JP7765064B2 - Material containing ε-polylysine and reducing sugars - Google Patents

Description

The present invention relates to a material that combines ε-polylysine with a reducing sugar.

Conventionally, ε-polylysine has been known to have antibacterial activity against microorganisms, particularly bacteria, while exhibiting almost no toxicity to animals, and has been widely used as a food preservative. Furthermore, cationic polymers, including ε-polylysine, are known to strongly interact with the cell membranes of animal cells, and are used for surface modification of petri dishes for cell observation, etc. As another example of the use of ε-polylysine, for example, Patent Document 1 describes a carrier for endotoxin adsorption in which ε-polylysine is crosslinked with a crosslinking agent such as epichlorohydrin. Furthermore, Patent Document 2 describes a medical adhesive composed of ε-polylysine and aldehyde-modified α-glucan.
However, when used for these applications, it has been widely known that ε-polylysine is used as an aqueous solution or an amorphous material, or that ε-polylysine is added to or coated on moldable materials such as plastics or rubber. However, there have been almost no attempts to use ε-polylysine as a moldable material without complexing it with an anionic polymer. This is because ε-polylysine is a brittle and highly hygroscopic compound, and it is difficult to mold ε-polylysine alone.
Non-Patent Document 1 reports a moldable material having thermoplasticity by complexing ε-polylysine with an anionic surfactant. However, the material has a small breaking elongation, low flexibility, and a breaking strength that is only a fraction of that of existing plastic materials. Therefore, the composite of ε-polylysine and anionic surfactant described in Non-Patent Document 1 is limited to use as an antibacterial coating agent or additive, and it has not been disclosed that the moldable material comprising the composite can be used alone to form a molded product.
Furthermore, no attempt has been made to utilize ε-polylysine itself as a moldable material without forming a complex with other compounds.

Japanese Patent Application Publication No. 11-152330 Re-tabled publication No. 2006/080523

Biomacromolecules;2017;18;1387-1392 Chemical Science;2015;6(11);6385-6391

If ε-polylysine could be made moldable, it could be expected to be used as a high-performance material with properties such as antibacterial properties and cell affinity. Furthermore, because ε-polylysine is a natural compound derived from microorganisms, it could also be expected to be used as a biodegradable, moldable material. However, at present, ε-polylysine is a brittle and highly hygroscopic compound, making it difficult to mold by itself. The present invention aims to impart moldability to ε-polylysine and further to provide such a moldable material with excellent flexibility, elasticity, strength, and water resistance.

The present inventors have found that by mixing ε-polylysine with a saccharide exhibiting reducing properties in an aqueous solvent, a material that can be molded into various shapes and has excellent elasticity is produced in the mixed solution.
Specifically, the present inventors have found that a sheet-like material can be easily obtained by mixing ε-polylysine with a reducing saccharide such as fructose or glucose and drying the resulting solution on a flat plate such as a petri dish, and that the material exhibits good elasticity in a tensile test and has good flexibility, strength, and water resistance.
The present invention was made based on these findings by the present inventors.

That is, this application provides the following inventions.
[1] A material having moldability, comprising ε-polylysine, a derivative thereof, or a salt thereof, and a saccharide exhibiting reducing properties.
[2] The material described in [1], characterized by having elasticity.
[3] The material according to [1] or [2], wherein the ε-polylysine has a weight-average molecular weight of 500 to 1,000,000.
[4] The material according to any one of [1] to [3], wherein the reducing saccharide is fructose, arabinose, glucose, lactose, maltose, ribose, xylose, mannose, or galactose.
[5] A method for producing the material according to any one of [1] to [4], comprising: a step of mixing ε-polylysine, a derivative thereof, or a salt thereof with a saccharide exhibiting reducing properties in a solvent to react the polymer with the saccharide; and a step of removing the solvent from the mixed solution or suspension.

Specifically, they confirmed that by combining ε-polylysine with reducing sugars, it is possible to obtain a moldable material that also has excellent flexibility, elasticity, strength, and water resistance.

Photographs of a sheet sample made of ε-polylysine and fructose and the sheet sample when bent are shown. Photographs of a sheet sample made of ε-polylysine and fructose and a test piece obtained by punching out the sheet sample into the shape of a dumbbell-shaped test piece (No. 8, JIS K 6251) are shown. A photograph of a sample consisting of only ε-polylysine is shown. 1 shows photographs of elasticity tests of samples made of ε-polylysine and fructose. 1 shows photographs of the results of a water resistance test of a sample made of ε-polylysine and fructose.

The present invention relates to a moldable material that contains ε-polylysine, a derivative thereof, or a salt thereof, and a reducing saccharide.

The ε-polylysine used in the present invention is not particularly limited, and may be, for example, one produced by a microorganism or one obtained by chemical synthesis.
The production method using a microorganism is not particularly limited as long as it can produce ε-polylysine, but for example, a production method using ε-polylysine-producing bacteria belonging to Streptomyces alblus or Streptomyces noursei can be used, and ε-polylysine can be isolated and purified from the culture medium of these bacteria. As a chemical synthesis method, for example, the chemical synthesis method described in Non-Patent Document 2 can be used, and ε-polylysine can be obtained by this chemical synthesis method. Moreover, ε-polylysine can also be obtained by using an ε-polylysine synthase derived from ε-polylysine-producing bacteria.

In addition, some of the amino groups or carboxyl groups of the ε-polylysine may be chemically modified via covalent bonds or the like.
Examples of the chemically modified compound include a compound in which an amino group of an ε-polylysine is modified via an amide bond with a carboxyl group-containing compound such as a carboxylic acid or an amino acid, and a compound in which a carboxyl group at the end of an ε-polylysine is modified via an amide bond with an amino group-containing compound such as an amine or an amino acid, or a hydroxyl group-containing compound such as an alcohol via an ester bond.
Moreover, examples of the derivatives of ε-polylysine include ε-polylysine obtained by dehydration condensation of ε-polylysine molecules to increase the molecular weight.
Moreover, from the viewpoint of water solubility, the ε-polylysine used in the present invention may be an inorganic acid salt such as a hydrochloride or an organic acid salt such as an acetate of the above-mentioned ε-polylysine.

The structure of lysine forming the ε-polylysine is not particularly limited, and any of those consisting of only L-lysine, only D-lysine, and those containing both can be used. In addition, the ratio of L-lysine to D-lysine is not particularly limited.
The degree of polymerization of ε-polylysine is not particularly limited, but is preferably a pentamer or more, and the weight-average molecular weight is preferably 500 to 1,000,000, and more preferably 1,000 to 10,000.

Examples of reducing sugars used in the present invention include fructose, arabinose, glucose, lactose, maltose, ribose, xylose, mannose, galactose, dihydroxyacetone, ribulose, psicose, and erythrose, as well as oligosaccharides and polysaccharides containing these sugars. Fructose, arabinose, glucose, lactose, maltose, ribose, xylose, mannose, or galactose is preferred, fructose, arabinose, glucose, xylose, galactose, or lactose is more preferred, and fructose or arabinose is particularly preferred. In the present invention, oligosaccharides refer to oligosaccharides, including, but not limited to, those formed by 2 to 10 monosaccharides linked by glycosidic bonds, and polysaccharides are those formed by at least two monosaccharides linked by glycosidic bonds. Oligosaccharides are included in the category of polysaccharides.

The phrase "comprising ε-polylysine, a derivative thereof, or a salt thereof, and a saccharide exhibiting reducing properties" can further include any other components other than "ε-polylysine, a derivative thereof, or a salt thereof, and a saccharide exhibiting reducing properties." It can also consist solely of "ε-polylysine, a derivative thereof, or a salt thereof, and a saccharide exhibiting reducing properties."

The composition ratio of ε-polylysine, its derivatives, or salts thereof to the reducing saccharide in the material is not particularly limited, but the weight ratio of the weight of ε-polylysine, or the weight of the ε-polylysine derivative, ε-polylysine salt, or the ε-polylysine equivalent value of the salt of the ε-polylysine derivative to the weight of the reducing saccharide is preferably 99:1 to 5:95, more preferably 95:5 to 50:50, and even more preferably 90:10 to 60:40.

The moisture content of the material is 3 to 95% by weight, preferably 5 to 40% by weight, and more preferably 10 to 30% by weight of the total weight of the material. This moisture content allows the material to have elasticity.
The water content is adjusted by maintaining a solution or suspension containing ε-polylysine, a derivative thereof, or a salt thereof, and a reducing saccharide in the atmosphere or under controlled humidity conditions. In this case, evaporation of water may be promoted by heating using a thermostatic bath or a hot plate. Alternatively, the solution or suspension may be added dropwise to an organic solvent to obtain a precipitate of the complex, and the resulting complex may then be maintained in the atmosphere or under controlled humidity conditions, as necessary, to adjust the water content.

In the present invention, "having formability" or "formable" refers to the property of being able to prepare a visually uniform sheet sample (thickness 0.5 mm or more), punch out a dumbbell-shaped test piece (No. 8, JIS K 6251) from the sheet sample, and then conduct a tensile test to evaluate the mechanical properties.

In the present invention, "having elasticity" refers to the property that when a dumbbell-shaped test piece (No. 8, JIS K 6251) is cut from a material, tension is applied to the test piece to cause deformation, and the tension is then completely removed and the test piece is allowed to stand for one hour, the amount of deformation after standing is 50% or less of the maximum amount of deformation when tension is applied. It is preferable that the amount of deformation after standing is 30% or less of the maximum amount of deformation when tension is applied.

In the present invention, "flexible" means, for example, that the test specimen can be bent to any angle or more without breaking, and it is preferable that the test specimen can be bent to 45° or more, 90° or more, 135° or more, or 180°.

In the present invention, "having excellent strength" means, for example, that a dumbbell-shaped test piece can be made without breakage using the material of the present invention molded into a sheet, and preferably that a tensile test can be performed on the made dumbbell-shaped test piece to calculate the breaking strength.

The material of the present invention can be molded, for example, into a sheet. There are no particular limitations on the thickness of the sheet, but it is preferable that it be 0.5 to 10 mm.

Another aspect of the present invention is a method for producing a material, specifically, the method includes a step of mixing ε-polylysine, a derivative thereof, or a salt thereof with a reducing saccharide in a solvent to react the polymer with the saccharide, and a step of removing the solvent from the mixed solution or suspension.
The step of reacting a polymer with a saccharide by mixing ε-polylysine, a derivative thereof, or a salt thereof with a saccharide exhibiting reducing property in a solvent is not particularly limited, and may be, for example, a step of reacting a polymer with a saccharide by mixing a solid made of ε-polylysine, a derivative thereof, or a salt thereof with a solid of a saccharide exhibiting reducing property in a solvent, or a step of reacting a polymer with a saccharide by mixing a solution containing ε-polylysine, a derivative thereof, or a salt thereof with a solution containing a saccharide exhibiting reducing property.
In this step, the order in which the components are mixed and the mixing conditions for the components, such as temperature, humidity, and time, are not particularly limited as long as a significantly non-uniform composition is not formed.
The step of removing the solvent may be, for example, a step of removing the solvent from the solution or suspension by volatilizing it, or, when the solvent is water, a step of pouring the aqueous solution or aqueous suspension into an organic solvent to precipitate a mixture of ε-polylysine, a derivative thereof, or a salt thereof, and a reducing saccharide in the organic solvent, followed by removal of the solvent. The step of removing the solvent by volatilizing it is not particularly limited as long as it can reduce the amount of solvent in the material to a predetermined content, and may be performed, for example, at room temperature, preferably 20°C to 40°C; at humidity preferably 40% to 60%; and for a period of time preferably at least one day or more.

The solvent used in this embodiment is not particularly limited as long as it can dissolve the ε-polylysine and the saccharides. However, preferred examples include water, organic solvents that are miscible with water, such as methanol, ethanol, isopropyl alcohol, ethylene glycol, glycerin, polyethylene glycol, formic acid, acetic acid, propionic acid, acetone, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, and dioxane, and mixed solvents of water and the above-mentioned organic solvents, and water is particularly preferred.

By performing tensile testing on the resulting material, the maximum stress, elongation at break, and Young's modulus can be measured.

The level of water resistance of the resulting material can be determined by conducting a water resistance test. That is, a test piece of a desired mass is cut out from the material, immersed in pure water, and shaken for a desired period of time, such as 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 5 days, 1 week, or more. The more the test piece in the pure water maintains its shape without disintegrating or dissolving after the desired time has passed, the less the material dissolves in the pure water, and therefore the higher the water resistance of the material.

Next, the present invention will be explained in more detail using examples, but the present invention is not limited to these examples.

Example 1: Preparation of a Material Composed of ε-Polylysine and Fructose. D-(-)-Fructose (Wako Pure Chemical Industries, Ltd., special grade) was mixed with purified water at the ratio shown in Table 1 and stirred until the D-(-)-fructose was completely dissolved. ε-Polylysine (JNC Corporation, molecular weight approximately 3,200-4,500, Lot No. 2160204, 25 wt % aqueous solution) was added to this solution at the ratio shown in Table 1 and mixed uniformly. This mixture was transferred to a fluororesin Petri dish (size: 6 cm x 6 cm) and the solvent was evaporated to obtain sheet samples with thicknesses of 1 mm or more for all compositions listed in Table 1 (sample names: PL-1 to PL-6). Figure 1 shows a sheet sample prepared with the composition designated "PL-2" in Table 1, as well as a photograph of the sheet sample bent by hand. As shown in Figure 1, the sample did not crack even when bent by hand nearly 180 degrees, demonstrating the flexibility of this material. Figure 2 shows photographs of a sheet sample prepared with the composition indicated as sample name "PL-3" in Table 1, and a test piece punched out from the same sheet sample into the shape of a dumbbell test piece (No. 8, JIS K 6251).

Example 2 Preparation of a Material Composed of ε-Polylysine and Arabinose 1.0 g of D-(-)-arabinose (Tokyo Chemical Industry Co., Ltd.) was mixed with 4 mL of pure water and stirred until the D-(-)-arabinose was completely dissolved. 16 mL of the ε-polylysine (JNC Corporation, molecular weight approximately 3,200-4,500, Lot No. 2160204, 25 wt % aqueous solution) used in Example 1 was added to this solution and mixed uniformly. This mixture was transferred to a fluororesin Petri dish (size: 6 cm x 6 cm) and the solvent was evaporated to obtain a sheet-like sample with a thickness of 1 mm or more. The appearance of the obtained sample was similar to that of Example 1.
Furthermore, even when the obtained sample was bent by hand nearly 180 degrees, no cracks were observed in the sample, demonstrating that this material is flexible.

Example 3: Preparation of a Material Composed of ε-Polylysine and Xylose. 1.0 g of D-(+)-xylose (Wako Pure Chemical Industries, Ltd., special grade) was mixed with 4 mL of purified water and stirred until the D-(+)-xylose was completely dissolved. 16 mL of the same ε-polylysine used in Example 1 (JNC Corporation, molecular weight approximately 3,200-4,500, Lot No. 2160204, 25 wt % aqueous solution) was added to this solution and mixed uniformly. This mixture was transferred to a fluororesin Petri dish (size: 6 cm x 6 cm), and the solvent was evaporated to obtain a sheet-like sample with a thickness of 1 mm or more. The appearance of the resulting sample was nearly identical to that of the sample obtained in Example 1. Furthermore, the sample did not crack even when bent by hand nearly 180 degrees, demonstrating the material's flexibility.

Example 4: Preparation of a Material Composed of ε-Polylysine and Galactose. 1.0 g of D-(+)-galactose (Wako Pure Chemical Industries, Ltd., special grade) was mixed with 4 mL of purified water and stirred until the D-(+)-galactose was completely dissolved. 16 mL of the same ε-polylysine used in Example 1 (JNC Corporation, molecular weight approximately 3,200-4,500, Lot No. 2160204, 25 wt % aqueous solution) was added to this solution and mixed uniformly. The mixture was transferred to a fluororesin Petri dish (size: 6 cm x 6 cm), and the solvent was evaporated to obtain a sheet-like sample with a thickness of 1 mm or more. The appearance of the resulting sample was nearly identical to that of the sample obtained in Example 1. Furthermore, the sample did not crack even when bent by hand nearly 180 degrees, demonstrating the material's flexibility.

Example 5: Preparation of a Material Composed of ε-Polylysine and Glucose. 1.0 g of D-(+)-glucose (Wako Pure Chemical Industries, Ltd., special grade) was mixed with 4 mL of purified water and stirred until the D-(+)-galactose was completely dissolved. 16 mL of the same ε-polylysine used in Example 1 (JNC Corporation, molecular weight approximately 3,200-4,500, Lot No. 2160204, 25 wt % aqueous solution) was added to this solution and mixed uniformly. This mixture was transferred to a fluororesin Petri dish (size: 6 cm x 6 cm), and the solvent was evaporated to obtain a sheet-like sample with a thickness of 1 mm or more. The appearance of the obtained sample was almost identical to that of the sample obtained in Example 1. Furthermore, the obtained sample did not crack even when bent by hand nearly 180 degrees, demonstrating the flexibility of this material.

Comparative Example 1 Preparation of Material Composed Only of ε-Polylysine 20 mL of the ε-polylysine (JNC Corporation, molecular weight approximately 3,200 to 4,500, Lot No. 2160204, 25 wt % aqueous solution) used in Example 1 was transferred to a fluororesin Petri dish (size: 6 cm × 6 cm), and the solvent was evaporated in the same manner as in Example 1. The dried sample was hard and brittle, and many cracks had occurred, so that a uniform sheet-like sample large enough to punch out a dumbbell-shaped test specimen (No. 8, JIS K 6251) could not be obtained ( FIG. 3 ).

Comparative Example 2: Preparation of a material consisting of ε-polylysine and sucrose (non-reducing sugar) 1.0 g of sucrose (Wako Pure Chemical Industries, Ltd., special grade) was mixed with 4 mL of purified water and stirred until the sucrose was completely dissolved. 16 mL of the ε-polylysine used in Example 1 (JNC Corporation, molecular weight approximately 3,200-4,500, Lot No. 2160204, 25 wt % aqueous solution) was added to this solution and mixed uniformly. This mixture was transferred to a fluororesin Petri dish (size: 6 cm x 6 cm), and the solvent was evaporated in the same manner as in Example 1. The dried sample was brittle and had multiple cracks, making it impossible to obtain a flexible sample.

The sheet-like samples (sample names: PL-1 to PL-6) prepared in Example 1 were allowed to stand for 5 days in a thermo-hygrostat at 30°C and a relative humidity of 50%, and then punched into the shape of dumbbell-shaped test pieces (No. 8, JIS K 6251) to prepare test pieces for evaluation. Tensile tests (in air, tensile speed of 50 mm/min) were conducted using these test pieces, and the results are shown in Table 2 (average values of 3 to 5 tests).
As shown in Table 2, it can be seen that all of the test pieces in Example 1 can be molded into dumbbell-shaped test pieces, and have sufficient moldability and strength to enable tensile testing.
On the other hand, in Comparative Examples 1 and 2, the samples were brittle, making it difficult to prepare and test the dumbbell-shaped test specimens.
This shows that by combining ε-polylysine with a saccharide exhibiting reducing properties, it is possible to impart moldability and flexibility to ε-polylysine, which is difficult to mold by itself.

Example 7 Elasticity Test of a Material Composed of ε-Polylysine and Fructose Figure 4 shows a photograph comparing the dumbbell-shaped test piece (Table 2, sample name "PL-3") after the tensile test in Example 6 with a rubber sheet cut into the same shape as the dumbbell-shaped test piece used in the test. Despite being deformed by approximately 90% (31.5 mm) in the tensile test, the deformation of the dumbbell-shaped test piece after the test was less than 5%. This indicates that the material recovers from deformation when the tension is completely removed, i.e., the material is elastic.

Example 8 Water Resistance Test of Material Composed of ε-Polylysine and Fructose A 60 mg test piece was cut out from the sheet-like sample prepared in Example 1. This test piece was immersed in 1.2 mL of pure water and shaken at room temperature for 30 minutes or 24 hours to perform a water resistance test. The more the test piece in the pure water maintains its shape without disintegrating or dissolving after shaking, the less the material dissolves in the pure water, which means that the material has high water resistance.
The results of the water resistance test using the sample named "PL-3" in Table 1 as the test piece are shown in Figure 5. When the test piece was immersed in pure water and shaken for 24 hours, neither disintegration of the test piece nor coloration of the supernatant was observed. On the other hand, when the same test was performed using a sample consisting of only ε-polylysine prepared in Comparative Example 1, the test piece was completely dissolved after 30 minutes. Furthermore, when the same test was performed using a sample consisting of ε-polylysine and sucrose (a non-reducing sugar) prepared in Comparative Example 2, the test piece was also completely dissolved after 30 minutes. In other words, it was found that the combination of ε-polylysine and fructose, a reducing sugar, made the material exhibited a remarkable effect in terms of water resistance.

The material of the present invention can be molded into a variety of shapes, is highly elastic, and is water-resistant. It can also be used as a raw material for molded products for a variety of applications that require the properties of ε-polylysine, such as antibacterial properties, cell affinity, and biodegradability.

Claims (5)

ε-polylysine, a derivative thereof, or a salt thereof, characterized by having moldability;
Materials containing reducing sugars (however, excluding materials in liquid form, materials used as binder compositions, and materials in the form of thermosets thereof ). The material described in claim 1, characterized by its elasticity. 3. The material according to claim 1, wherein the ε-polylysine has a weight-average molecular weight of 500 to 1,000,000. The material described in any one of claims 1 to 3, wherein the reducing sugar is fructose, arabinose, glucose, lactose, maltose, ribose, xylose, mannose, or galactose. A method for producing the material according to any one of claims 1 to 4, comprising: a step of mixing ε-polylysine, a derivative thereof, or a salt thereof with a saccharide exhibiting reducing properties in a solvent to react the polymer with the saccharide; and a step of removing the solvent from the mixed solution or suspension.