Gelling agents, gel-like compositions, methods for producing gelling agents, methods for producing gel-like compositions, foods, chemical products, pharmaceuticals and cosmetics

Abstract

The present invention provides a polysaccharide-based gelling agent having a novel chemical structure, a gel-like composition, a method for producing the gelling agent, a method for producing the gel-like composition, and applications in food, chemical products, pharmaceuticals, and cosmetics. [Solution] A gelling agent represented by the following formula (1). [C1] JPEG2026114619000006.jpg61134 (In equation (1), n ​​is a natural number.)

Description

Technical Field

[0001] The present invention relates to a gelling agent, a gel composition, a method for producing a gelling agent, a method for producing a gel composition, foods, chemical products, pharmaceuticals, and cosmetics.

Background Art

[0002] Gelling agents are additives used in various fields such as foods, chemical products, pharmaceuticals, or cosmetics, and play a role in adjusting the viscosity of liquids and semi-solids or hardening them to improve the texture and stability of products.

[0003] For example, typical natural polysaccharide gelling agents (hydrogel-forming polysaccharides) include agar derived from seaweed, carrageenan, alginic acid, pectin derived from plants, glucomannan, galactomannan, gellan gum derived from bacteria (Non-Patent Document 1), curdlan (Non-Patent Document 2), and the like.

[0004] [→3)D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→]n-structured gellan gum is obtained from Sphingomonas elodea and Shingomonas paucimobilis, etc., and curdlan, which is a β1,3-glucan, is obtained from various bacteria such as the genus Agrobacterium, Rhizobium, Alcaligenes, Gluconacetobacter, and Bacillus.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] Conventionally, various gelling agents have been known, each producing a unique hydrogel, and the effects of temperature, pH, salt, etc., on texture and syneresis vary. Furthermore, the presence or absence of thermal reversibility also differs depending on the gelling agent. The required properties differ depending on the application, and it cannot be said that current gelling agents satisfy all applications. There is a constant need for novel gelling agents that can better suit existing applications or be used in new applications.

[0007] The present invention aims to solve these problems by providing a novel polysaccharide-based gelling agent having a novel chemical structure, a gel-like composition, a method for producing the gelling agent, a method for producing the gel-like composition, and products such as food, chemical products, pharmaceuticals, and cosmetics. [Means for solving the problem]

[0008] The above problems are solved by the present invention, which is defined as follows. 1. A gelling agent represented by the following formula (1). [ka] (In equation (1), n ​​is a natural number.) 2. A gel-like composition comprising the gelling agent and solvent described in item 1 above. 3. A step of culturing Sphaerotilus hippei in a culture medium to which a nitrogen source or carbon source has been added, The process involves adding an alkaline solution containing a surfactant to the cultured Sphaerotilus hippei and heating it, The process involves washing and filtering the heated treated material, and then drying the resulting residue. A method for producing the gelling agent described in 1 above, including the method described in 1 above. 4. A method for producing a gel-like composition, comprising the step of adding a solvent to the gelling agent described in item 1 and heating it. 5. Foods, chemical products, pharmaceuticals, or cosmetics containing any of the gelling agents described in items 1 to 4 above. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a polysaccharide-based gelling agent having a novel chemical structure, a gel-like composition, a method for producing the gelling agent, a method for producing the gel-like composition, and products such as food, chemical products, pharmaceuticals, and cosmetics. [Brief explanation of the drawing]

[0010] [Figure 1] This is a photograph showing the appearance of the sponge-like gelling agent obtained in Test Example 1. [Figure 2] The images above show a phase-contrast microscope photograph of the bacterial cells in Test Example 1, and a phase-contrast microscope photograph of the sheath (hollow filamentous structure: obtained gelling agent) in the lower figure. [Figure 3] This is a chromatograph related to Analysis 1. [Figure 4] This is a chromatograph related to Analysis 2. [Figure 5] This is the 1D-1H NMR spectrum of the partial decomposition product of the gelling agent related to Analysis 3. [Figure 6] This is the 2D-1H,13C NMR spectrum of the partial decomposition product of the gelling agent related to Analysis 3. [Figure 7]It is a graph showing the relationship between the gel concentration and the water separation rate of the gelling agent obtained in Test Example 1 and agar. [Figure 8] It is a graph showing the relationship between the pH of the gelling agent and agar obtained in Test Example 1 and the water separation rate.

Mode for Carrying Out the Invention

[0011] Next, the mode for carrying out the present invention will be described in detail while referring to the drawings. The present invention is not limited to the following embodiments, and it should be understood that design changes, improvements, etc. can be appropriately made based on the ordinary knowledge of those skilled in the art without departing from the gist of the present invention.

[0012] <Gelling Agent> The gelling agent according to the embodiment of the present invention is represented by the following formula (1). The natural number n in formula (1) is not particularly limited, but may be 1000 to 100000.

Chemical Formula

[0013] The gelling agent according to the embodiment of the present invention is prepared from cultured Sphaerotilus hippei as described later. Sphaerotilus hippei is a safe filamentous bacterium discovered from marsh water. Since its cells are rod-shaped and have flagella, it is basically a dispersible bacterium. On the other hand, it can form a microtubular extracellular structure (sheath) that wraps around the cell chain to form filaments, aggregates, or adhere to solid surfaces. The sheath of Sphaerotilus hippei is formed of an insoluble polysaccharide with a novel chemical structure consisting of equimolar amounts of galactose and mannose. Furthermore, it was discovered that this sheath, being composed of polysaccharides, dissolves upon heating and yields a hydrogel upon cooling. Conventionally, it was not known that Sphaerotilus hippei produces a gelling agent, and the gelling agent according to the embodiment of the present invention is a novel gelling agent in terms of both the species that produces it and its chemical structure.

[0014] The gelling agent according to the embodiment of the present invention has the same composition as fenugreek gum (structural formula shown in formula (2) below), which is a type of galactomannan derived from plants. However, while galactomannan, including fenugreek gum, has a mannose polymer as its main chain, the gelling agent derived from Sphaerotilus hippei according to the embodiment of the present invention has a main chain copolymerized of β-D-galactose (β-D-Gal) and α-D-mannose (α-D-Man) in a molar ratio of 1:2, as shown in formula (3) below. Furthermore, β-D-galactose (β-D-Gal) is bonded to the 6-position of the α-D-mannose (α-D-Man) that constitutes the main chain. In other words, the gelling agent according to the embodiment of the present invention is a novel polysaccharide-based gelling agent having a chemical structure that has not been known before. [ka] (In equations (2) and (3), n is a natural number.)

[0015] The hydrogel produced by the gelling agent derived from Sphaerotilus hippei according to the embodiment of the present invention exhibits unique thermoplasticity, dissolving rapidly even at relatively low temperatures (around 50°C) despite being polysaccharide-based, similar to the peptide-based gelling agent gelatin. Furthermore, it is readily soluble in warm water and does not require the presence of polyvalent cations or other substances for gelation.

[0016] <Method for manufacturing gelling agents> A method for producing a gelling agent according to an embodiment of the present invention will be described in detail below. First, Sphaerotilus hippei is cultured in a culture medium to which a nitrogen source or carbon source has been added. The culture medium is not particularly limited as long as it is used for bacterial culture, but fish peptone can be used as the nitrogen source, and Bacterio-N-KN (Maruha Nichiro Corporation) is particularly preferred from the viewpoint of its high growth effect in culturing Sphaerotilus hippei.

[0017] Preferred nitrogen sources to add to the culture medium include fish peptone, yeast extract, meat extract, and tryptone. Preferred carbon sources to be added to the culture medium include ethanol, butanol, isobutanol, acetate, citrate, lactate, arabinose, sucrose, fructose, glucose, glycerol, histidine, peptone, and yeast extract, with glucose being particularly preferred.

[0018] Culturing Sphaerotilus hippei is performed by inoculating Sphaerotilus hippei into a culture medium and then allowing it to stand at 20-30°C, preferably around 25°C. The duration of this standing culture is not particularly limited, but it is preferably around 3-8 days. After culturing, lysis treatment using lysozyme or similar agents may be performed.

[0019] Next, the cultured Sphaerotilus hippei cells are collected by filtration or centrifugation, and an alkaline solution containing a surfactant is added and heated. Preferred alkaline solutions include aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, sodium carbonate, and potassium carbonate. Preferred surfactants include sodium dodecyl sulfate and sodium laureth sulfate. The heating temperature is preferably 40-95°C, and the heating time is preferably 30 minutes to 3 days.

[0020] By adding an alkaline solution containing a surfactant to the cultured Sphaerotilus hippei and heating it, only the bacterial cells are dissolved, and a gelling agent fraction is obtained as the residue.

[0021] Next, the treated material (gelling agent fraction) obtained by the above-described heat treatment is washed with water or the like to remove soluble impurities, and then the residue obtained by filtering it with a mesh filter or the like is dried. The drying method is not particularly limited, but freeze-drying is preferred. This yields a gelling agent according to the embodiment of the present invention.

[0022] Previously, it was not known that Sphaerotilus hippei produced a gelling agent that yielded hydrogels. In the fermentation production of polysaccharides, the increase in viscosity of the culture medium during growth and the removal of microbial cells after cultivation are often challenges. Because the gelling agent of Sphaerotilus hippei covers the surface of the cell rows as a thin film in an aggregated state, the culture medium does not become viscous. In other words, it does not require a large amount of power for aeration and stirring of the fermentation tank. The microbial cells are filamentous, so they have low dispersibility and high sedimentation. Therefore, microbial cell recovery by centrifugation or filtration is easy. If the microbial cells are heated under alkaline conditions, only the cells dissolve, and the gelling agent is obtained as a residue. This ease of preparation is another excellent feature of the gelling agent of Sphaerotilus hippei.

[0023] <Gel-like composition and method for producing the gel-like composition> A gel-like composition according to an embodiment of the present invention comprises a gelling agent and a solvent according to an embodiment of the present invention, and is obtained by adding the solvent to the gelling agent and heating it. The solvent may be pure water, or an aqueous solution of KCl, NaCl, Mg2SO4, CaCl2, NaHCO3, etc. Alternatively, an aqueous solution containing various organic acids such as acetic acid, lactic acid, citric acid, ascorbic acid, tartaric acid, malic acid, succinic acid, or fumaric acid may be used. Furthermore, the pH of the solvent can be set to 4-10. The gelling agent according to the embodiment of the present invention can be gelled by heating at 50 to 100°C.

[0024] While known gellan gums do not gel in alkaline conditions, the gelling agent derived from Sphaerotilus hippei according to the embodiment of the present invention does not require polyvalent cations for gelation and, as described above, has a wide pH range in which gelation is possible. Furthermore, the gelling agent derived from Sphaerotilus hippei according to the embodiment of the present invention has superior solubility compared to known curdlans, and moreover, it exhibits thermoplasticity and gels even at low temperatures (around 50°C) compared to gellan gum and curdlans.

[0025] <Application> The gelling agents and gel-like compositions according to embodiments of the present invention are useful as materials for food (for gelling, imparting elasticity, improving water retention, dietary fiber, etc.), chemical products (for gelling, improving moisturizing properties, preventing syneresis, etc.), pharmaceuticals (for dispersants, sustained-release substrates, drug delivery substrates, cell scaffolds, solid culture medium substrates, etc.), and cosmetics (for improving moisturizing properties, etc.). The constituent monosaccharides are galactose and mannose, which are not acylated and do not have dissociating groups such as carboxyl groups or amino groups. Therefore, the presence of acids, alkalis, salts, etc., for charge neutralization or reduction of electrostatic repulsion is not required for gelation. Consequently, except in the presence of a strong alkali, it is less affected by pH, and hydrogel formation is possible without being significantly affected by salt concentration and osmotic pressure. Therefore, it has the characteristic of being very easy to use as a material for the aforementioned food, chemical products, pharmaceuticals, and cosmetics. [Examples]

[0026] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0027] (Test Example 1) Sphaerotilus hippei was cultured statically at 25°C for 6 days in a culture medium supplemented with 15 g / L of Bacterio-N-KN (nitrogen source) and 2 g / L of glucose (carbon source). Next, lysozyme was used to perform cell lysis. Next, the cultured Sphaerotilus hippei cells were collected by centrifugation, suspended in 0.5 M sodium hydroxide containing 1% sodium dodecyl sulfate (surfactant), and heated at 95°C for 1 hour. This lysed only the bacterial cells, and a gelling agent fraction was obtained as the residue. Next, after cooling, the gelling agent fraction was collected using a mesh filter (nylon mesh), the gelling agent on the mesh filter was washed with water, and freeze-dried. As a result, as shown in Figure 1, approximately 0.3 g of sponge-like gelling agent was obtained per liter of culture medium. Figure 2 also shows a phase-contrast microscope image (top) of the bacterial cells obtained by culturing Sphaerotilus hippei at 25°C for 5 days with shaking, and a phase-contrast microscope image (bottom) of the sheath (hollow filamentous structure: obtained gelling agent).

[0028] (Analysis 1: Analysis of sugar composition by algitol acetate formation) The gelling agent obtained in Test Example 1 was hydrolyzed with trifluoroacetic acid, and the resulting hydrolysate (free constituent monosaccharides) was treated with a reducing agent and acetic anhydride to convert it to algitol acetate. This was subjected to gas chromatography (GC-2010Plus: Shimadzu Corporation) to obtain the chromatograph shown in Figure 3. Monosaccharides were identified from the retention time of the detected peaks, and the molar ratio of constituent monosaccharides (galactose:mannose = 1:1) was calculated from the area ratio.

[0029] (Analysis 2: Analysis of absolute conformation of constituent monosaccharides by butyl glycoside formation) The gelling agent (sample) obtained in Test Example 1 was treated with (-)-2-butanol in the presence of acetyl chloride to convert the constituent monosaccharides (galactose, mannose) into butyl glycosides. The resulting glycosides were subjected to trimethylsisilylation and then subjected to gas chromatography. Commercially available D-galactose and D-mannose were subjected to the same procedure and gas chromatography as standards for trimethylsisilylated butyl glycosides. Figure 4 shows the obtained chromatograms. From the similarity between the standard chromatogram and the sample chromatogram, it was confirmed that the constituent monosaccharides of the gelling agent (sample) obtained in Test Example 1 are D-isomers (D-galactose, D-mannose).

[0030] (Analysis 3: Analysis of the anomeric structure, ring structure, arrangement, and binding position of constituent monosaccharides by NMR spectroscopy) The gelling agent (sample) obtained in Test Example 1 was solubilized by treatment with dilute hydrochloric acid. The sugar composition of the solubilized product was the same as that of the untreated sample. That is, it was thought that the basic structure was maintained while being solubilized, so the solubilized product was dissolved in heavy water and its structure was determined using an NMR spectrometer (AVANCE III HD 600 spectrophotometer: Bruker Japan Co., Ltd.). When proton 1D NMR measurements were performed, four signals were detected in the anomeric proton region, as shown in Figure 5, so it was predicted that the sheath-forming polysaccharide consists of repeating units of 4 sugars (A, B, C, D). Of the anomeric proton signals (proton signals at position 1), the two on the low-field side (A1, B1) were not split and were therefore predicted to be derived from mannose residues. The two signals on the high-field side (C1, D1) were split (7.6~7.7 Hz) and were thought to be galactose residues. Four correlation signals were detected in the anomeric region during proton-carbon two-dimensional NMR (Edited-HSQC) measurements, indicating that the repeating unit consists of two mannose residues and two galactose residues. All proton and carbon signals (positions 1-6) originating from each sugar residue were assigned using proton-proton two-dimensional NMR (COSY and TOCSY) and proton-carbon two-dimensional NMR (Edited-HSQC and HMBC) measurements (Figure 6). Based on the CH coupling constants of each anomeric position obtained by HSQC measurements without decoupling, the anomeric structures of each sugar residue (A, α-D-Man; B, β-D-Man; C, β-D-Gal; D, β-D-Gal) were predicted. In HMBC measurements, intraresidial correlation signals indicating the ring structure (all 6-membered rings) of each sugar residue were detected. Furthermore, inter-residue correlation signals indicating the binding site and sequence of each residue ([→4A1→3B1→3C1→]n and D1→A6) were also detected. Based on analyses 1-3 above and their results, it was confirmed that the gelling agent (sheath-forming polysaccharide) in Test Example 1 has the structure shown in formula (1) above.

[0031] (Test Example 2) A gel-like composition (hydrogel) was obtained by adding 2 mg of the gelling agent obtained in Test Example 1 to 1 mL of water and heating it at 50°C for 20 minutes. Similarly, when 2 mg of the gelling agent obtained in Test Example 1 was added to KCl aqueous solution, NaCl aqueous solution, Mg2SO4 aqueous solution, CaCl2 aqueous solution, and NaHCO3 aqueous solution (1 mL of each 1 M solution), and heated at 50°C for 20 minutes, a gel-like composition (hydrogel) was obtained in all cases.

[0032] (Test Example 3) The following tests were conducted to investigate the effect of organic acids used as food additives on gelation. When 2 mg of the gelling agent obtained in Test Example 1 was added to 1 M acetic acid, 1 M lactic acid, 1 M citric acid, 1 M ascorbic acid, 0.5 M tartaric acid, 0.5 M malic acid, 0.1 M succinic acid, and 40 mM fumaric acid (1 mL each), and heated at 50°C for 20 minutes, a gel-like composition (hydrogel) was obtained in all cases.

[0033] (Test Example 4) The following tests were conducted to compare the relationship between gel concentration and syneresis rate between the gelling agent obtained in Test Example 1 and agar. First, the gelling agent obtained in Test Example 1 was placed in water and heated at 50°C for 20 minutes, then allowed to cool to obtain a gelled composition. Separately, agar was placed in water and heated at 100°C for 30 minutes, then allowed to cool to obtain a gel-like composition. Next, after measuring the mass of each gel-like composition, centrifugation was performed at 3000 rpm for 15 minutes, followed by removal of the liquid phase, and then the mass of the solid phase was measured. Next, the syneresis rate was calculated by subtracting the mass of the solid phase from the mass of the gel-like composition measured initially, and then dividing the resulting difference by the mass of the gel-like composition measured initially. Furthermore, the above measurements were performed at various gel concentrations. The results are shown in Figure 7. As shown in Figure 7, the gelling agent obtained in Test Example 1 did not gel at a gel concentration of less than 0.025%, and the agar did not gel at a gel concentration of less than 0.2%. Thus, it can be seen that the gelling agent obtained in Test Example 1 had a lower syneresis rate and was more stable as a gel.

[0034] (Test Example 5) The following tests were conducted to compare the relationship between pH and syneresis rate of the gelling agent and agar obtained in Test Example 1. The gelling agent and agar obtained in Test Example 1 were placed in water containing CHES (2-cyclohexylaminoethanesulfonic acid), MES (2-morpholinoethanesulfonic acid), and HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) at a concentration of 0.1 M, respectively, as buffers, and heated at 50°C for 20 minutes, followed by cooling to obtain a gel-like composition. The water containing CHES, MES, and HEPES at a concentration of 0.1 M as buffers had its pH adjusted as shown in Figure 8 below. Next, after measuring the mass of each gel-like composition, centrifugation was performed at 3000 rpm for 15 minutes, followed by removal of the liquid phase, and then the mass of the solid phase was measured. Next, the water separation rate was calculated using the same procedure as in Test Example 4. The results are shown in Figure 8. As shown in Figure 8, the gel-like composition prepared using the gelling agent obtained in Test Example 1 was stable as a gel over a wide pH range of 4 to 10.

Claims

1. A gelling agent represented by the following formula (1). 【Chemistry 1】 (In equation (1), n ​​is a natural number.)

2. A gel-like composition comprising the gelling agent and solvent described in claim 1.

3. A step of culturing Sphaerotilus hippei in a culture medium to which a nitrogen source or carbon source has been added, The process involves adding an alkaline solution containing a surfactant to the cultured Sphaerotilus hippei and heating it, The process involves washing and filtering the heated treated material, and then drying the resulting residue. A method for producing a gelling agent according to claim 1, including the method described in claim 1.

4. A method for producing a gel-like composition, comprising the step of adding a solvent to the gelling agent described in claim 1 and heating it.

5. A food product, chemical product, pharmaceutical product, or cosmetic product comprising the gelling agent described in any one of claims 1 to 4.

Images