that, as molecular weight increased, water absorption rate of the prepared poly(gamma-glutamic acid) hydrogel increased proportionally.

[89]

[90] Experimental example 3: Measurement of water absorption rate of poly(gamma-glutamic acid) hydrogel according to salt concentration

[91]

[92] Water absorption rate of poly(gamma-glutamic acid) hydrogel according to salt concentration was measured with the same method as described in Experimental example 1, using poly(gamma-glutamic acid) hydrogels prepared by adding KH2PO4 to poly- gamma-glutamic acid having a molecular weight of 7,00OkDa at concentrations of

0.01%, 0.05%, 0.1%, 0.5% and 1.0%, respectively, among the poly(gamma-glutamic acid) hydrogels prepared in Example 2.

[93]

[94] As a result, as shown in FIG. 3, it was found that, as the added salt concentration was increased, water absorption rate of the hydrogels was increased. Especially, when the concentration of the salt was 0.1%, the highest water absorption rate was shown.

[95]

[96] Experimental example 4: Safety analysis for poly(gamma-glutamic acid) hydrogel

[97]

[98] In order to analyze the safety of poly(gamma-glutamic acid) hydrogel containing poly-gamma-glutamic acid) with various molecular weights, viscosity changes with time were compared and analyzed. O.lg of each of the hydrogels containing various molecular weights of poly-gamma-glutamic acid, prepared in Example 2 was dissolved in 100 ml of distillated water and left to stand at room temperature, thus measuring changes in viscosity reduction.

[99]

[100] As a result, as shown in FIG. 4, it was confirmed that, as molecular weight of poly- gamma-glutamic acid increased, viscosity safety of the prepared poly(gamma-glutamic acid) hydrogel proportionally increased, which suggests that the crosslinking density of the hydrogel increase proportional to molecular weight of poly-gamma-glutamic acid, thus making it possible to increase the safety of hydrogel.

[101]

Industrial Applicability

[102] As described above, poly (gamma- glutamic acid) hydrogel according to the present invention is safe and shows high hygroscopic property, and thus it can be used as highly moisturizing cosmetics and water absorptive gels, as well as applied to high value-added new materials having various uses for moisturizing.

[103]

[104] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.