JP2008120725A - External preparation for skin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stably provide an external preparation for skins, having desired qualities. <P>SOLUTION: This external preparation for skins is characterized by containing at least one of poly-γ-L-glutamic acid and a poly-γ-L-glutamic acid cross-linked product. Since the poly-γ-L-glutamic acid is produced from only L-glutamic acid, the quality of the poly-γ-L-glutamic acid and the quality of the L-glutamic acid cross-linked product obtained from the poly-γ-L-glutamic acid are stabilized without having heterogeneous qualities for productions. The external preparation for the skins can suitably be used as a moisturizing agent or a cosmetic, because of having an excellent moisturizing property. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an external preparation for skin. More specifically, the present invention relates to an external preparation for skin which contains at least one of poly-γ-L-glutamic acid and poly-γ-L-glutamic acid crosslinked body having a high moisturizing effect and is suitable as a moisturizing agent and cosmetic.

  There are two types of rough skin: exfoliation of keratinocytes, and deterioration of skin health due to dryness, leading to hardening and damage of the epidermis. In particular, rough skin due to exfoliation of keratinocytes may evaporate stratum corneum by elution of intercellular keratinocyte lipids such as cholesterol, ceramide, and fatty acids, degeneration of keratinocytes by ultraviolet rays, detergents, etc., epidermal cell proliferation balance and / or disruption of keratinization balance. It occurs due to barrier formation failure.

  Conventionally, for the purpose of preventing or healing rough skin, studies have been conducted on synthesizing and supplying a keratin intercellular lipid component or a similar keratin intercellular lipid to the skin. The stratum corneum intercellular lipid is a layered plate granule biosynthesized by cells in the spinous layer and the granule layer, and is released between the cells immediately below the stratum corneum and extends to form a layered plate (lamellar) structure. A substance that spreads between cells.

  The lamellar granule is composed of glucosylceramide, cholesterol, ceramide, phospholipid, etc., but the horny layer intercellular lipid contains almost no glucosylceramide. That is, it is considered that glucosylceramide in the lamellar granule is hydrolyzed by β-glucocerebrosidase and converted to ceramide. Then, it is considered that ceramide forms a lamellar structure, thereby improving the formation of a stratum corneum permeation barrier as a stratum corneum intercellular lipid and functioning as a barrier for preventing rough skin. In particular, it has been reported that supplementation with ceramide is effective for rough skin caused by detergents and the like, and shows a high effect in improving rough skin (Non-Patent Document 1).

  On the other hand, in order to prevent rough skin due to hardening or damage of the epidermis, conventionally, skin external preparations such as cosmetics having a moisturizing effect have been used. By using an external preparation for skin having a moisturizing effect, moisture evaporation from the skin is prevented, and moisture is retained in the epidermis and the stratum corneum. Thereby, skin homeostasis is maintained, moisture retention and flexibility can be maintained, and fresh skin can be maintained.

  Conventionally reported lipophilic substances having a moisturizing effect on the skin include vegetable oils such as olive oil and animal-derived lipids such as lanolin. Further, as hydrophilic substances having a moisturizing effect on the skin, water-soluble polyhydric alcohols such as glycerin, 1,3-butylene glycol, propylene glycol and sorbitol, polysaccharides such as hyaluronic acid and xanthan gum, polyethylene glycol and the like Examples thereof include water-soluble polymers, pyrrolidone carboxylates, low molecular weight natural moisturizing factors represented by amino acids, plant extracts and the like.

  As described above, various substances exist as substances having a moisturizing effect on the skin. However, in recent years, animal-derived substances and chemically synthesized products tend to be avoided due to the trend of emphasizing safety. And the substance derived from a natural product and the fermentation product by microorganisms are considered preferable. Furthermore, biodegradable materials that are less harmful not only to living bodies but also to the environment are expected and attracting attention.

  Among biodegradable materials, biopolymers produced by microorganisms are promising. Among biopolymers, various functions have been found in a group of biopolymers called polyamino acids constituted by condensation polymerization of amino acids, and attention has been focused on their potential. Conventionally, three types of polyamino acids have been identified: poly-γ-glutamic acid (hereinafter referred to as “PGA”), poly-ε-lysine and cyanophysin.

  PGA is a polyamino acid in which an α-amino group and a γ-carboxyl group of glutamic acid are amide-bonded. PGA is a water-absorbing polyamino acid known as the main material of stringing of natto, which has been familiar to Japanese people for a long time, but as a background of this familiarity, it is due to its attractive functionality. large. As an attractive function of PGA, it is known that it has both biodegradability and high water absorption. Utilizing these functions, it is expected to be used in various fields and applications including the above-described cosmetics, medical products, foods and the like.

  The PGA is generally PGA formed by randomly binding both optical isomers of L-glutamic acid and D-glutamic acid, but PGA formed by binding only L-glutamic acid (Patent Document 1, Non-Patent Document). 1-3), and PGA formed by binding only D-glutamic acid (Non-patent Document 4) has also been reported. Moreover, in patent document 2, the crosslinked body of PGA is used as a water absorbing resin.

In the present specification, for convenience of explanation, PGA formed by combining D-glutamic acid and L-glutamic acid is referred to as “DL-PGA”. A PGA consisting only of D-glutamic acid is denoted as “D-PGA”, and a PGA consisting solely of L-glutamic acid is denoted as “poly-γ-L-glutamic acid” or “L-PGA”.
Special Table 2002-517204 (announced June 18, 2002) Japanese Patent Laid-Open No. 10-251402 (published on September 22, 1998) Aono, R., M. Ito, and T. Machida, Contribution of the Cell Wall Component Teichuronopeptide to pH Homeostasis and Alkaliphily in the Alkaliphile Bacillus lentus C-125, Journal of Bacteriology, 1999, Vol. 181, 6600-6606. Weber, J., Poly (gamma-glutamic acid) s are the major constituents of nematocysts in Hydra (Hydrozoa, Cnidaria), Journal of Biological Chemistry, 1990, Vol. 265, 9664-9669. Hezayen, FF, BHA Rehm, BJ Tindall and A. Steinbuchel, Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp. Nov. And description of Natrialba aegyptiaca sp. from Egypt that produces extracellular poly (glutamic acid), International Journal of Systematic and Evolutionary Microbiology, 2001, 51, 1133-1142. Makino, S., I. Uchida, N. Terakado, C. Sasakawa, and M. Yoshikawa, Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis, Journal of Bacteriology, 1989, 171, 722 -730.

  However, the above-mentioned conventional skin external preparation containing PGA has a problem that it is difficult to stably produce a skin external preparation having a desired quality, and a problem that moisture retention is insufficient.

  As described above, the currently commercialized DL-PGA is a chemically heterogeneous polymer. Specifically, PGA is produced from Bacillus natto and its related bacteria, but D-glutamic acid and L-glutamic acid are irregularly bound, and the content ratio and sequence thereof are different for each culture of the producing bacteria. fluctuate. In general, the structural characteristics of polyamino acids (optical activity and type of constituent amino acids, molecular size, binding mode, etc.) strongly influence their functions. Since the DL-PGA has a different structure for each molecule, its property also differs for each molecule. This makes it difficult to stably produce DL-PGA having a desired quality.

  Moreover, since the moisture retention of DL-PGA is inadequate, a big subject remains in practical use as skin external preparations, such as cosmetics.

  By the way, there is no report that the skin external preparation containing L-PGA was manufactured conventionally. The following reasons can be considered.

  Generally, when producing an external preparation for skin using PGA, moisture retention is required, and thus high molecular weight PGA is indispensable. On the other hand, conventionally, L-PGA having a large average molecular weight has not been obtained in liquid culture. This makes it difficult to even conceive of producing a humectant containing L-PGA.

  Further, PGA for industrial use is required to be able to be produced by liquid culture. This is because it is difficult to culture a large amount of microorganisms at once in plate culture, and it is inefficient to recover L-PGA from the plate medium.

  Specifically, as an organism for synthesizing L-PGA, Non-Patent Document 1 discloses an alkaliphilic bacterium Bacillus halodurans, and Non-Patent Document 2 discloses hydra. However, the molecular weight of L-PGA synthesized by these organisms is about 100,000, which is extremely small.

  Patent Document 1 and Non-Patent Document 3 report that Natrialba aegyptiaca, which is a halophilic archaea, produces L-PGA having a molecular weight of about 100,000 to 1,000,000 when cultured in a plate medium. . However, L-PGA synthesized by the Natrialba aegyptiaca under liquid culture conditions has a molecular weight of about 100,000 and its synthesis efficiency is extremely low.

  On the other hand, the D-PGA obtained so far is not suitable for industrial use.

  This is because the bacterium that synthesizes D-PGA disclosed in Non-Patent Document 4 is Bacillus anthracis having strong pathogenicity. It is extremely inappropriate to use anthrax for industrial production of PGA.

  By the way, although the crosslinked body of DL-PGA is used as a water absorbing resin in Patent Document 2, it is difficult to use the crosslinked body of DL-PGA as a skin external preparation.

  DL-PGA, which is a raw material of the DL-PGA crosslinked body disclosed in Patent Document 2, is synthesized by Bacillus subtilis and other natto bacteria and their related bacteria. Since the quality of DL-PGA used as a raw material is not constant, it is difficult to stably obtain a crosslinked product. Even in the study by the present inventors, a crosslinked product of DL-PGA was not obtained. This is probably because DL-PGA has a different structure for each molecule as described above. That is, the cross-linking efficiency in producing a PGA cross-linked body depends on the structure of the molecule, and when the structure of each molecule is irregularly different, the cross-linking efficiency is significantly reduced. Therefore, it is difficult to crosslink DL-PGA having a different structure for each molecule, and the yield of the crosslinked product is extremely low.

  Therefore, it is difficult to stably produce a skin external preparation of a desired quality even if a DL-PGA crosslinked product is used.

  On the other hand, there has been no report that a cross-linked product of L-PGA has been obtained.

This is because as described above, L-PGA having a large average molecular weight is not obtained in liquid culture. Since it is technically common knowledge that it is extremely difficult to obtain a crosslinked product of a low molecular weight organic compound, it is difficult for those skilled in the art to even conceive of obtaining a crosslinked product of low molecular weight L-PGA. Therefore, there is no report that an attempt was made to obtain a crosslinked product of L-PGA. Even if a crosslinked product of D-PGA could be obtained, as described above, Since it is only anthrax, it is not suitable for industrial use.

  This invention is made | formed in view of said problem, The objective is to provide the skin external preparation which has desired quality stably.

  In order to solve the above-mentioned problems, the present inventors have intensively studied to obtain a natural product-derived biodegradable material that has high moisture retention and can stably produce desired quality. As a result, L-PGA having a high molecular weight was independently developed. Since the L-PGA is formed by binding only L-glutamic acid, the optical activity is uniform and the molecular weight is high, so that the L-PGA has an excellent moisturizing property. And it discovered that the said subject could be solved by containing the crosslinked body of L-PGA.

  That is, the external preparation for skin according to the present invention is characterized by containing at least one of poly-γ-L-glutamic acid and a poly-γ-L-glutamic acid crosslinked product in order to solve the above problems.

  In the external preparation for skin according to the present invention, the external preparation for skin is more preferably a cosmetic.

  In the external preparation for skin according to the present invention, the external preparation for skin is more preferably a moisturizing agent.

  As described above, the external preparation for skin according to the present invention contains at least one of L-PGA and L-PGA cross-linked product. Therefore, the skin external preparation having the desired quality can be stably provided.

  Moreover, since L-PGA and L-PGA crosslinked body have the outstanding moisturizing property, there exists an effect that the skin external preparation useful especially as cosmetics and a moisturizing agent can be provided.

  An embodiment of the present invention will be described as follows. It should be noted that the scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

[External preparation for skin according to the present invention]
The skin external preparation according to the present invention may include at least one of L-PGA and L-PGA cross-linked product, and other specific configurations are not particularly limited.

  Since L-PGA is formed by binding L-glutamic acid, the optical activity is uniform and the properties of each molecule are uniform. Therefore, the L-PGA crosslinked body obtained from L-PGA can also be stably produced with desired quality. Therefore, by using at least one of L-PGA and L-PGA crosslinked product, a skin external preparation having a desired quality can be stably provided.

  Furthermore, since L-PGA and L-PGA crosslinked body are excellent in moisturizing property, the skin external preparation according to the present invention can be suitably used as a moisturizing agent and / or a cosmetic.

  When the skin external preparation according to the present invention is used as a moisturizer, specifically, face care products, hand care products, body care products, foot care products, head care products, hair care products, nail care products, mouse care products, etc. Can be suitably used.

  In addition, when using the external preparation for skin according to the present invention as a cosmetic, specifically, a face care product such as a milky lotion, a cosmetic liquid, a cream, a lotion, a face wash, a makeup remover, a hand care product, a body care product, It can be suitably used as a foot care product, a head care product, a hair care product, a nail care product, a mouse care product, or the like.

  In this specification, “skin” is intended to be skin of the face, neck, chest, back, arms, legs, hands and scalp. Further, in this specification, the “skin external preparation” is intended to be used for improving the skin condition such as dry or rough skin or suppressing the deterioration of the skin condition.

(L-PGA)
L-PGA contained in the external preparation for skin according to the present invention is a homopolymer formed by binding L-glutamic acid, and has a structure represented by the following formula (1).

(In formula (1), n represents the number of polymerizations)
The average molecular weight of L-PGA contained in the external preparation for skin according to the present invention may be appropriately selected according to the use of the external preparation for skin, etc., but preferably 1.3 million or more, more preferably 2 million or more, Preferably it is 3.5 million or more.

  The higher the average molecular weight of L-PGA, the better the moisture retention of the external preparation for skin containing the L-PGA. Therefore, the upper limit value of the average molecular weight of L-PGA is not particularly limited. In addition, according to the manufacturing method of L-PGA mentioned later, L-PGA with an average molecular weight of 6 million and a maximum of 15 million can be obtained, for example.

  In the present specification, the “average molecular weight” means the number average molecular weight (Mn) calculated in terms of the molecular weight of the pullulan standard substance.

  As L-PGA contained in the external preparation for skin according to the present invention, L-PGA obtained by various conventionally known methods may be used. For example, a microorganism producing L-PGA (hereinafter simply referred to as “L-PGA”). L-PGA obtained by using “production microorganism”) may be used.

(L-PGA producing microorganism)
The L-PGA-producing microorganism is not limited as long as it is a microorganism that synthesizes L-PGA. L-PGA-producing microorganisms can be produced by wild-type L-PGA-producing microorganisms, mutants thereof, or gene recombination techniques. What is necessary is just to use the microorganisms which gave or strengthened production capacity. Among these, halophilic bacteria or mutants thereof are preferable. Thermophilic bacteria, hyperthermophilic bacteria, psychrophilic bacteria, acidophilic bacteria, thermophilic bacteria, low-temperature-growing bacteria, and the like may be used as long as they have properties as halophilic bacteria. The halophilic bacteria are low halophilic bacteria (growing at 0.2 to 0.5 M NaCl concentration), moderate halophilic bacteria (growing at 0.5 to 2.5 M NaCl concentration), and high halophilic bacteria (growing at a NaCl concentration of 0.5 to 2.5 M). Any of which may be used, but highly halophilic bacteria are preferred. Since halophilic bacteria can grow even under high salt conditions where other microorganisms cannot grow, they can be cultured without aseptic manipulation.

  The L-PGA producing microorganism may be an archaea. Examples of archaea include thermophilic archaea classified as the above-mentioned highly halophilic bacteria, thermophilic archaea, methane bacteria (methane-producing archaea), etc., but L-PGA can be produced. As long as it is, any archaea may be used. Among archaea, highly halophilic archaea are preferred.

  Examples of highly halophilic archaea include, for example, the genus Halobacterium (Halobacterium), the genus Haloarcula, the genus Haloferax, the genus Halococcus, the genus Halorubrum, the genus Halobaculum, Examples include the genus Natrialba, the genus Natronomonas, the genus Natronobacterium, and the genus Natronococcus. The genus Natrialba is preferable, and the Natrialba aegyptiaca is more preferable. N. aegyptiaca is thought to produce L-PGA to skillfully protect itself from the dehydration phenomenon that occurs in high salt environments (eg FF Hezayen, BHA Rehm, R. Eberhardt, A. Steinbuchel, Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor, Applied Microbiology and Biotechnology, 2000, 54,319). Among N. aegyptiaca, Natrialba aegyptiaca (Natrialba edipithiakia) 0830-82 strain (Accession number: FERM P-20872), Natrialba aegyptiaca (Natrialba edeputiacia) 0830-243 strain (Accession number: FERM P-20873), and It is more preferable to use at least one strain selected from the group consisting of Natrialba aegyptiaca 0831-264 (accession number: FERM P-20874). When N. aegyptiaca is used, L-PGA having a higher molecular weight can be obtained. In particular, N. aegyptiaca FERM P-20873, N. aegyptiaca FERM P-20873, and N. aegyptiaca FERM P-20874 all synthesize L-PGA having an average molecular weight of 1.3 million or more under liquid culture conditions. Can do. Therefore, the yield of the cross-linked product of L-PGA is good, and the production efficiency of L-PGA is also good.

  N. aegyptiaca FERM P-20873, N. aegyptiaca FERM P-20873, and N. aegyptiaca FERM P-20874 are based on the mutation treatment method and screening method described in Japanese Patent Application No. 2006-142585. It is a mutant strain of N. aegyptiaca that was found uniquely. In the present specification, the expression “N. aegyptiaca” simply includes a mutant strain of N. aegyptiaca.

  As described above, as the L-PGA to be included in the external preparation for skin according to the present invention, a microorganism producing L-PGA having a large average molecular weight may be selected and used by the screening method, and further, the mutation treatment described above may be used. The microorganism selected by the above screening method may be used after the microorganism is subjected to mutation treatment by the method.

(Mutation processing method)
Here, an embodiment in which a mutant strain of a microorganism with improved L-PGA productivity is produced by the mutation treatment method described in Japanese Patent Application No. 2006-142585 will be described.

  Examples of the mutation treatment method include, for example, a method by genetic recombination, a method of contacting a cell or spore with a mutagenic agent, a method of irradiating radiation such as X-rays or γ-rays, ultraviolet rays, etc. A conventionally known method may be used. Examples of the mutagenic agent include alkylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonic acid (EMS). The present inventors performed mutation treatment on L-PGA producing microorganisms using NTG. In addition, when performing the above-mentioned mutation treatment on the L-PGA-producing microorganism, it is preferable to carry out the strain at a strength such that the survival rate of the strain after the mutation treatment is 1% or less, but there is a particular limitation. It is not a thing.

  For example, the present inventors first cultured N.aegyptiaca (JCM11194) as a parent strain in a medium for producing PGA, then cultured in an NTG solution, and further cultured in a medium for producing PGA. At this time, conditions, such as the density | concentration of the NTG solution from which a survival rate will be 1% or less, were discovered, and the surviving microbial cell was obtained as a mutant.

Here, the medium for PGA production is not limited as long as the strain to be used in the mutation treatment method is a medium capable of producing PGA. The present inventors have described that 22.5 wt% NaCl, 2 A liquid medium for PGA production having a composition of wt% MgSO ₄ .7H ₂ O, 0.2 wt% KCl, 3 wt% Trisodium Citrate, 1 wt% Yeast Extract, 0.75 wt% Casamino acid was used. The culture conditions are not particularly limited. For example, the present inventors cultured in an 18 ml test tube containing 3 ml of the above-mentioned liquid medium for PGA production at 37 ° C. and 300 rpm for 3 days, and then obtained the culture solution. 0.5 ml was inoculated into a 500 ml Sakaguchi flask containing 50 ml of the above liquid medium for PGA production and cultured at 37 ° C. and 180 rpm for 5 days.

  The composition of the NTG solution is not particularly limited as long as it is set as appropriate so that the survival rate of the strain to be subjected to the mutation treatment method is 1%. For example, the present inventors suspended a strain obtained by culturing a saturated solution of NTG (Tokyo Kasei Co., Ltd.), an aqueous solution of 70% by weight, 50% by weight, 20% by weight, and 10% by weight in the above liquid medium for PGA production. 1/10 amount of the suspension was added to the liquid, and the suspension was cultured. When preparing the suspension, the liquid in which the strain is suspended is not particularly limited, but the present inventors used a 100 mM citrate buffer (pH 6.0). Specifically, after centrifuging at 3000 rpm for 5 minutes from the culture solution obtained by culturing with the above-described PGA production liquid medium to collect the cells, 100 mM citrate buffer solution (pH 6.0) was added and suspended. It became cloudy. This operation from centrifugation to suspension was repeated three times to obtain a suspension. The culture conditions using the NTG solution are not particularly limited. For example, the present inventors cultured at 42 ° C. and 150 rpm for 1 hour.

The medium for PGA production for culturing the strain after culturing with the NTG solution is not particularly limited, and the above-described liquid medium for PGA production may be used. (10% NaCl, 2% MgSO 4 · 7H 2 O, 0.2% KCl, 3% trisodium Citrate, 1% Yeast Extract, 0.75% casamino acid, 2% Agar) seeding the 5 days at 37 ° C. Cultured. Thus, when N.aegyptiaca (JCM11194) was used as a parent strain and subjected to the mutation treatment method, the survival rate was 1% or less when a 70 wt% NTG solution was used.

(Screening method)
Next, the screening method will be described. In addition, the strain to be subjected to the screening method may be a strain that has been subjected to mutation treatment by the mutation treatment method, or may be a wild strain that has not been subjected to the mutation treatment method.

  The screening method is performed by selecting L-PGA producing microorganisms with high salt sensitivity. Specifically, the L-PGA-producing microorganism is usually cultured under a salt concentration where it is difficult to produce L-PGA, and a colony showing a mucoid shape is selected as a guide. . As the salt, conventionally known ones such as sodium, potassium, magnesium, manganese, calcium, zinc and iron may be used, and sodium is preferred.

  In the present specification, the term “mucoid” means a state where the colony is viscous, and specifically, the microorganism has a monosaccharide or a polysaccharide chain side chain covalently bonded to the main column of the polypeptide chain. A state in which a macromolecule is produced and the colony of the microorganism is viscous is intended. That is, in the above screening method, colonies that show a viscous state, that is, a mucoid shape by binding L-PGA and polysaccharide may be selected.

  Moreover, in this specification, "salt sensitivity" intends the salt concentration from which microorganisms start production of L-PGA. And "high salt sensitivity" intends that the salt concentration with which microorganisms start production of L-PGA is low. That is, in the above screening method, a strain that produces L-PGA with a lower salt concentration is selected. Specifically, it is preferable to select a strain that produces L-PGA with NaCl of 5% by weight to 20% by weight, preferably 7% by weight to 15% by weight.

  Conventionally, it has been difficult to screen for PGA-producing microorganisms under liquid culture conditions. This is because, for example, N. aegyptiaca was difficult to separate single colonies because mucoid colonies formed on the surface of the solid medium fused with each other. Furthermore, even if single colonies were separable, enormous time and labor were required for liquid culture one by one and confirming the presence or absence of L-PGA production. That is, the skin external preparation according to the present invention is a completely new skin external preparation that has been made possible for the first time by using a high molecular weight L-PGA-producing bacterium obtained by the screening method uniquely found by the present inventors. It is.

  Here, an example of a method for screening a strain having excellent L-PGA production ability from the mutant strain of N. aegyptiaca obtained by the above-described mutation treatment method will be described, but the present invention is not limited thereto.

First, the mutant strain to be subjected to the above screening method may be cultured in a conventionally known nutrient medium. The nutrient medium is not particularly limited. For example, a medium containing gravy, peptone, soybean flour, Yeast Extract, Casamino acid, amino acids or a mixture thereof, or inorganic synthesis containing necessary nutrients. An agar plate medium such as a medium may be used. Moreover, you may use the culture medium in which PGA production microorganisms are easy to produce PGA. The medium that can easily produce the PGA is not particularly limited. For example, 22.5 wt% NaCl, 2 wt% MgSO ₄ .7H ₂ O, 0.2 wt% KCl, 3 wt% Trisodium Citrate, An agar medium composed of 1% by weight Yeast Extract, 0.75% by weight Casamino acid, 2% by weight Agar (hereinafter referred to as “Agar medium for PGA production”) may be used. The culture period is not particularly limited, but is preferably 2 to 4 days.

  Next, the mutant strain may be cultured using a medium in which L-PGA producing microorganisms are difficult to produce L-PGA, and a strain that produces L-PGA in the medium may be selected. The medium in which L-PGA is difficult to produce is not limited as long as the L-PGA producing microorganism has a composition that makes it difficult to produce L-PGA. Specifically, the salt concentration in the medium may be lowered. For example, the inventors used a solid medium containing 10% by weight NaCl. This is because N. aegyptiaca can itself grow on a medium containing 10% by weight or more of a salt, but unless it is cultured on a medium containing 20% by weight or more of a salt, a strain that does not produce L-PGA Because there are many. N. aegyptiaca does not show mucoid under solid culture conditions with a NaCl concentration of 10% by weight. Furthermore, the solid culture is higher by 10 times or more in the production of L-PGA per cell than in the liquid culture.

More specifically, we have 10 wt% NaCl, 2 wt% MgSO ₄ .7H ₂ O, 0.2 wt% KCl, 3 wt% Trisodium Citrate, 1 wt% Yeast Extract, 0.75 wt%. An agar medium comprising% Casamino acid and 2% by weight Agar (hereinafter referred to as “Agar medium for PGA production”) was used. The culture conditions at this time are not particularly limited. For example, the temperature may be 37 ° C., and the culture period may be 4 to 6 days.

  In addition, while culturing the strain using a medium that is difficult to produce L-PGA such as PGA production agar medium B, a medium that is easy to produce L-PGA such as PGA production agar medium A is also used. It is preferable to confirm in parallel the L-PGA production ability of the strain to be subjected to screening.

  The selected strain is preferably cultured in a medium that can easily produce L-PGA to confirm reproducibility of L-PGA production ability. For the confirmation, for example, the above-described PGA production agar medium A may be used for culture.

  In this way, a strain having a high salt sensitivity can be selected.

  Next, the selected strain with high salt sensitivity is cultured in a liquid medium for PGA production, the production amount of L-PGA is measured, and the strain with a higher L-PGA production amount is selected again.

  At this time, since L-PGA producing microorganisms that produce L-PGA well have already been selected, selection by liquid culture can be easily performed. This is one of the points where the method for selecting a strain having high salt sensitivity is superior to the method for selecting other PGA-producing microorganisms. That is, it is not easy for those skilled in the art to obtain a microorganism or a mutant thereof capable of producing L-PGA only by selection by liquid culture without performing the selection method. This is because it is necessary to culture each colony obtained by solid culture and confirm the production amount of L-PGA. Those skilled in the art can easily understand that this operation is astronomical and is virtually impossible. The inventors of the present invention have made diligent efforts and found a method for easily obtaining a microorganism or a mutant thereof capable of producing L-PGA by liquid culture.

  Although it does not specifically limit as a liquid culture medium for PGA production used when re-selecting a strain with a high L-PGA production amount, If the liquid culture medium for PGA production used in the above-mentioned mutation treatment method is used, Good. The culture conditions are not particularly limited. For example, the present inventors have excellent L-PGA production ability by culturing at 37 ° C. and 1180 rpm for 4 days using the above-described liquid medium for PGA production. Strains were selected. Moreover, after culturing once in a small system, the system may be enlarged and cultured. For example, the present inventors inoculated a single colony of a strain into an 18 ml test tube containing 1 platinum ear scrape and 3 ml of the above liquid medium for PGA production and cultured at 37 ° C. and 300 rpm for 3 days. Then, 0.5 ml of the obtained culture solution was inoculated into a 500 ml Sakaguchi flask containing 50 ml of the above-mentioned liquid medium for PGA production and cultured at 37 ° C. and 180 rpm for 3 days.

  Next, L-PGA in the liquid medium may be measured to select a strain that produced more L-PGA.

  The method for measuring the amount of L-PGA in the liquid medium is not particularly limited. For example, L-PGA is precipitated using copper sulfate or ethanol, and the weight of the precipitate is measured, or A method of measuring total nitrogen by the Kijderder method (M. Bovarnick, J. Biol. Chem., 145, 415, 1942), a method of measuring the amount of glutamic acid after hydrochloric acid hydrolysis (RDHousewrigt, CBThorne, J. Bacteriol., 60, 89, 1950), a colorimetric method using quantitative binding with a basic dye (M. Bovarnick et al., J. Biol. Chem., 207, 593) , 1954). Among these, it is preferable to use a colorimetric method utilizing quantitative binding with a basic dye.

  Examples of the basic dye used in the colorimetric method include crystal violet, aniline blue, safranin o, methylene blue, methyl violet, toluidine blue, congo red, azocarmine, thionine, and hematoxylin, and safranin o is preferable. The present inventors measured the amount of L-PGA in a liquid medium using 1 / 5-fold diluted safranin o, and selected strains having higher L-PGA production ability than the parent strain.

  The present inventors screened 30,000 strains by the mutation treatment method and screening method described so far, and as a result, three L-PGA production mutant strains were obtained.

  The bacterial strain obtained in this way is registered with the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary at Natrialba aegyptiaca 0830-82 (trusted organization: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit). Center, date of receipt: April 4, 2006, accession number: FERM P-20872), Natrialba aegyptiaca 0830-243 strain (trusted organization: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center) Date of receipt: April 4, 2006, accession number: FERM P-20873), or Natrialba aegyptiaca 0831-264 shares (trusted institution: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary) Date: April 4, 2006, accession number: FERM P-20874 It has been deposited as.

  All of the three strains have an L-PGA production ability superior to the parent strain JCM11194, and can produce high molecular weight L-PGA on an industrial scale. For example, according to the culture divided into the above two stages, JCM11194 strain produced 0.61 g of L-PGA per liter of culture solution, while 0831-264 strain produced 4.99 g of L-PGA. .

(Manufacturing method of L-PGA)
Next, as a method for producing L-PGA, a case where N. aegyptiaca is used is exemplified, but the method for obtaining L-PGA contained in the external preparation for skin according to the present invention is not limited thereto.

  The medium for culturing N. aegyptiaca is not particularly limited as long as the medium can grow N. aegyptiaca and can synthesize L-PGA, but is preferably a liquid medium. If a liquid medium is used, N. aegyptiaca can be cultured in large quantities at a time, so that the production efficiency of L-PGA is greatly improved.

The components of the medium used for culturing N. aegyptiaca may include a carbon source and inorganic salts that can be ingested by N. aegyptiaca, and other nutrients such as Yeast Extract may be added as necessary. For example, in the examples described below, the inventors have described 22.5% NaCl, 2% MgSO ₄ .7H ₂ O, 0.2% KCl, 3% Trisodium Citrate, 1% Yeast Extract, 0.75% Casamino. N. aegyptica FERM P-20874 is cultured using an acid medium. In addition, when adding Yeast Extract to a culture medium, it is preferable that the density | concentration is 0.1 to 10 weight%, and 0.5 to 5.0 weight% is further more preferable.

  Since N. aegyptiaca is a highly halophilic bacterium, a salt may be added to the medium depending on the growth characteristics of N. aegyptiaca used for the production of L-PGA. The culture may be performed at a salt concentration of 10% by weight to 30% by weight, preferably 15% by weight to 25% by weight.

  The pH of the medium used for culturing N. aegyptiaca is not particularly limited, but is preferably 5.0 or more and 10 or less, and more preferably 6.0 or more and 8.5 or less. The pH may be adjusted using sodium hydroxide, potassium hydroxide, ammonia, hydrochloric acid, sulfuric acid, an aqueous solution thereof, or the like, but is not limited as long as the pH can be adjusted.

  After the medium is prepared, it is sterilized by a normal method, and then N. aegyptiaca used for L-PGA production may be added and cultured. The medium may be sterilized by a conventionally known method, for example, 110 to 140 ° C. for 8 to 20 minutes. Note that the sterilization step can be omitted by setting the NaCl concentration of the medium to a saturated concentration. As described above, because N. aegyptiaca is a highly halophilic bacterium, it can grow even in the presence of saturated NaCl, but other microorganisms cannot grow.

  When N. aegyptiaca is subjected to liquid culture, it is preferable to perform shaking culture, aeration-agitation culture, or the like. The culture temperature is not particularly limited, but is preferably 25 ° C. or higher and 50 ° C. or lower, and more preferably 30 ° C. or higher and 45 ° C. or lower.

  The culture period of N. aegyptiaca is not particularly limited as long as it is appropriately set according to other culture conditions and the target L-PGA production amount, and may be, for example, about 2 to 4 days.

  When N. aegyptiaca is cultured based on the above culture conditions and the like, L-PGA is mainly accumulated outside the cells.

  The method for separating and recovering L-PGA from the medium after culturing N. aegyptiaca is not particularly limited, and a conventionally known method may be used. For example, (1) a method of extracting and separating from a solid culture with 20% or less of saline (for example, see JP-A-3-30648), (2) a precipitation method using copper sulfate (for example, Throne.BC, CCGomez, NENoues and RDHousevright: J. Bacteriol., 68, 307, 1954), (3) Alcohol precipitation method (eg RMVard, RF Anderson and FKDean: Biotechnology and Bioengineering, 5, 41, 1963), (4) Chromatographic method using a crosslinked chitosan molded product as an adsorbent (see, for example, JP-A-3-244392), (5) Molecular ultrafiltration using a molecular ultrafiltration membrane And (6) a method in which the above (1) to (5) are appropriately combined. These methods can also be applied when a microorganism other than N. aegyptiaca is used for the production of L-PGA. The L-PGA separated and recovered in this way may be used as an L-PGA-containing liquid for the production of an L-PGA crosslinked product described later, and conventionally known methods such as spray drying and freeze-drying as necessary. Or powder.

  Below, although an example of the method of isolate | separating and collect | recovering L-PGA from the culture medium after culture | cultivating N. aegyptiaca is demonstrated, it is not limited to this.

  First, by removing the cells from the culture solution after culturing N. aegyptiaca by centrifugation or the like, and then adding lower alcohol such as ethanol from the obtained supernatant, L-PGA is precipitated. Good. The precipitate is preferably dissolved in a buffer solution as appropriate, and impurities are removed by dialysis or the like. In addition, as shown also in the Example mentioned later, the present inventors added and diluted 3 times the amount of water to the supernatant after collect | recovering microbial cells, and also adjusted pH to 3.0. Then, after stirring at room temperature for 5 hours, the precipitate was collected by adding 3 times the amount of ethanol. The precipitate was dissolved in 0.1 mM Tris-HCl buffer (pH 8.0) and dialyzed to remove impurities.

  Even if dialysis is performed, it is considered that nucleic acids and proteins are mixed, and therefore, it is preferable to perform DNase treatment, RNase treatment, proteinase treatment, and the like. Further, after these treatments, further purification treatment such as dialysis can be performed to obtain L-PGA of higher purity.

  As described above, a solution containing L-PGA can be obtained. Furthermore, a powdery L-PGA crosslinked product can be obtained by lyophilizing the obtained solution. Moreover, you may refine | purify the said solution further as needed. Purification may be performed by a conventionally known method. For example, the above-described dialysis may be performed, and an anion exchange resin may be used.

(L-PGA crosslinked product)
The L-PGA crosslinked body contained in the external preparation for skin according to the present invention is not particularly limited as long as it has a crosslinked structure of L-PGA molecules.

  In this specification, “crosslinked structure” intends a structure in which molecules of a linear polymer compound are linked by a physical or chemical method. In addition, in the present specification, the “crosslinked body” intends a polymer compound having a crosslinked structure and having changed physical and chemical properties.

  That is, in the L-PGA crosslinked body, L-PGA molecules are linked in a three-dimensional manner by covalent bonds. Specifically, L-PGA molecules are linked three-dimensionally by covalently bonding any element other than H in the above formula (1) between the L-PGA molecules. In other words, the L-PGA cross-linked product according to the present invention is a polymer in which L-PGA molecules are three-dimensionally connected, that is, a network polymer having L-PGA as a constituent molecule. In addition, between the molecules of L-PGA, N shown in the above formula (1) in one L-PGA molecule and C shown in the rightmost end of the above formula (1) in the other L-PGA molecule are bonded. This is a polymerization between molecules of L-PGA, and is not intended as a “crosslinked structure”.

  The average molecular weight of L-PGA constituting the L-PGA crosslinked body contained in the external preparation for skin according to the present invention is not limited as long as the molecules are crosslinked, but preferably 1 million or more. More preferably, it is 2 million or more, and more preferably 3.5 million or more. When the molecular weight is 1,000,000 or more, the gelation rate when the hydrogel is produced from the raw material L-PGA is improved, so that the yield of the hydrogel can be improved.

  The higher the average molecular weight of L-PGA, the higher the water absorption capacity of the resulting L-PGA crosslinked product. Therefore, the upper limit value of the average molecular weight of L-PGA constituting the L-PGA crosslinked body according to the present invention is not particularly limited.

  The water absorption capacity of the L-PGA crosslinked body contained in the skin external preparation according to the present invention is not particularly limited, but according to the method for producing an L-PGA crosslinked body according to the present invention described later, for example, 10 times. More than 5000 times, especially 1900 times or more and 4400 times or less can be suitably obtained. In particular, as a water-absorbing resin made of PGA, a resin having a water absorption ratio larger than 3300 times could not be obtained even if DL-PGA was used in Patent Document 2 above, which was an innovative PGA biodegradability. It is a water absorbent resin.

  In the present specification, the “water absorption capacity” intends a rate of increase in weight due to swelling of a substance containing a hydrophilic liquid such as water. What is necessary is just to calculate the water absorption magnification of a L-PGA crosslinked body as follows, for example. That is, after the powder of the L-PGA crosslinked body is sufficiently swollen by placing it in a sufficient amount of water for the L-PGA crosslinked body to swell, it is allowed to stand at 4 ° C. for 1 week, and then 80 mesh. The value obtained by subtracting the dry weight of the powder of the L-PGA crosslinked product from the wet weight of the L-PGA after draining with a wire mesh is divided by the dry weight of the powder of the L-PGA crosslinked product. it can.

  The L-PGA crosslinked body contained in the external preparation for skin according to the present invention is preferably a crosslinked body consisting only of L-PGA, but may contain DL-PGA molecules or D-PGA molecules. . However, in order to stabilize the quality of each L-PGA crosslinked product to be produced, the content is preferably 0% by weight or more and 20% by weight or less.

(Manufacturing method of L-PGA crosslinked body)
The method for producing the L-PGA crosslinked body contained in the external preparation for skin according to the present invention only needs to include a crosslinking step of crosslinking L-PGA molecules. By cross-linking L-PGA consisting only of L-glutamic acid and having uniform optical activity as a raw material, the properties of the L-PGA cross-linked product are uniform. Therefore, it is possible to stably produce an L-PGA crosslinked product having a desired quality.

  L-PGA may be subjected to a crosslinking reaction after dissolving in a solvent to prepare a solution of L-PGA. The solvent for dissolving L-PGA is not limited as long as L-PGA can be dissolved, and examples thereof include water, alcohol, acetone, methyl acetate, ethyl acetate, and the like. Methyl alcohol and ethyl alcohol are preferable, and water is more preferable. The concentration of L-PGA when dissolved in these solvents is not particularly limited, but is preferably 1% by weight to 10% by weight, more preferably 2% by weight to 8% by weight. Particularly preferably, it is 2% by weight or more and 7% by weight or less. The pH of the L-PGA solution is not particularly limited, but is preferably 5.0 or more and 9.0 or less, and more preferably pH 6.0 or more and 8.0 or less.

  When a cross-linking reaction is performed on the L-PGA solution, a cross-linked product of L-PGA is formed in the solution, and the L-PGA cross-linked product swells containing the solvent, whereby a hydrogel is obtained. This is one of the aspects of the hydrogel comprising the L-PGA crosslinked product described later. Furthermore, L-PGA crosslinked body which does not contain the said solvent component can be obtained by removing the solvent component by freeze-drying the said hydrogel.

  According to the above-described method for producing a crosslinked L-PGA, L-PGA can be obtained in the crosslinking reaction at a gelation rate of, for example, 50% to 100%, particularly 70% to 100%.

  In the present specification, the “gelation rate” intends the percentage of the weight of L-PGA formed by a crosslinking reaction with respect to the weight of L-PGA used as a raw material. In other words, the “gelation rate” represents the yield of the obtained L-PGA crosslinked body and thus the hydrogel with respect to the L-PGA used as a raw material. Specifically, the value obtained by dividing the dry weight of the hydrogel obtained by the crosslinking reaction by the dry weight of L-PGA subjected to the crosslinking reaction is calculated by multiplying by 100.

  The method for the crosslinking reaction of L-PGA is not limited as long as L-PGA molecules can be crosslinked with each other, and may be performed by a conventionally known method. For example, a cross-linking agent may be used and radiation may be used, but it is preferable to use radiation. If radiation is used, an operation for removing the crosslinking agent is not required after the crosslinking reaction, and a highly pure L-PGA crosslinked product can be produced.

  The usable radiation in the crosslinking reaction of L-PGA is not particularly limited, and α rays, β rays, γ rays, electron beams, neutron rays, X rays and the like may be used. Among these, γ rays are preferable. The γ rays may be generated using a conventionally known method and apparatus such as an irradiation apparatus using cobalt 60 as a radiation source.

  Further, the radiation dose to the L-PGA is preferably 0.5 kGy or more and 20 kGy or less, more preferably 2 kGy or more and 10 kGy or less, and particularly preferably 3 kGy or more and 7 kGy or less. What is necessary is just to set suitably according to the use etc. of a body. In general, a hard hydrogel can be obtained if the irradiation dose is large, and a soft hydrogel can be obtained if the irradiation dose is small.

  Moreover, when using a radiation for bridge | crosslinking of L-PGA, what is necessary is just to put and use the solution of L-PGA in a radiation transparent container. The radiation transmissive container is not particularly limited, and for example, a glass container such as a glass vial can be used.

  After putting the L-PGA solution in the radiation transmissive container, radiation may be applied as it is, but it is preferable to bubble the solution beforehand using nitrogen. By removing oxygen contained in the solution, inhibition of the crosslinking reaction can be prevented.

  In addition, when using a crosslinking agent for crosslinking of L-PGA, a conventionally known crosslinking agent such as an epoxy compound, a polysaccharide containing a carboxylic acid group and / or a carboxylate group, an amino acid, etc. may be used, and is particularly limited. is not. For example, the epoxy compound includes glycerin triglycidyl ether, di-glycerin polyglycidyl ether, poly-glycerin polyglycidyl ether, polyoxyethylene sorbitol polyglycidyl ether, and the polysaccharide includes glucose, fructose, galactose and glucuronic acid. Mixtures, mixtures of rhamnose, glucose, galactose, and glucuronic acid, and polycarboxylic acids based on hyaluronic acid, amino acids include polyaspartic acid, polylysine, aspartic acid, lysine, arginine, and mixtures thereof It is done. These may be used alone or in combination of two or more.

  Moreover, L-PGA used in order to manufacture the L-PGA crosslinked body contained in the external preparation for skin according to the present invention may be in a salt state, for example, sodium salt, potassium salt, magnesium salt, calcium salt, etc. Can be used. Of these, sodium salts are preferred.

  As L-PGA used for producing the L-PGA crosslinked body contained in the external preparation for skin according to the present invention, L-PGA obtained by various conventionally known methods may be used. PGA may be used.

  In the case of producing an L-PGA crosslinked product containing DL-PGA molecules and D-PGA molecules, the DL-PGA molecules and / or D-PGA molecules are mixed with the above-mentioned L-PGA solution. Thus, the solution may be subjected to the above-described crosslinking reaction.

  A hydrogel comprising an L-PGA crosslinked body may be used as the L-PGA crosslinked body contained in the external preparation for skin according to the present invention.

  In the present specification, “hydrogel” means a gel formed by swelling when a polymer contains a solvent such as water. In other words, it is a solvent swelling body of a polymer mainly composed of a polymer and water such as a solvent. A hydrogel contains a large amount of water, is a substance in an intermediate state between a liquid and a solid, and is different from a liquid in that it does not have fluidity. Moreover, even if it presses and pressurizes, the solvent in hydrogel does not ooze out.

  That is, it can be said that the hydrogel comprising the L-PGA crosslinked body is a solvent swelled body mainly composed of the L-PGA crosslinked body and the solvent.

  A hydrogel comprising an L-PGA crosslinked body is obtained by subjecting a solution obtained by dissolving L-PGA in a solvent such as water to a crosslinking reaction as described in the above-described method for producing an L-PGA crosslinked body. be able to.

  Moreover, the hydrogel which comprises a L-PGA crosslinked body can be obtained by adding solvents, such as water, to the above-mentioned powdery L-PGA crosslinked body. At this time, if a hydrogel is obtained after reducing the amount of the solvent, a hydrogel excellent in water absorption capable of further absorbing a solvent such as water can be obtained. Moreover, since the solvent absorbed by the hydrogel containing a L-PGA crosslinked body does not ooze out from the hydrogel, it is excellent in moisture retention.

  The hydrogel containing L-PGA may be uniformly granulated into a predetermined shape, and may be in an irregularly crushed shape, a spherical shape, or the like. Moreover, it can be used not only in the hygiene field but also in various fields. For example, it can be suitably used as a soil conditioner for not only external preparations for skin but also toiletries such as paper diapers, medical products such as body fluid absorbers, and the like.

(Composition of external preparation for skin)
The concentration of at least one of the L-PGA and the L-PGA cross-linked product contained in the external preparation for skin according to the present invention is not particularly limited, but when it contains only L-PGA, 0.00001 to 30 % By weight, more preferably 0.0001 to 20% by weight. When only the L-PGA crosslinked product is contained, 0.00001 to 30% by weight is preferable, and more preferably 0.0001 to When it is 20% by weight and contains L-PGA and L-PGA cross-linked product, the total amount is preferably 0.00001-30% by weight, more preferably 0.0001-20% by weight. If it is this range, there is little smell and a color tone is also preferable. Furthermore, since a high moisturizing property is exhibited, a skin external preparation more useful as a moisturizing agent and / or cosmetic can be obtained.

  The skin external preparation according to the present invention may be prepared by dissolving at least one of L-PGA and L-PGA crosslinked product in a conventionally known solvent. Although it does not specifically limit as a solvent used for preparation of the skin external preparation which concerns on this invention, What is necessary is just to use water.

  In addition, the skin external preparation according to the present invention is generally used for skin external preparations such as cosmetics, quasi-drugs, and pharmaceuticals as long as the effects of the present invention are not impaired as appropriate according to the purpose of use. Additives used in, for example, oily components such as hydrocarbons, fats and oils, waxes, silicones, alcohols, fatty acids, antioxidants, antibacterial agents, ultraviolet absorbers, drugs, aqueous components such as purified water, Add additives such as moisturizers, thickeners, preservatives, surfactants, fragrances, colorants, various skin nutrients other than plant extracts, neutralizers, L-PGA and L-PGA cross-linked products May be.

  Although the specific example of these additives is given to the following, it is not limited to this. Moreover, these additives may be used independently and may mix and use 2 or more types.

  Examples of the hydrocarbons include liquid paraffin, squalane, microcrystalline wax, ceresin wax, paraffin wax, petrolatum and the like.

  Examples of the fats and oils include avocado oil, camellia oil, macadamia nut oil, olive oil, lanolin, castor oil, olive oil, grape seed oil, cacao oil, coconut oil, tree wax, jojoba oil and the like. .

  Examples of the waxes include jojoba oil, carnauba wax, candelilla wax, beeswax, and whale wax.

  Examples of the silicones include dimethylpolysiloxane and methylphenylsiloxane.

  Examples of the alcohols include higher alcohols such as capryl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, cholesterol, phytosterol, cetanol, stearyl alcohol, hexyl decanol, and octyldodecanol, and lower alcohols such as ethanol.

  Examples of the fatty acid include higher fatty acids such as capric acid, myristic acid, palmitic acid, stearic acid, behenic acid, lanolin fatty acid, linoleic acid, linolenic acid, lauric acid, oleic acid, and isostearic acid.

  Examples of the antioxidant include butylhydroxytoluene, tocopherol, and phytin.

  Examples of the antibacterial agent include the benzoic acid, salicylic acid, sorbic acid, paraoxybenzoic acid alkyl ester, and hexachlorophene.

  Examples of the ultraviolet absorber include paraaminobenzoic acid ultraviolet absorbers, anthranilic acid ultraviolet absorbers, salicylic acid ultraviolet absorbers, cinnamic acid ultraviolet absorbers, benzophenone ultraviolet absorbers, sugar ultraviolet absorbers, 3- (4′-methylbenzylidene) -d-camphor, 3-benzylidene-d, 1-camphor, urocanic acid, urocanic acid ethyl ester, 2-phenyl-5-methylbenzoxazole, 2,2′-hydroxy- 5-methylphenylbenzotriazole, 2- (2′-hydroxy-5′-t-octylphenyl) benzotriazole, 2- (2′-hydroxy-5′-methylphenylbenzotriazole), dibenzalazine, dianisoylmethane, 4- Methoxy-4′-tert-butyldibenzoylmethane, 5- (3,3-di Chill 2-norbornylidene) -3-pentan-2-one, and the like.

  Examples of the drug include glycine, alanine, valine, leucine, threonine, phenylalanine, tyrosine, aspartic acid, asparagine, glutamine, taurine, arginine, histidine, and other amino acids, or alkali metal salts and hydrochlorides thereof; acyl sarcosine acid (For example, lauroyl sarcosine sodium), glutathione, citric acid, malic acid, tartaric acid, lactic acid and other organic acids; nicotinic acid amide, benzyl nicotinate, γ-oryzanol, allantoin, glycyrrhizic acid (salt), glycyrrhetinic acid and its derivatives, hinokitiol , Saponins such as bisabolol, eucalptone, thymol, inositol, psychosaponin, carrot saponin, loofah saponin, muclodisaponin, pantothenyl ethyl ether, ethinyl ester Toraji ol, tranexamic acid, arbutin, cepharanthine, placenta extract, and the like.

  Examples of the various skin nutrients include vitamin A and its derivatives, vitamin B2, pantothenic acid and its derivatives, niacin, biotin, and mixtures thereof.

  The neutralizing agent is not particularly limited. For example, potassium hydroxide, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate, sodium acetate, 2-amino-2-methyl- Examples include 1-propanol, 2-amino-2-methyl-1,3-propanediol, and triethanolamine.

  Examples of the surfactant include polyoxyethylene lauryl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene Alkyl allyl ether, polyoxyethylene distyrenated phenyl ether, polyoxyethylene derivative, sorbitan monolaurate, sorbitan monooleate, sorbitan sesquioleate, sorbitan tanylate, polyoxyethylene monolaurate, polyoxyethylene sorbitan Laurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, 1-polyoxyethylene Rubitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tetraoleate, polyethylene glycol monolaurate, polyethylene glycol monooleate, polyoxyethylene hydrogenated castor oil, polyoxythielen castor oil, polyoxyethylene Nonionic surfactants such as lanolin and amphoteric surfactants such as glycine type, alkylaminobetaine, imidazoline type, L-arginine type, and L-lysine type can be mentioned.

  Examples of the humectant other than the L-PGA and the L-PGA crosslinked product include polyhydric alcohols such as glycerin, propylene glycol, 1,3-butylene glycol, and polyethylene glycol, glucose, sorbitol, dextrin, trehalose, and lactose. Saccharides and derivatives thereof, amino acids such as glutamate, keratin derivatives, collagen derivatives, trimethylglycine and derivatives thereof, carboxyvinyl polymer, sodium chondroitin sulfate, sodium hyaluronate, sodium pyrrolidone carboxylate, sodium lactate and the like, Seaweed extract, yeast extract, various plant extracts having moisturizing action, and mixtures thereof, such as isopropyl palmitate, isopropyl myristate, octyldodecyl myristate, olei Octyldodecyl, esters such as cholesteryl oleate, sodium polyacrylate, cellulose, various plant essential oils and mixtures thereof. In addition, the above-mentioned vegetable oils and fats, waxes, fatty acids, and higher alcohols can also be used as a humectant.

  Examples of the thickener include water-soluble polysaccharides such as xanthan gum, water-soluble celluloses such as sodium hydroxymethylcellulose, methylcellulose, and hydroxyethylcellulose, water-soluble polymers such as pullulan and sodium polyacrylate.

  Examples of the preservative include paraben, salicylic acid, benzoate, phenoxyethanol, chlorhexidine gluconate, and the like.

  Examples of the fragrances include vanillin, orange flavor, lemon flavor, milk flavor geraniol, and linalool.

  Examples of the colorant include water-soluble tar dyes, water-insoluble tar dyes, gardenia dyes, safflower dyes, turmeric dyes, paprika dyes, anato dyes, cochineal dyes and other natural dyes, acidic and basic dyes. Is mentioned.

  In addition, for example, borage, clara, corn, orange, sage, yarrow, mallow, assembly, thyme, pearl, spruce, birch, horsetail, loofah, maroonie, yukinoshita, arnica, lily, mugwort, peonies, aloe, gardenia, sawara, An extract of a plant such as white lily may be added.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. In the following examples, “%” is “% by weight”.

[Example 1; Production of poly-γ-L-glutamic acid]
Natrialba aegyptica (Accession No .: FERM P-20874) L dry ampoule with 0.4 ml liquid medium for PGA production (22.5% NaCl, 2% MgSO ₄ .7H ₂ O, 0.2% KCl, 3% Trisodium Citrate, 1% Yeast Extract, 0.75% Casamino acid) was added to obtain a suspension. 0.2 ml of the suspension was added to PGA agar medium (10% NaCl, 2% MgSO ₄ .7H ₂ O, 0.2% KCl, 3% Trisodium Citrate, 1% Yeast Extract, 0.75% Casamino acid, 2% Agar) and cultured at 37 ° C. for 3 days to obtain a single colony.

Next, in each of five 18 ml test tubes, 3 ml of PGA production liquid medium (22.5% NaCl, 2% MgSO ₄ .7H ₂ O, 0.2% KCl, 3% Trisodium Citrate, 1% Yeast Extract, 0.75% Casamino acid, pH 7.2) was added, and the single colony was scraped and inoculated with one platinum ear with a platinum ear. The test tube after inoculation was cultured at 37 ° C. and 300 rpm for 3 days, and then 0.5 ml of the obtained culture solution was inoculated into 10 500 ml Sakaguchi flasks each containing 50 ml PGA production liquid medium. And cultured at 37 ° C. for 5 days. After culturing, the obtained culture solution was centrifuged, the cells were removed, and the supernatant was collected.

  Next, 3 times the amount of water was added to the collected supernatant for dilution, and then the pH was adjusted to 3.0 with 1N sulfuric acid. After adjusting the pH, the mixture was stirred at room temperature for 5 hours. Thereafter, 3 times the amount of ethanol was added and centrifuged, and the precipitate was collected. This precipitate is L-PGA.

The collected L-PGA was dissolved in 0.1 mM Tris-HCl buffer (pH 8.0), and dialyzed to remove impurities such as low molecular weight substances. Next, in order to remove nucleic acid contained in the liquid after dialysis, MgCl ₂ is 1 mM, DNase I (TAKARA) is 10 U / ml, and RNase I (NIPPON GENE) is 20 μg / ml. And incubated at 37 ° C. for 2 hours. Next, in order to remove the protein, Proteinase K (manufactured by TAKARA) was added to the liquid after removing the nucleic acid so as to be 3 U / ml, followed by incubation at 37 ° C. for 5 hours to perform Proteinase K treatment.

  After proteinase K treatment, dialyzed with ultrapure water to remove low molecular weight substances. Next, L-PGA was adsorbed on an anion exchange resin (Q sepharose Fast Flow, manufactured by GE Healthcare Bioscience), washed with 0.5 M NaCl aqueous solution, and then eluted with 1 M NaCl aqueous solution. The obtained solution was further dialyzed with ultrapure water, and the solution after dialysis was freeze-dried to obtain a sodium salt of L-PGA (hereinafter referred to as “L-PGA / Na salt”). The ultrapure water was produced with MilliQ (pure water production apparatus manufactured by Millipore).

[Example 2; Molecular weight analysis of poly-γ-L-glutamic acid-1]
The average molecular weight of the L-PGA · Na salt obtained in Example 1 was measured by GPC analysis. As a result, it was confirmed that Mw = 7,522,000, Mn = 3,704,000, and Mw / Mn = 2.031 (in pullulan conversion).

  The GPC analysis was performed under the following conditions. Apparatus: HLC-8220GPC (manufactured by Tosoh Corporation), column: TSKgel α-M (manufactured by Tosoh Corporation), flow rate: 0.6 ml / min, eluent: 0.15 M NaCl aqueous solution, column temperature: 40 ° C., injection volume: 10 μl , Detector: differential refractometer.

[Example 3; Molecular weight analysis of poly-γ-L-glutamic acid-2]
In Example 1, L-PGA obtained by performing the same operation as in Example 1 except that after eluting with 1.0 M NaCl aqueous solution, the pH was adjusted to 2.0 using 1N HCl. -The average molecular weight of the Na salt was measured by GPC analysis. As a result, it was confirmed that Mw = 2,888,000, Mn = 1,327,000, and Mw / Mn = 2.176 (in pullulan conversion). The GPC analysis in this example was performed in the same manner as in Example 2.

[Example 4; Production of crosslinked poly-γ-L-glutamic acid]
A 5% aqueous solution of the L-PGA · Na salt obtained in Example 1 was prepared.

  Next, an aqueous L-PGA / Na salt solution was bubbled with nitrogen for 3 minutes, and then 2 ml was taken into a 10 ml sample bottle with a lid and the lid was closed.

  Next, the sample bottle was irradiated with γ-rays using a γ-ray irradiation device using cobalt 60 as a radiation source. Irradiation dose was 5 kGy. The product obtained after γ-ray irradiation was taken out of the sample bottle, excess water was drained with an 80-mesh wire mesh, and freeze-dried to obtain L-PGA crosslinked powder. The excess water contains uncrosslinked L-PGA, and the main purpose of draining is to remove uncrosslinked L-PGA.

[Example 5: Evaluation of moisturizing property of poly-γ-L-glutamic acid by dry skin roughness model]
A test skin (Toyobo Co., Ltd .: Code No. LSE-002) kit was taken out, and LSE (Living Skin Equivalent) tissue was taken out. Next, the LSE tissue was set in an assay plate (supplied with the above test skin kit) and allowed to stand for 7 hours in a dry state (CO ₂ incubator adjusted to a temperature of 37 ° C. and a relative humidity of 15% RH). . As a result, a dry rough skin model (hereinafter referred to as “dry LSE structure”) in which water in the stratum corneum evaporated was obtained.

  Next, 70 μl each of pure water, 0.5% DL-PGA aqueous solution, 2.5% DL-PGA aqueous solution, 0.5% L-PGA aqueous solution, and 2.5% L-PGA aqueous solution with a micropipette , And dropped onto the surface of the dry LSE tissue. In addition, L-PGA * Na salt obtained in Example 1 was used as L-PGA. In addition, DL-PGA / Na salt manufactured by Wako Pure Chemical Industries was used as DL-PGA.

Next, 600 μl of assay medium (attached to the above test skin kit) is added to the lower part of the assay plate on which the dry LSE tissue is set, and then left in a CO ₂ incubator adjusted to a temperature of 37 ° C. and a relative humidity of 15% RH. And incubated for 24 hours. Thereafter, the dry LSE tissue is removed from the CO ₂ incubator, and a mixed solution of an assay medium (attached to the test skin kit) containing tetrazolium salt (MTT) reagent 0.333 g / ml according to the instruction attached to the test skin kit. The dry LSE tissue was subjected to MTT treatment by placing 600 μl in an assay tray and incubating in a CO ₂ incubator adjusted to a temperature of 37 ° C. and a relative humidity of 15% RH for 3 hours.

  After the MTT treatment, the central portion of the dry LSE tissue was cut out together with the polycarbonate film below the dry LSE tissue using a biopsy punch (Toyobo Co., Ltd .; diameter 8 mm). Next, the cut section was transferred to a test tube, and 0.04N hydrochloric acid-isopropanol (300 μl) was added, and the mixture was allowed to stand in the dark for 2 hours. Next, the solution in the test tube was stirred and mixed well, and then centrifuged at 3,000 rpm for 5 minutes to obtain a supernatant. Next, the amount of blue-violet formazan contained in 200 μl of the supernatant was calculated by measuring the absorbance at 572 nm.

  The results are shown in FIG. FIG. 1 is a diagram showing the results of moisturizing evaluation, and the vertical axis shows the amount of formazan (absorbance) when the amount of formazan extracted from the LSE tissue not subjected to the drying treatment is 100%. The horizontal axis indicates the type of each sample. In FIG. 1, “dry untreated” indicates an LSE structure that has not been subjected to a drying process, and “no sample” refers to pure water or an aqueous DL-PGA solution for the LSE structure that has been subjected to a drying process. Neither the L-PGA aqueous solution nor the L-PGA aqueous solution was dropped. That is, FIG. 1 shows the results of measuring the amount of formazan by directly performing MTT treatment on the untreated sample and the amount of formazan by directly performing MTT treatment on the above-mentioned dry LSE tissue. The measurement results are also shown.

  The absorbance (amount of formazan) obtained by the method described in this example is closely related to the effect of improving rough skin, and human dry skin roughness is evaluated quantitatively, simply and economically. It is an effective dry skin roughness improvement evaluation method.

  As is clear from FIG. 1, in the case of commercially available DL-PGA that has been conventionally used as a moisturizing agent, the amount of formazan, that is, the recovery rate of rough skin was 30% as a result of dropping 2.5% aqueous solution. On the other hand, as a result of dropping 2.5% aqueous solution of L-PGA, 60% rough skin recovery rate was shown. Thereby, L-PGA showed the rough skin recovery rate of about 2 times compared with the conventional commercial item, and it was shown that it has high moisture retention.

Example 6 Moisturizing Effect of Poly-γ-L-glutamic Acid Crosslinked Hydrogel by Human Skin Roughness Test
The skin was roughened by subjecting the human upper arm to SDS treatment with a 0.5% SDS solution for 10 minutes. On the other hand, the L-PGA crosslinked powder obtained in Example 4 was mixed with water to obtain 0.15% to obtain a hydrogel.

  Next, the hydrogel was applied to the inside of the above-mentioned rough human upper arm, and left in a constant temperature and humidity room at room temperature (23 ° C.) and humidity of 45% for 1 hour. Next, the skin keratin water content inside the human upper arm (sample D) after standing was measured with a stratum corneum moisture measuring device (product name: skicon, manufactured by IBS Co., Ltd.).

  After measuring the same amount of skin keratin moisture, human upper arm before SDS treatment (sample A), after SDS treatment, human upper arm inside (sample B) before applying the hydrogel, after SDS treatment, Water was applied instead of the hydrogel, and it was also performed on the inside of the human upper arm (sample C) after standing in the constant temperature and humidity room under the same conditions as described above.

  The results are shown in FIG. FIG. 2 is a diagram showing the results of a human rough skin test, in which the vertical axis represents the skin keratin water content and the horizontal axis represents the type of sample. In FIG. 2, A to D correspond to the samples A to D, respectively.

  As shown in FIG. 2, Sample D showed that a high skin keratin water content was obtained and the keratin water content was recovered.

  As mentioned above, although this invention was demonstrated based on the Example, this invention is not limited to embodiment mentioned above, A various change is possible in the range shown to the claim. That is, embodiments obtained by combining technical means appropriately changed within the scope of the claims are also included in the technical scope of the present invention.

  According to the present invention, it is possible to provide a skin external preparation having a higher moisturizing property than conventional products by using a crosslinked product of poly-γ-L-glutamic acid and poly-γ-L-glutamic acid having a uniform optical purity and high molecular weight. It is expected to make a significant contribution to the cosmetics industry.

In Example 5 of this invention, it is a figure which shows the result of moisture retention evaluation. In Example 6 of this invention, it is a figure which shows the result of a human rough skin test.

Claims (3)

  An external preparation for skin comprising at least one of poly-γ-L-glutamic acid and a crosslinked poly-γ-L-glutamic acid.   The skin external preparation according to claim 1, wherein the skin external preparation is a cosmetic.   The skin external preparation according to claim 1, wherein the skin external preparation is a moisturizing agent.

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