JPWO2007034795A1 - γ-polyglutamic acid cross-linked product and method for producing the same - Google Patents

Abstract

To provide a poly-γ-glutamic acid gel having excellent swelling degree and moisture retention and biodegradability. In cross-linking poly-γ-glutamic acid with polyamine, water-soluble carbodiimide (eg, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) and N-hydroxysuccinimide (eg, N-hydroxy By using succinimide) as a condensing agent and a condensing aid, a poly-γ-glutamic acid hydrogel is obtained in a very high yield. In particular, when poly-γ-glutamic acid having a molecular weight of 1 million or more is used as a raw material, a poly-γ-glutamic acid hydrogel having excellent swelling and biodegradability is provided.

Description

  The present invention relates to a method for producing a polyamine-crosslinked poly-γ-glutamic acid gel.

  Poly-γ-glutamic acid hydrogel has attracted attention as a material for cosmetics and the like that is biodegradable and excellent in moisture retention. Hereinafter, in the present specification, poly-γ-glutamic acid is also referred to as “PGA”.

  Japanese Patent Laid-Open No. 2002-233391 (Patent Document 1) discloses a Bacillus strain that produces high molecular weight poly-γ-glutamic acid.

  International Publication No. WO2004-7593 (Patent Document 2) discloses poly-γ-glutamic acid having an average molecular weight of 5,000 kDa or more.

  JP-A-2005-179534 (Patent Document 3) discloses a method for obtaining a poly-γ-glutamic acid hydrogel using polyglycidyl ether as a crosslinking agent.

  However, the method described in Patent Document 3 requires a relatively high temperature (for example, 55 ° C.) and a long time (for example, 20 hours) in order to perform a ring-opening reaction of glycidyl ether without a catalyst. There is a disadvantage that PGA is easily decomposed due to high temperature.

Kunioka et al. (Non-Patent Document 1) discloses a method of cross-linking poly-γ-glutamic acid with polyamine, and water-soluble carbodiimide (for example, 1-ethyl-3- (3-dimethylaminopropyl) as a condensing agent is disclosed. Disclosed is a method for obtaining a poly-γ-glutamic acid hydrogel using -carbodiimide hydrochloride). In the specification of the present application, water-soluble carbodiimide is also referred to as “WSC” (acronym for Water-Soluble Carbideimide).

  However, this method has a drawback that the yield is very low. In addition, there is a drawback that only gels with a high polyamine content can be obtained. Furthermore, the degree of swelling of the gel is low, and there is a disadvantage that it is inferior in moisture retention when used as a hydrogel.

  Specifically, Non-Patent Document 1 describes the following formulation and data of the obtained gel in Table I on page 1892.

  In Table I of Non-Patent Document 1, there are no columns for the blending number and the amount of PGA (polyglutamic acid), but for the convenience of explanation, a column for the blending number and the amount of PGA is added in the following table. And describe. In the table, PGA is polyglutamic acid, and 1,3-PD is 1,3-propanediamine.

注（１）：非特許文献１の表Ｉ中にはＰＧＡの量は記載されていないが、表の下の脚注に１００ｍｇのＰＧＡが使用されたことが記載されている。
（２）：非特許文献１の表Ｉ中においては「Ｄｒｙ Ｇｅｌ Ｗｅｉｇｈｔ」と記載されている。
（３）：非特許文献１の表Ｉ中においては「Ｓｐｅｃｉｆｉｃ Ｗａｔｅｒ Ｃｏｎｔｅｎｔ」と記載されている。
（４）：非特許文献１の表Ｉ中においては「ｇｌｕ」と記載されている。
（５）：非特許文献１の表Ｉ中においては「１，３−ＰＤ」と記載されている。
（６）：上記表中、１，３−ＰＤの配合量は体積で示されている。１，３−ＰＤの比重（０．８８５ｇ／ｍＬ）を用いて重量に換算することが可能である。

Note (1): Although the amount of PGA is not described in Table I of Non-Patent Document 1, it is described that 100 mg of PGA was used in the footnote below the table.
(2): In Table I of Non-Patent Document 1, it is described as “Dry Gel Weight”.
(3): In Table I of Non-Patent Document 1, “Specific Water Content” is described.
(4): In Table I of Non-Patent Document 1, “glu” is described.
(5): In Table I of Non-Patent Document 1, “1,3-PD” is described.
(6): In the above table, the blending amount of 1,3-PD is indicated by volume. It is possible to convert to weight using the specific gravity of 1,3-PD (0.885 g / mL).

  No. in Table 1 above. 4 and no. 6-No. Comparing 9 it is understood that this method cannot produce a gel with a low polyamine mole fraction.

  That is, no. 4 and no. 8-No. In the blending of No. 9, polyamine was blended in a large amount, and 2.5 to 6.5 mg of gel was obtained. In 6, the yield of the dried gel has become zero. That is, the method of Non-Patent Document 1 has a drawback that a gel cannot be obtained when the amount of polyamine is small. For this reason, in order to obtain a gel, it is necessary to use a large amount of diamine. When a large amount of diamine is used, the diamine content in the gel obtained is inevitably increased. According to the data in Table 1 above, no. The diamine mole fraction of 7 is 17%.

  As described above, in the method of Non-Patent Document 1, it is necessary to add a large amount of diamine, and as a result, only a gel having a high diamine content (for example, 17 mol% or more) is obtained. I couldn't. That is, according to the method of Non-Patent Document 1, a gel having a low polyamine content (for example, 10 mol% or less) could not be obtained.

  And the gel whose polyamine content rate is too high has the fault that swelling degree is low. Specifically, in Table 1, the degree of swelling is only 834 times (No. 7) at the maximum. In general, it is known that a poly-γ-glutamic acid gel should have as high a degree of swelling as possible in order to exhibit good properties such as moisture retention, and specifically, for example, about 1000 times It was considered preferable to have the above degree of swelling. For this reason, the degree of swelling shown in Table 1 is not a desirable value as a poly-γ-glutamic acid gel, and a gel having a higher degree of swelling has been desired.

  In the first place, the method of Non-Patent Document 1 has a drawback that the yield is extremely low. Specifically, when the ratio of the dry weight of the obtained gel to the sum of the weight of polyglutamic acid and the weight of polyamine used in the experiment of Table 1 above was calculated as the yield, No. 1 in Table 1 was calculated. In the formulation of No. 5, a yield of about 10% is obtained. For 3, 4, 7, 8, and 9, the yield is only about 1 to 3%. And No. For 1, 2, and 6, it is not necessary to calculate and the yield is 0%. That is, in the method of Non-Patent Document 1, the yield is only about 10% at the maximum among various formulations adjusted as shown in Table 1.

  Thus, the method of Non-Patent Document 1 is far from being put into practical use as an industrial production method because of its extremely low yield.

Further, as described above, it has been attempted to use polyamine as a crosslinking agent, but it has not been considered to use a polymer as a crosslinking agent. In particular, it is known that poly (ethyleneimine) which is a cationic polymer (abbreviated as “PEI” in the present specification) is easily insolubilized by forming a polyion complex with PGA which is an anionic polymer. Therefore, it has not been thought that PEI can be used as a crosslinking agent for PGA.
JP 2002-233391 A International Publication No. WO2004-7593 JP 2005-179534 A J. et al. Appl. Polym. Sci. Vol. 65, pp. 1889-1896, 1997, Poly (γ-glutamic acid) Hydrogen Prepared from Microbial Poly (γ-glutamate acid) and Alkadiamine with Water-Solubil Carbodiimid, et al.

  The present invention is intended to solve the above problems, and provides a gel having a high degree of swelling, in which PGA is crosslinked with a small amount of polyamine, and a method for obtaining the gel in high yield. For the purpose.

  As a result of intensive studies, the present inventors have found that the above problem can be solved by adopting a method using a specific condensation auxiliary agent, and based on this, the present invention has been completed.

  Specifically, according to the present invention, the following method and gel are provided.

  (1) A method for producing a poly-γ-glutamic acid gel, comprising a step of crosslinking poly-γ-glutamic acid or a salt thereof with a polyamine in the presence of a water-soluble carbodiimide and N-hydroxyimide.

  (2) The method according to Item 1, wherein the N-hydroxyimide is N-hydroxysuccinimide.

  (3) The method according to Item 1, wherein the poly-γ-glutamic acid or a salt thereof has a weight average molecular weight of 1,000,000 or more.

(4) The method according to Item 1, wherein the water-soluble carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride.

  (5) The method according to Item 1, wherein the polyamine is alkylene diamine or poly (ethyleneimine).

  (6) The method according to Item 1, wherein the polyamine is an alkylenediamine having 2 to 8 carbon atoms.

  (7) The method according to item 1, wherein the polyamine is poly (ethyleneimine) having a weight average molecular weight of 100 to 20,000.

  (8) The method according to Item 1, wherein the polyamine is 1,3-propanediamine.

  (9) A poly-γ-glutamic acid gel in which poly-γ-glutamic acid is crosslinked with a polyamine, wherein the polyamine is the sum of the number of moles of glutamic acid residues and the number of moles of polyamine residues in the dried gel A poly-γ-glutamic acid gel, wherein the ratio of the number of moles of residues is 10 mol% or less.

  (10) The poly-γ-glutamic acid gel according to Item 9, wherein the ratio of the number of moles of polyamine residues in the dry gel is 3 mol% or less.

  (11) A poly-γ-glutamic acid gel in which poly-γ-glutamic acid is crosslinked with poly (ethyleneimine).

  According to the present invention, a gel in which PGA is crosslinked with polyamine in a very high yield is provided. According to the present invention, a gel having a low polyamine residue content, a low crosslinking density, and a high degree of swelling is provided.

  Hereinafter, the present invention will be described in detail.

(PGA)
Poly-γ-glutamic acid (PGA) refers to a compound obtained by polymerizing D, L-glutamic acid. PGA can also be used in the present invention as a salt thereof. For example, a sodium salt of PGA can be suitably used.

  The molecular weight of PGA is not particularly limited. However, in view of physical properties and the like, the weight average molecular weight is preferably 10,000 or more, and more preferably 100,000 or more. More preferably, it is 500,000 or more, more preferably 1 million or more, particularly preferably 1.5 million or more, and particularly preferably 2 million or more. Further, in terms of synthesis difficulty, the weight average molecular weight is preferably 13 million or less, and more preferably 10 million or less.

  For example, Patent Document 1 (Japanese Patent Application Laid-Open No. 2002-233391) describes PGA having a weight average molecular weight of about 13 million, and this PGA can be suitably used in the present invention.

  In addition, as described in the conventional technical column of Patent Document 1, a PGA having a molecular weight of 100,000 to 2,000,000 has been conventionally used, and a PGA having this molecular weight range can be used in the present invention. is there.

  The PGA used in the present invention can be produced by any known production method. For example, the method described in Patent Document 1 can be used.

(Polyamine)
In the present specification, polyamine refers to a compound having a plurality of amino groups. The amino group may be a primary amino group (—NH ₂ ) or a secondary amino group (—NH—). Primary amino groups are preferred. The number of amino groups is not particularly limited, but in one preferred embodiment, there are two in the molecule (ie, diamine).

  Preferably, the polyamine is an alkylene diamine or polyethylene imine.

  The number of carbon atoms in the alkylene diamine is not particularly limited. Preferably, it is 2 or more. Moreover, Preferably it is 10 or less, More preferably, it is 8 or less, More preferably, it is 6 or less. Further, the alkylene group in the alkylene diamine may be linear or branched. A straight chain is preferred.

  Specifically, preferable alkylene diamines include 1,2-ethylenediamine, 1,3-propylenediamine, 1,4-butanediamine, 1,5-heptanediamine, 1,6-hexanediamine, and the like.

  Polyethyleneimine is a polymer obtained by polymerizing ethyleneimine, and any conventionally known polyethyleneimine can be used in the method of the present invention. Preferably, it is a polymer containing 1: 2: 1 primary amine: secondary amine: tertiary amine.

  The weight average molecular weight of polyethyleneimine is not particularly limited. Preferably, it is 100 or more, more preferably 200 or more, still more preferably 400 or more, still more preferably 500 or more, and most preferably 600 or more. Moreover, Preferably it is 100,000 or less, More preferably, it is 50,000 or less, More preferably, it is 10,000 or less, Most preferably, it is 5000 or less.

  As the polyethyleneimine, a commercially available polymer can be used as it is. Alternatively, it may be obtained by polymerization from ethyleneimine by a known method.

  The amount of polyamine used is not particularly limited, but it is preferably blended so that the total of primary amino groups of the polyamine used is 0.0001 mol or more with respect to 1 mol of glutamic acid residues in PGA. 0.001 mol or more is more preferable, 0.003 mol or more is more preferable, and 0.005 mol or more is particularly preferable. The total of primary amino groups of the polyamine used is preferably 1 mol or less, more preferably 0.2 mol or less, and even more preferably 0.1 mol or less with respect to 1 mol of glutamic acid residue in PGA. 0.06 mol or less is more preferable, and 0.04 mol or less is particularly preferable.

  On a weight basis, the polyamine used is preferably blended so that the weight of the polyamine used is 0.00002 g or more, more preferably 0.0002 g or more, still more preferably 0.0005 g or more, and particularly preferably 0.001 g or more. 001 g or more. Further, it is preferably 0.25 g or less, more preferably 0.05 g or less, still more preferably 0.25 g or less, even more preferably 0.015 g or less, and particularly preferably 0.01 g or less with respect to 1 g of PGA.

  When the amount of polyamine used is too large, the crosslinking density tends to be high, and a PGA gel having a low degree of swelling is likely to be obtained. If the amount of polyamine used is too small, the crosslinking density tends to be low, and it may be difficult to obtain a gel state.

(Water-soluble carbodiimide)
In the method of the present invention, water-soluble carbodiimide is used as a condensing agent. In the present specification, the water-soluble carbodiimide is a compound having a carbodiimide group (—N═C═N—) in the molecule and having water solubility. In the present specification, water-soluble carbodiimide is also referred to as WSC. Specific examples of WSC that can be used in the present invention include 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide or a salt thereof, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-meth- Examples thereof include p-toluene sulfuric acid or a salt thereof. 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-meth-p-toluene sulfate (CMC) can be preferably used. is there.

  The amount of WSC used is not particularly limited, but is preferably 2 moles or more, more preferably 3 moles or more, still more preferably 6 moles or more, even more preferably 12 moles or more, and 24 moles per mole of the polyamine used. The above is particularly preferable. Moreover, 50 mol or less is preferable with respect to 1 mol of polyamines used, 40 mol or less is more preferable, and 30 mol or less is further more preferable.

  Further, as described above, the amount of WSC used is preferably determined based on the amount of polyamine. However, as a guideline of the blending amount based on the amount of PGA, 1 mol of glutamic acid residue in PGA is used. On the other hand, 0.001 mol or more is preferable, 0.01 mol or more is more preferable, 0.03 mol or more is further preferable, and 0.06 mol or more is particularly preferable. Moreover, 1 mol or less is preferable with respect to 1 mol of glutamic acid residues in PGA, 0.3 mol or less is more preferable, and 0.1 mol or less is more preferable.

(N-hydroxyimide)
The N-hydroxyimide used in the method of the present invention has an N-hydroxyimide group in the molecule:
-(C = O)-(N-OH)-(C = O)-
It is a compound which has this. That is, this compound is represented by the following general formula.
^{R A - (C = O)} - (N-OH) - (C = O) -R B
Here, a ring structure may be formed by combining R ^A and R ^{B. A} compound in which R ^A and R ^B are combined to form a 5-membered ring with two carbons in R ^A and R ^B and an N-hydroxyimide group is preferred. Moreover, it is preferable that N-hydroxyimide is water-soluble.

Specific examples of N-hydroxyimide that can be used in the present invention include the following compounds:
N-hydroxysuccinimide,
N-hydroxymaleic acid imide,
N-hydroxyhexahydrophthalic imide,
N, N'-dihydroxycyclohexanetetracarboxylic imide,
N-hydroxyphthalic acid imide,
N-hydroxytetrabromophthalimide,
N-hydroxytetrachlorophthalimide,
N-hydroxyhetic acid imide,
N-hydroxyhymic acid imide,
N-hydroxytrimellitic acid imide,
N, N′-dihydroxypyromellitic imide,
N, N′-dihydroxynaphthalenetetracarboxylic imide N-hydroxyimide can be a commercially available reagent as it is, and may be synthesized by a conventionally known synthesis method. For example, you may synthesize | combine by the method of Unexamined-Japanese-Patent No. 2002-47270.

  Among N-hydroxyimides, N-hydroxysuccinimide is most preferable.

  As N-hydroxysuccinimide, commercially available reagents and the like can be used as they are. In the present specification, N-hydroxysuccinimide is also referred to as NHS.

  Although the usage-amount of N-hydroxyimide is not specifically limited, 2 mol or more is preferable with respect to 1 mol of polyamines used, 3 mol or more is more preferable, 6 mol or more is further more preferable, and 12 mol or more is still more preferable. 24 mol or more is particularly preferable. Moreover, 50 mol or less is preferable with respect to 1 mol of polyamines used, 40 mol or less is more preferable, and 30 mol or less is further more preferable.

  The amount of N-hydroxyimide used is preferably equimolar to the amount of WSC used.

  Further, the amount of N-hydroxyimide used is preferably determined based on the amount of polyamine as described above. However, as a guideline of the blending amount based on the amount of PGA, a glutamic acid residue in PGA is used. 0.001 mol or more is preferable with respect to 1 mol, 0.01 mol or more is more preferable, 0.03 mol or more is more preferable, and 0.06 mol or more is particularly preferable. Moreover, 1 mol or less is preferable with respect to 1 mol of glutamic acid residues in PGA, 0.3 mol or less is more preferable, and 0.1 mol or less is more preferable.

(solvent)
In the method of the present invention, the solvent is not particularly limited. Preferably, it is water.

(Other ingredients)
The method of the present invention is preferably carried out in a solvent using only the above materials. However, in addition to each of the above materials, an appropriate amount of a known additive or the like may be blended as necessary.

  Specifically, for example, condensing agents other than WSC and NHS, condensation auxiliary agents, catalysts, fillers, colorants (pigments or dyes), reinforcing agents (for example, fibers), antioxidants, mold release agents, solvents, A thickener or the like can be added depending on the application.

(Crosslinking reaction conditions)
The conditions for the crosslinking reaction are not particularly limited. It may be room temperature or may be heated. However, if the temperature is too low, the curing reaction takes a very long time, so it is preferable to perform heating. Specifically, the temperature during the crosslinking reaction is preferably 10 ° C or higher, more preferably 15 ° C or higher, and further preferably 20 ° C or higher. Moreover, Preferably it is 100 degrees C or less, More preferably, it is 50 degrees C or less. If it is too high, the PGA tends to decompose. Therefore, it is preferable to carry out at around room temperature.

The pH during the crosslinking reaction is not particularly limited, but is preferably 6 or more, more preferably 7 or more. Moreover, Preferably it is 11 or less, More preferably, it is 10 or less.

(procedure)
A gel in which PGA is crosslinked is obtained by crosslinking reaction of the above-described reaction materials (PGA, polyamine, WSC and N-hydroxyimide) in a solvent. The order in which each material is charged is not particularly limited, and all materials may be charged at once, or each material may be charged in any order, but preferably WSC is charged last. That is, it is preferable in terms of yield to add WSC after preparing a reaction solution containing PGA, polyamine and N-hydroxyimide. It is preferable that the time until the WSC is added after the addition of N-hydroxyimide is 1 minute or longer, more preferably 5 minutes or longer, and further preferably 10 minutes or longer. Moreover, it is preferably 3 hours or less, more preferably 1 hour or less, and even more preferably 30 minutes or less.

  When all the reaction materials are charged, the crosslinking reaction starts. The reaction time for the crosslinking reaction is preferably 5 minutes or more, more preferably 10 minutes or more, still more preferably 20 minutes or more, and still more preferably in order to sufficiently perform the crosslinking reaction. Is 30 minutes or more, and particularly preferably 1 hour or more. However, in order to shorten the overall length of the process, it is preferably 5 days or less, more preferably 2 days or less, and even more preferably 1 day or less. More preferably, it is 12 hours or less. Particularly preferably, it is 6 hours or less.

  In the cross-linking reaction, the reaction solution may be stirred or allowed to stand as necessary. Preferably, it is left still.

  After sufficient time for the crosslinking reaction, a gel is obtained in the reaction solution. By washing this reaction solution with water (preferably distilled water), WSC and N-hydroxyimide in the reaction solution are removed, and a gel in which PGA is crosslinked with polyamine is obtained.

(Product)
The gel obtained by the above-described method has a structure in which polyamine is crosslinked to PGA. WSC and N-hydroxyimide are substantially absent in the final gel. In the obtained gel, the number of moles of polyamine residues is preferably 15% or less, more preferably 10% or less, based on the sum of the number of moles of glutamic acid residues and the number of moles of polyamine residues. 5% or less is further preferable, 3% or less is more preferable, and 1% or less is particularly preferable. Moreover, 0.001% or more is preferable, 0.01% or more is more preferable, and 0.1% or more is further more preferable.

(Manufacture of hydrogel)
The gel obtained by the above-described method becomes a hydrogel form by adding water. It is possible to obtain a hydrogel product containing a necessary amount of water depending on the application.

(Performance)
The gel of the present invention exhibits an excellent degree of swelling. For example, it is possible to obtain a gel having a degree of gel swelling of 1000 times or more after being immersed in distilled water for 2 days at room temperature. By appropriately adjusting the blending, 2000 times or more, 3000 times or more, 4000 times or more As described above, a gel having a degree of swelling of 5000 times or more, 5500 times or more, or 6000 times or more can be produced. The higher the degree of swelling, the better. However, specifically, for example, it is possible to obtain a gel having a degree of swelling of up to about 6500 times or up to about 7000 times.

(Use)
The hydrogel of the present invention is expected to be applied in a wide range of fields. For example, in the cosmetic field, it can be used as a moisturizer or humectant. In the field of agricultural and horticultural supplies, it is used as a soil modifier, seed coating, water retention agent for plant cultivation, animal compost fixative, compost additive, faint and urine additive be able to. In the civil engineering field, it can be used as water conditioning sludge for water treatment sludge, sewage sludge, river sewer sludge, solidifying agent, modifier, coagulant, or soil for reservoirs. In the medical / hygiene field, it can be used as an absorbent for blood, body fluids, disposable diapers and tampons, or as a deodorant or controlled release drug carrier. In the field of bioengineering, it can be used as a medium substrate for culturing microorganisms, plant cells, or animal cells, or as a fixing material for a bioreactor.

(Examples 1-4: Preparation of PGA crosslinked product using 1,3-PD as a crosslinking agent)
Examples using 1,3-propylenediamine will be described below.

(Example 1)
Put 0.453 g of PGA sodium salt (PGANa) and 120 μL of 1,3-PD aqueous solution [0.1853 g / 10 mL (= 0.25 mmol / mL)] (corresponding to 2.2236 mg) in 3 mL of water, and slowly The mixture was dissolved by stirring for about 30 minutes, and 0.0207 g of NHS dissolved in 1 mL of water was added thereto. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.88 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL.

The crosslinking reaction was allowed to proceed sufficiently (one day), then immersed in 2 L of distilled water, and the gel was swollen while exchanging water along the way. The gel from which the crosslinking agent was removed by swelling (for one day) was freeze-dried to measure the degree of gel swelling and the gel yield, which were 780 times and 97%, respectively. Moreover, the gel swelling degree and the gel yield when immersed in distilled water for 2 days for sufficient swelling were 6620 times and 92%, respectively. In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time (time until the elastic modulus and the viscosity modulus cross over) obtained from the viscoelasticity measurement is 7.8 minutes, and the gel elastic modulus G in a steady state. 'Was 130 Pa. From the result of the compression test, the initial elastic modulus E ₀ was obtained and was 1.9 kPa.

  As described above, since the gel was obtained with a yield of almost 100%, the composition of the obtained gel naturally had a blending ratio of PGA and 1,3-PD in the starting material almost unchanged. It is thought to have a composition. That is, it is understood that the 1,3-PD residue in the gel is about 1%.

  That is, it was found that a gel having a low crosslinker density that could not be obtained by the technique of Non-Patent Document 1 was obtained in Example 1.

Example 1A
A gel crosslinked product was prepared using PGA having a weight average molecular weight of 500,000 instead of PGA having a molecular weight of 2 million in Example 1. As a result, the degree of gel swelling and the gel yield were 2440 times and 79%, respectively.

(Example 1A ')
In Example 1A, the amount of 1,3-PD aqueous solution was changed to 240 μL (= 4.4472 mg equivalent), and the experiment was performed in the same manner as in Example 1A except that. As a result, a good gel cross-linked product was obtained.

  As explained in the background section above, the yield in Non-Patent Document 1 was only about several percent to 10%. Compared to this prior art, the gel yield of 97% in Example 1 above and the gel yield of 79% in Example 1A are very high yields that could not be expected from the prior art. Understood.

  In the first place, in Non-Patent Document 1, when the amount of 1,3-PD added is small, a gel could not be obtained. Specifically, for example, when 10 μL of 1,3-PD (about 9 μg, about 9 parts by weight with respect to 100 parts by weight of PGA) was added to 100 mg of PGA, a gel could not be obtained. That is, the yield is 0%. On the other hand, in the above Example 1 and Example 1A, gels were obtained with a yield of 97% and 79% in a composition in which only 1 part by weight of 1,3-PD was added to 100 parts by weight of PGA. There is nothing but to evaluate that it is a surprisingly high yield.

(Example 1B)
In Example 1, a gel crosslinked product was prepared using PGA having a weight average molecular weight of 5 million instead of PGA having a molecular weight of 2 million. As a result, a good gel cross-linked product was obtained.

(Example 1B ′)
In Example 1B, the amount of 1,3-PD aqueous solution was changed to 240 μL (= 4.4472 mg equivalent), and the experiment was performed in the same manner as in Example 1B except that. As a result, a good gel cross-linked product was obtained.

(Example 2)
0.453 g of PGANa [molecular weight: 2 million] in 3 mL of water and 240 μL of 1,3-PD aqueous solution [0.1853 g / 10 mL (= 0.25 mmol / mL)] instead of 120 μL in Example 1 (= 4 .4472 mg) was added, dissolved by slowly stirring at room temperature for about 30 minutes, and 0.0207 g of NHS dissolved in 1 mL of water was added thereto. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.76 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL. The crosslinking reaction was allowed to proceed sufficiently (one day), then immersed in 2 L of distilled water, and the gel was sufficiently swollen while exchanging water along the way. The gel in a state where the crosslinking agent was removed by swelling (for 2 days) was freeze-dried to measure the degree of gel swelling and the gel yield, which were 2400 times and 95%, respectively. In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time obtained from the viscoelasticity measurement (the time until the elastic modulus and the viscosity modulus cross over) is 7.7 minutes, and the gel elastic modulus G in a steady state. 'Was 590 Pa. From the result of the compression test, the initial elastic modulus E ₀ was obtained and was 2.6 kPa.

(Example 3)
0.453 g of PGANa [molecular weight: 2 million] in 3 mL of water and 60 μL of 1,3-PD aqueous solution [0.1853 g / 10 mL (= 0.25 mmol / mL)] instead of 120 μL in Example 1 (= 1 0.1118 mg equivalent) was added and dissolved by slowly stirring at room temperature for about 30 minutes, and 0.0207 g of NHS dissolved in 1 mL of water was added thereto. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.76 mL of water. In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time (time until the elastic modulus and the viscosity modulus cross over) obtained from the viscoelasticity measurement is 11 minutes, and the gel elastic modulus G ′ in the steady state is It was 52 Pa. From the result of the compression test, the initial elastic modulus E ₀ was obtained and was 0.24 kPa.

(Example 4)
0.453 g of PGANa [molecular weight: 2 million] in 3 mL of water and 120 in Example 1
Add 30 μL of 1,3-PD aqueous solution [0.1853 g / 10 mL (= 0.25 mmol / mL)] (corresponding to 0.5559 mg) instead of μL, and dissolve it by slowly stirring at room temperature for about 30 minutes. 0.0207 g NHS dissolved in 1 mL water was added thereto. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.76 mL of water. Moreover, as a result of evaluating the physical properties of the obtained gel, the gelation time (time until elastic modulus and viscosity crossover over) obtained from viscoelasticity measurement is 16 minutes, and the gel elastic modulus G ′ in the steady state is It was 20 Pa.

  The formulation of Examples 1-4 above is shown in the table below.

ａ）０．０１８５３ｇ／ｍＬ（＝０．２５ｍｍｏｌ／ｍＬ）水溶液を調製の上、添加した。
ｂ），ｃ）水溶液とした上で添加することで、全量が反応に関与するようにした。
ｄ） 溶媒の全量
＊） ＰＧＡＮａのモル数はモノマーユニット換算。

a) A 0.01853 g / mL (= 0.25 mmol / mL) aqueous solution was prepared and added.
b), c) The solution was added in the form of an aqueous solution so that the entire amount was involved in the reaction.
d) Total amount of solvent *) The number of moles of PGANa is in terms of monomer units.

  The molecular weight of PGA is a weight average molecular weight.

  As described above, no special pH adjustment work was performed, but it was possible to prepare the gel with good reproducibility and high yield.

The following table shows measurement results such as gelation time and elastic modulus of Examples 1, 1A and 1B.

ａ）架橋反応開始後、十分な時間（１日程度）放置したものをサンプルとして使用。弾性率測定の際に周波数依存がないことを確認の上、１Ｈｚの周波数を用いてレオメーターにより測定した。

a) A sample that was allowed to stand for a sufficient time (about 1 day) after the start of the crosslinking reaction was used as a sample. It was measured with a rheometer using a frequency of 1 Hz after confirming that there was no frequency dependence when measuring the elastic modulus.

(Discussion)
The following can be understood from the above experimental results.

  The gelation time can be controlled by the amount of the crosslinking agent.

  The influence of the amount of the crosslinking agent is larger than the molecular weight of PGA on the gelation time.

  From the results of elastic modulus measurement, it was confirmed that the cross-linked product strength increased with the increase in the amount of cross-linking agent and the molecular weight of PGA (that is, for the same cross-linked product strength, the concentration of the cross-linking agent was decreased if high molecular weight PGA was used. Can be).

  The change in the initial elastic modulus derived from the compression test results showed a high homology with the viscoelasticity measurement results measured using a rheometer.

(Comparative Example 1)
The experiment was performed in the same manner as in Example 1 except that NHS was not added. However, a crosslinked product was not obtained, and the yield was 0%.

(Comparative Example 2)
In Comparative Example 1, an experiment was performed in the same manner as Comparative Example 1 except that the pH of the reaction solution was adjusted to 6. Since WSC is known to have high activity at about pH 6, the pH was adjusted. As a result, the viscosity of the reaction solution increased, but no crosslinked product was obtained, and the yield was 0%.

(Examples 5 to 9: Preparation of high molecular weight PGA crosslinked product using PEI as a crosslinking agent)
Hereinafter, examples using poly (ethyleneimine) will be described.

(Example 5)
0.453 g of PGANa (weight average molecular weight: 2 million) and 120 μL of PEI (weight average molecular weight: 600, manufactured by Wako Pure Chemical Industries, Ltd., reagent grade) aqueous solution [0.88 g / 10 mL (= 0.25 mmol) in 3 mL of water / ML)] (= 2.64 mg equivalent) (0.25 mol% PEI relative to PGA monomer unit) was added and dissolved by slowly stirring for about 30 minutes, and dissolved in 1 mL of water there. 0.0207 g NHS was added. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.88 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL.

In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time (time until the elastic modulus and the viscosity modulus cross over) obtained from the viscoelasticity measurement is 2.6 minutes, and the gel elastic modulus G in a steady state. 'Was 400 Pa. From the result of the compression test, the initial elastic modulus E ₀ was obtained and was 2.3 kPa.

(Example 6)
0.453 g of PGANa [weight average molecular weight: 2 million] and 120 μL of PEI (weight average molecular weight: 1800, manufactured by Wako Pure Chemical Industries, Ltd., reagent grade) aqueous solution [0.88 g / 10 mL (= 0.25 mmol) in 3 mL of water / ML)] (= 2.64 mg equivalent) (0.25 mol% PEI relative to PGA monomer unit) was added and dissolved by slowly stirring for about 30 minutes, and dissolved in 1 mL of water there. 0.0207 g NHS was added. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.88 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL. In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time obtained from the viscoelasticity measurement (the time until the elastic modulus and the viscosity modulus cross over) is 2.3 minutes, and the gel elastic modulus G in a steady state. 'Was 200 Pa.

(Example 7)
0.453 g of PGANa [weight average molecular weight: 2 million] and 120 μL of PEI (weight average molecular weight: 10,000, manufactured by Wako Pure Chemical Industries, Ltd., reagent grade) in 3 mL of water [0.88 g / 10 mL (= 0.25 mmol) / ML)] (= 2.64 mg equivalent) (0.25 mol% PEI relative to PGA monomer unit) was added and dissolved by slowly stirring for about 30 minutes, and dissolved in 1 mL of water there. 0.0207 g NHS was added. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.88 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL. In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time (time until the elastic modulus and the viscosity modulus cross over) obtained from the viscoelasticity measurement is 11 minutes, and the gel elastic modulus G ′ in the steady state is It was 39 Pa.

(Example 8)
0.453 g of PGANa [molecular weight: 2 million] and 60 μL of PEI (weight average molecular weight: 600) aqueous solution [0.88 g / 10 mL (= 0.25 mmol / mL)] (corresponding to 1.32 mg) in 3 mL of water (PGA 0.125 mol% of PEI) was added to the monomer unit, and the mixture was dissolved by slowly stirring for about 30 minutes, and 0.0207 g of NHS dissolved in 1 mL of water was added thereto. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.88 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL. Moreover, as a result of evaluating the physical properties of the obtained gel, the gelation time obtained from the viscoelasticity measurement (time until the elastic modulus and the viscous modulus cross over) is 4.0 minutes, and the gel elastic modulus G in a steady state is obtained. 'Was 52 Pa. From the result of the compression test, the initial elastic modulus E ₀ was obtained and was 1.5 kPa.

Example 9
0.453 g of PGANa [molecular weight: 2 million] and 240 μL of PEI (weight average molecular weight: 600) aqueous solution [0.88 g / 10 mL (= 0.25 mmol / mL)] (= 5.28 mg equivalent) (PGA) 0.5 mol% of PEI) was added to the monomer unit and dissolved by slowly stirring for about 30 minutes, and 0.0207 g of NHS dissolved in 1 mL of water was added thereto. After 15 minutes, a PGA cross-linked product was prepared by adding 0.0346 g of WSC dissolved in 0.88 mL of water. The total amount of water contained in the PGA crosslinked product was 5 mL. The swelling degree of the obtained gel was 2000 times. In addition, as a result of evaluating the physical properties of the obtained gel, the gelation time (time until the elastic modulus and the viscosity modulus cross over) obtained from the viscoelasticity measurement is 1.8 minutes, and the gel elastic modulus G in a steady state. 'Was 1100 Pa. From the result of the compression test, the initial elastic modulus E ₀ was obtained and was 6.4 kPa.

  The formulations of Examples 5-9 are shown in the table below.

調製条件
架橋剤：ＰＥＩ （重量平均分子量：６００，１８００，１００００）
ＰＧＡの重量平均分子量：２００万
ａ） ポリエチレンイミンの１モノマーユニットの分子量は４４。ポリエチレンイミン中、一級アミン：二級アミン：三級アミン＝１：２：１の比率で含まれている。一級アミンが２個含まれる繰り返し単位をユニットとすると、８個のモノマーユニットからなる１ユニット中に２個の一級アミンが含まれる計算になり、１ユニットの分子量は３５２となる。この計算方法により、他のジアミン類との比較およびＰＥＩの分子量の影響を検討することが可能となった。
ｂ），ｃ） 水溶液とした上で添加することで、全量が反応に関与するようにした。
ｄ） 溶媒の全量
＊） ＰＧＡＮａのモル数はモノマーユニット換算

Preparation conditions Crosslinking agent: PEI (weight average molecular weight: 600, 1800, 10000)
Weight average molecular weight of PGA: 2 million a) The molecular weight of one monomer unit of polyethyleneimine is 44. Polyethyleneimine is contained in a ratio of primary amine: secondary amine: tertiary amine = 1: 2: 1. When a repeating unit including two primary amines is defined as a unit, the calculation includes two primary amines in one unit of eight monomer units, and the molecular weight of one unit is 352. This calculation method makes it possible to compare with other diamines and to study the influence of the molecular weight of PEI.
b) and c) By adding the solution after making it into an aqueous solution, the entire amount was involved in the reaction.
d) Total amount of solvent *) The number of moles of PGANa is in terms of monomer units.

ａ）レオメーターを用いて貯蔵弾性率（弾性率）Ｇ’の値と損失弾性率（粘性率）Ｇ’’の値を測定し、液体（Ｇ’＜Ｇ’’）が架橋反応の進行に伴ってＧ’＞Ｇ’’（固体）となるまでの時間をゲル化時間とした。（測定不能領域は、各サンプルともおよそ３０分程度であった）
ｂ）架橋反応開始後、十分な時間（１日程度）放置したものをサンプルとして使用。弾性率測定の際に周波数依存がないことを確認の上、１Ｈｚの周波数を用いてレオメーターにより測定した。
ｃ）作製したゲルを用いて圧縮試験により測定を行った。

a) Using a rheometer, measure the storage elastic modulus (elastic modulus) G ′ and the loss elastic modulus (viscosity) G ″, and the liquid (G ′ <G ″) Accordingly, the time until G ′> G ″ (solid) was taken as the gelation time. (The non-measurable area was about 30 minutes for each sample)
b) A sample left for a sufficient time (about 1 day) after the start of the crosslinking reaction is used as a sample. It was measured with a rheometer using a frequency of 1 Hz after confirming that there was no frequency dependence when measuring the elastic modulus.
c) Using the prepared gel, measurement was performed by a compression test.

(Comparative Example 3)
The experiment was performed in the same manner as in Example 5 except that NHS was not added. However, a crosslinked product was not obtained, and the yield was 0%.

(Comparative Example 4)
In Comparative Example 3, the experiment was performed in the same manner as Comparative Example 3 except that the pH of the reaction solution was adjusted to 6. Since WSC is known to have high activity at about pH 6, the pH was adjusted. As a result, the viscosity of the reaction solution increased, but no crosslinked product was obtained, and the yield was 0%.

(Discussion)
By using N-hydroxyimide (NHS in the above examples) as a crosslinking activation aid in addition to WSC as a crosslinking activator, a gel composed of PGA crosslinked rapidly with PEI could be prepared.

  It can be understood from the above results that when the molecular weight of PEI as a crosslinking agent is large, the strength of the crosslinked product tends to decrease. Accordingly, it is understood that it is advantageous to use a lower molecular weight polyamine as a crosslinking agent when a high strength crosslinked product is desired.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. Those skilled in the art can understand the scope equivalent to the claims based on the description of the specific preferred embodiments of the present invention and the common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Since the gel of this invention is excellent in swelling degree, it can be used effectively for the use in which the conventionally well-known hydrogel was used. For example, since it is excellent in moisture retention, application is expected in the field of cosmetics and the like. In addition, since the water absorption rate is high, application is expected in the field of sanitary products.

Claims (11)

A method for producing a poly-γ-glutamic acid gel, comprising the step of cross-linking poly-γ-glutamic acid or a salt thereof with a polyamine in the presence of a water-soluble carbodiimide and N-hydroxyimide. The method of claim 1, wherein the N-hydroxyimide is N-hydroxysuccinimide. The method according to claim 1, wherein the poly-γ-glutamic acid or a salt thereof has a weight average molecular weight of 1 million or more. The method of claim 1, wherein the water-soluble carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. The method of claim 1, wherein the polyamine is an alkylene diamine or poly (ethyleneimine). The method according to claim 1, wherein the polyamine is an alkylene diamine having 2 to 8 carbon atoms. The method according to claim 1, wherein the polyamine is poly (ethyleneimine) having a weight average molecular weight of 100 to 20,000. The method of claim 1, wherein the polyamine is 1,3-propanediamine. A poly-γ-glutamic acid gel in which poly-γ-glutamic acid is cross-linked with a polyamine, wherein the polyamine residue of the sum of the number of moles of glutamic acid residues and the number of moles of polyamine residues in the dried gel is A poly-γ-glutamic acid gel having a molar ratio of 10 mol% or less. The poly-γ-glutamic acid gel according to claim 9, wherein a ratio of the number of moles of polyamine residues in the dry gel is 3 mol% or less. A poly-γ-glutamic acid gel in which poly-γ-glutamic acid is crosslinked with poly (ethyleneimine).