WO2010061454A1 - Process for production of high-purity chitin slurry - Google Patents

Abstract

Disclosed is a process for producing a hydrated chitin slurry, which comprises the following steps (a) to (c): (a) dissolving a chitin having a protein content of less than 0.14 wt% and a degree of acetylation of 96% or more in a solvent comprising an alcohol selected from methyl alcohol and ethyl alcohol and a halogenated calcium salt or a halogenated magnesium salt to produce a chitin solution: (b) filtering the chitin solution to remove any insoluble matter; and (c) diluting the filtered chitin solution with water, optionally washing a produced precipitate of chitin or carrying out dialysis to substitute a calcium ion or a magnesium ion and the alcohol contained in the solution by a water molecule, thereby producing the hydrated chitin slurry.

Description

Manufacturing method of high purity chitin slurry

The present invention relates to a method for producing a high purity chitin slurry having a low content of impurities such as proteins.

Chitin is a natural mucopolysaccharide obtained from crustacean shells such as crabs and shrimps, insect epidermis, and bacterial cell walls such as mushrooms, and is a biological resource that exists in large quantities on the earth. Such chitin has been found to have various excellent properties such as moisture retention, physiological activity, and biocompatibility. For this reason, many studies have been made in order to utilize the excellent characteristics of chitin in the fields of fibers, films, cosmetics, foods, medicines, medical materials and the like.

However, chitin is known to exhibit poor solubility because it forms a hard crystal structure due to a strong hydrogen bond derived from its chemical structure. Due to this poor solubility, it is impossible to form a solution or dispersion in which chitin is dissolved or well dispersed, and the application field thereof is to physically knead chitin powder into a substrate to form a molded body. It was extremely limited in use.

The inventors of the present invention have made extensive studies by paying attention to the fact that the above-mentioned excellent characteristics of chitin can be easily used if chitin, which is hardly soluble as described above, can be dissolved or well dispersed in a solvent. However, it has been found that chitin can be dissolved by using a specific solvent comprising alcohol (particularly methanol) and a calcium halide salt or magnesium halide salt (particularly calcium chloride dihydrate) (Patent Literature). 1 and Non-Patent Document 1). As a result, various mixtures and molded articles using the chitin solution have been developed (Patent Documents 2 to 4).

It was also found that by diluting the chitin solution with water, a hydrated chitin gel (hydrated chitin slurry) in which the special solvent is replaced with water molecules can be obtained (Patent Documents 5 and 6, Non-Patent Documents). 2). The hydrated chitin slurry can be applied to various uses without causing the problem of environmental pollution by methanol.

Chitin as a raw material for the hydrated chitin slurry is usually derived from the raw material, and a small amount of protein is bound as an impurity to the chitin polymer. Proteins can cause antigen-antibody reactions and cause health hazards. In particular, in the case of chitin derived from salmon, crustacean allergen (tropomyosin) remains, which may cause crustacean allergy.
In addition, chitin as a raw material contains chitosan (a polymer of N-acetylglucosamine and glucosamine having an acetylation degree of 50% or less) as an impurity, and has a relatively low acetylation degree and is close to chitosan. There is also low quality chitin. There are people who do not fit the constitution of chitosan, and if a large amount of chitosan is ingested, it may adversely affect the human body.

Furthermore, in the conventional method for producing a hydrated chitin slurry, when insoluble matter in the chitin solution is removed in the filtration step, chitin with a high amount of protein combined with insoluble matter such as chitosan that has been confirmed so far, low quality It has been found that impurities such as chitin form an insoluble gel and cause clogging of the filter cloth. Furthermore, when filtering using external force, such as vacuum filtration, the said gel passed the filter cloth unlike insoluble substances, such as chitosan, and caused the quality of the obtained hydrated chitin slurry to deteriorate.

As described above, in the conventional method for producing a hydrated chitin slurry, the removal of impurities such as proteins and chitosan that may adversely affect the human body is not sufficient, and there is a problem in the quality and safety of the obtained hydrated chitin slurry. It was. In addition, its production efficiency was low and not satisfactory.
Japanese Patent Laid-Open No. 06-179702 Japanese Unexamined Patent Publication No. 07-316202 JP 08-120173 A Japanese Patent Laid-Open No. 10-101811 JP 2005-035934 Japanese Unexamined Patent Publication No. 2005-036109 Akihiro Shirai, Kiyohisa Takahashi, Ratana Rujiravanit, Norio Nishi and Seiichi Tokura, Title: Chitin and Chitosan: The Versatile Environmentally Friendly Modern Materials “Regeneration of Chitin Using New Solvent System”, Ed. Mat B Zakaria, Wan Mohamed Wan Muda, Md Pauzi Abdullah, Pb. Universiti Kebangsaan Malaysia Press, 1995 "Kitin, Chitosan Experiment Manual", edited by Chitin, Chitosan Study Group, Gihodo Publishing Co., Ltd. (April 1991)

An object of the present invention is to provide a method for efficiently producing a hydrated chitin slurry having sufficiently low impurities such as protein and chitosan that can adversely affect the human body and improved quality and safety.

As described in detail below, the present inventors have found that the above-mentioned problems can be solved by using a specific chitin as a starting material and, if necessary, performing a specific step, The present invention has been completed.
That is, the present invention includes the following.
[1] (a) a solvent comprising chitin having a protein content of less than 0.14% by weight and an acetylation degree of 96% or more, an alcohol comprising methyl alcohol or ethyl alcohol, and a calcium halide salt or a magnesium halide salt To obtain a chitin solution by dissolving in
(B) filtering the chitin solution to remove insoluble matter;
(C) Dilute the filtered chitin solution with water, and if necessary, wash the produced chitin precipitate with water or dialyze it to replace calcium ions or magnesium ions and alcohol contained in the solution with water molecules. And a step of obtaining a hydrated chitin slurry.
[2] The method according to [1], further comprising the step of (d) adding carbon dioxide gas and / or sodium hydrogen carbonate to the chitin solution or hydrated chitin slurry to separate the remaining alcohol.
[3] The method according to [1] or [2], further comprising the step of (e) adding an organic acid to the chitin solution or hydrated chitin slurry to separate the remaining calcium ions or magnesium ions.
[4] The method according to [3] above, wherein the organic acid is selected from the group consisting of citric acid, succinic acid, succinic anhydride, glutamic acid, ethylenediamine acid and asurbic acid.
[5] The method according to [1] above, wherein the chitin used in the step (a) is dried at a temperature of 60 ° C. or higher.
[6] The method according to [1] above, wherein the chitin used in step (a) is regenerated chitin.
[7] The method according to [1] above, wherein the chitin used in step (a) is de-O-acylated.

According to the method for producing a chitin slurry of the present invention, it is possible to efficiently produce a hydrated chitin slurry having sufficiently low impurities such as protein and chitosan that can adversely affect the human body and improved quality and safety.

The method for producing the hydrated chitin slurry of the present invention comprises:
(A) A chitin having a protein content of less than 0.14% by weight and an acetylation degree of 96% or more is dissolved in a solvent containing methyl alcohol or ethyl alcohol and a calcium halide salt or a magnesium halide salt. Obtaining a chitin solution;
(B) a step of filtering the chitin solution to remove insoluble matter; and (c) diluting the chitin solution with water, and if necessary, washing the produced chitin precipitate with water to contain calcium ions or magnesium contained in the solution. Replacing ions and alcohol with water molecules to obtain a hydrated chitin slurry.

The chitin used as a starting material in the method for producing a hydrated chitin slurry of the present invention is generally a polysaccharide obtained from a shell of a crustacean such as a crab or shrimp, an insect epidermis, a skeleton such as a squid, or a bacterial cell wall such as a mushroom ( Poly (N-acetyl-D-glucosamine), which usually has powder properties. In the present invention, any commercially available chitin can be used, and it may be produced using a microorganism. Further, regenerated chitin obtained by N-acetylating chitosan or the like may be used. In particular, by using regenerated chitin, it is possible to solve the problem of purity reduction due to deacetylation that occurs in the process of protein removal and molecular weight change in normal chitin, and it is possible to create high purity chitin slurry with any molecular weight It becomes. Further, chitin may have any three-dimensional structure including α-type and β-type.

The weight average molecular weight of the starting material chitin is 2.3 × 10 ³ or more, preferably 5 × 10 ³ or more, more preferably 1 × 10 ⁴ to 5 × 10 ⁶ , and particularly preferably 5 × 10 ⁴ to 5 ×. 10 ⁵ . What is necessary is just to select the said weight average molecular weight suitably according to the use of a final product.
The value of the weight average molecular weight of the chitin is a value determined by a viscosity method described later.

In the present invention, the protein content (calculated as amino acid content) in chitin as the starting material is less than 0.14% by weight, preferably 0.1% by weight or less, more preferably 0.01% by weight or less, and The acetylation degree of the starting material chitin is 96% or more, preferably 97% or more, particularly preferably 98% or more.
By making the starting material chitin into a chitin having a low protein content and a high acetylation degree, the amount of gel-like substance that interferes with filtration in the filtration step (b) can be reduced. Therefore, the filtration can be performed at a high filtration rate, and the gel material does not pass through the filter cloth because it is not necessary to use an external force. As a result, the chitin in the filtrate becomes high quality (high purity).
Here, the gelatinous substance is chitin having a low degree of acetylation, chitin having a high protein content, and chitin containing methanol, water, and the like, which will be described later.
The protein content (amino acid content) in the chitin is 18 amino acids (lysine, histidine, phenylalanine, leucine, isoleucine, methionine, valine, threonine, tryptophan, arginine, tyrosine, alanine, glycine, proline, glutamic acid, Serine, aspartic acid, cystine) is a value determined by measuring by an amino acid automatic analysis method and a high performance liquid chromatography method.
The value of the degree of acetylation of chitin is a value determined by neutralizing and titrating chitin treated with 2 mol / l hydrochloric acid with 0.1 mol / l NaOH.

The chitin as the starting material is preferably dried in advance at a temperature of preferably 60 ° C. or higher, more preferably 100 ° C. or higher before being dissolved in the solvent in the step (a). Although the upper limit of drying temperature is not specifically limited, Preferably it is 200 degrees C or less, More preferably, it is 120 degrees C or less. A temperature of 80 ° C. to 100 ° C. is particularly preferable.
The drying time is not particularly limited, but is, for example, 1 hour to 48 hours, preferably 4 hours to 12 hours.
Further, the drying is preferably performed under reduced pressure to vacuum.
By drying in this way, it is possible to further reduce the amount of gel-like substance that interferes with filtration in the filtration step (b). Therefore, filtration can be performed without using external force at a higher filtration rate. As a result, the chitin in the filtrate is of higher quality (high purity).
In addition, since the moisture adhering to the chitin and the residue of the organic solvent (such as methanol) used in the manufacturing process and the washing process of the chitin (regenerated chitin) are removed from the chitin by such drying, the gel form It is believed that a decrease in the amount of material produced will occur.
The drying temperature, drying time, and drying pressure can be appropriately set in consideration of the degree of improvement in the filtration rate.

The starting chitin is preferably regenerated chitin. In the present invention, “regenerated chitin” means that glucosamine in chitosan (degree of acetylation of 50% or less) or chitin / chitosan (degree of acetylation of about 50%) is acetylated again to form N-acetylglucosamine, and the degree of acetylation is 50 It means chitin obtained by re-acetylating glucosamine in chitin made higher than% and low-quality chitin (acetylation degree of 90% or less).
Regenerated chitin can be produced, for example, by acetylating chitosan dissolved in acetic acid with acetic anhydride.

In the present invention, the starting chitin (particularly regenerated chitin) is preferably de-O-acylated. To produce regenerated chitin, the OH group is also acetylated (O-acylated) when N-acetylating chitosan, but the O-acylated chitin does not dissolve in the solvent in step (a) . As a result, the O-acylated chitin is removed as an insoluble substance in the filtration step (b), and the amount of chitin in the resulting hydrated chitin slurry is reduced. Such a decrease in the amount of chitin can be prevented by de-O-acylation.
De-O-acylation can be performed, for example, by heating at 20 ° C. to 95 ° C. (particularly 80 ° C.) under alkaline conditions of pH 10 to pH 11 (particularly pH 11).

In the step (a), chitin having a protein content of less than 0.14% by weight and an acetylation degree of 96% or more comprises an alcohol comprising methyl alcohol or ethyl alcohol and a calcium halide salt or a magnesium halide salt. Dissolve in solvent to obtain a chitin solution. By using such a solvent, it becomes possible to break down the hard crystalline structure of chitin, which is hardly soluble, and to allow the solvent to penetrate into the chitin molecule. As a result, solvation occurs and the molecular structure is expanded and dissolved. The state chitin can be obtained. This step can be performed, for example, as described in JP-A No. 06-179702.

Examples of the calcium halide salt in the solvent include calcium chloride dihydrate, calcium bromide dihydrate, calcium iodide dihydrate, and the like.
Examples of the magnesium halide salt in the above solvent include magnesium chloride and hexahydrate.

The concentration of the calcium halide salt or magnesium halide salt in the alcohol in the solvent is preferably a saturated concentration from the viewpoint of the amount of chitin dissolved. However, when calcium halide salt or magnesium halide salt is precipitated due to evaporation of alcohol or the like, the precipitated salt binds to chitin that has not yet dissolved, and not only prevents the chitin from dissolving, but also filters it. In step (b), a gel-like substance is formed, and filtration is hindered. Therefore, from the viewpoint of the yield of chitin and the efficiency of the filtration step (b) (filtration rate), it is preferable to adjust the concentration of the calcium halide salt or the magnesium halide salt so that these salts do not precipitate. .

In some embodiments, chitin is added to the above solvent at a concentration that is preferably 0.1-5% (w / v), more preferably 0.5-2% (w / v). The solution is preferably dissolved at a temperature of 20 ° C. to 70 ° C., more preferably 40 ° C. to 60 ° C. with sufficient stirring under reflux. It is preferable to adjust the chitin concentration in the solvent according to the molecular weight of the chitin used.

In the step (b), the chitin solution is filtered to remove insoluble matters. In the present invention, since the above-mentioned specific chitin is used as a starting material, the chitin solution can be filtered without using external force at a much higher filtration rate than when conventional chitin is used.

As the filter cloth used in the filtration step (b), those having various materials and various properties (thickness, pore diameter, pore density, etc.) can be used depending on the starting material chitin and the like.
For example, the average pore diameter is preferably 0.01 to 1 mm, more preferably 0.05 to 0.5 mm, especially 0.13 mm and 0.2 mm, and / or the average mesh number is preferably 0.5. Filter cloths of ˜50 / mm, more preferably 1˜10 / mm, especially 4 / mm can be used.
Here, the average pore diameter is a value measured by an optical measuring device such as a microscope or a microscope.
The average number of meshes (pieces / mm) indicates the average value of the number of meshes (holes) existing per 1 mm length of the filter cloth.

Moreover, the filtration in a filtration process (b) is not specifically limited, For example, the natural filtration and suction filtration etc. which pour a chitin solution on said filter cloth and filter by gravity are mentioned.
Moreover, although the yield of chitin is reduced, the gel which is an impurity can be allowed to stand and then subjected to precipitation separation or centrifugation, followed by filtration (natural filtration, suction filtration, etc.).

In the step (c), the filtered chitin solution is diluted with water, and if necessary, the produced chitin precipitate is washed with water or dialyzed to remove calcium ions or magnesium ions and alcohol contained in the solution. By replacing with water molecules, a hydrated chitin slurry can be obtained.

In an embodiment, after cooling the chitin solution to room temperature, the insoluble part is removed with a filter cloth (nel). Then, dilute with distilled water. The dilution concentration at that time is such that the volume ratio of the chitin solution to water (chitin solution: water) is preferably 1: 1 to 1: 4, more preferably 1: 2 to 1: 3. When diluted with distilled water, chitin usually precipitates, and this is thoroughly washed to remove alcohol and calcium ions or magnesium ions. Further, after dilution with water, dialysis may be performed in order to remove alcohol and calcium ions or magnesium ions. Further, washing with water and dialysis may be repeated.
Further, the hydrated chitin slurry having a desired chitin content can be obtained by adjusting the water content of the obtained hydrated chitin slurry by a centrifugal method or the like.

The method of the present invention includes a step (d) of adding carbon dioxide and / or sodium hydrogen carbonate to a chitin solution or a hydrated chitin slurry to remove residual alcohol, and / or a chitin solution, if necessary. Alternatively, a step (e) of adding an organic acid (for example, citric acid, succinic acid, succinic anhydride, glutamic acid, ethylenediamine acid, asurbic acid) to the hydrated chitin slurry to remove calcium ions or magnesium ions is included. By obtaining these steps, the resulting hydrated chitin slurry is of higher quality.

In the step (d), a chitin solution (for example, one obtained from the above step (b)) or a hydrated chitin slurry (for example, one obtained from the above step (c)) is diluted with water as necessary, and then Carbon dioxide and / or sodium hydrogen carbonate is added to the mixture to separate the remaining alcohol. By adding carbon dioxide and / or sodium bicarbonate, the chitin solution and hydrated chitin slurry (including dilutions with these waters) can cause precipitation (solid phase) of chitin and calcium carbonate or magnesium, water, Separate into a liquid phase consisting of alcohol and NaOH. Therefore, an alcohol-containing aqueous solution (liquid phase) can be separated from the chitin precipitate (solid phase) by known solid-liquid separation means such as filtration and centrifugation.
Subsequently, the chitin precipitate from which the alcohol has been separated is treated in the same manner as in the step (c), whereby a hydrated chitin slurry with a further reduced alcohol content can be obtained.

The amount of carbon dioxide and / or sodium hydrogen carbonate added to the chitin solution and the hydrated chitin slurry (including these water dilutions) in step (d) is adjusted to pH 4 to 6.5 (particularly pH 6). Adjust to

In the step (d), for example, 150 mL of the chitin solution (1.5 g of chitin content) is added to 150 mL of ion-exchanged water at a temperature from cooling (for example, 1 to 15 ° C.) to room temperature (about 20 to 30 ° C.). Then add 3 g of sodium bicarbonate and mix (in this case pH 4.5). Next, the obtained precipitate is filtered in the same manner as in the above step (b) to separate the liquid phase containing alcohol. Next, the filtered precipitate is treated in the same manner as in the above step (c) to obtain a hydrated chitin slurry with a further reduced alcohol content.

In the step (e), an organic acid is added to a chitin solution (for example, obtained from the step (b)) or a hydrated chitin slurry (for example, obtained from the step (c) or (d)). To separate any remaining calcium or magnesium ions. By adding such an acid to the chitin solution or hydrated chitin slurry, insoluble salts such as calcium carbonate or magnesium present in the chitin solution or hydrated chitin slurry are dissolved. Therefore, by performing dialysis or the like, calcium ions or magnesium ions can be separated from the chitin solution or hydrated chitin slurry.

Examples of the organic acid used in the above step (e) include citric acid, glutamic acid, ethylenediamine acid, asurbic acid, tartaric acid, malic acid, ascorbic acid, dicarboxylic acid group (oxalic acid, malonic acid, succinic acid, succinic anhydride). Acid, glutaric acid, adipic acid, pimelic acid, sebacic acid, phthalic acid, terephthalic acid, etc.). These may be used alone or as a mixture of two or more.
Among these, it is preferable to use an organic acid selected from citric acid, succinic acid, succinic anhydride, glutamic acid, ethylenediamine acid, and asurbic acid from the viewpoint of safety, availability, and the like.

The concentration of the aqueous solution of the organic acid in the hydrated chitin slurry in step (e) is 0.1 to 10% (w / v), preferably 1 to 3% (w / v), more preferably 1 to 2% ( w / v).

Moreover, it is preferable to apply the step (e) particularly to the hydrated chitin slurry (chitin precipitation) obtained from the step (d). In this case, the calcium carbonate or magnesium accumulated in the hydrated chitin slurry in step (d) is dissolved in the liquid and easily separated as calcium ions or magnesium ions by known solid-liquid separation means such as filtration and centrifugation. can do. As a result, a hydrated chitin slurry having a further reduced alcohol content, calcium ion content or magnesium ion content can be obtained.

Step (e) is a hydrated chitin slurry obtained by, for example, diluting 150 mL of the chitin solution (1.5 g of chitin content) with 150 mL of ion-exchanged water and then adding 3 g of sodium bicarbonate. (Chitin content 1.5 g; pH about 4.5) is added to 500 mL of 1% (w / v) aqueous citric acid solution at room temperature and mixed. Next, the obtained precipitate is filtered in the same manner as in the above step (b), and the liquid phase containing calcium ions or magnesium ions is separated. Next, the filtered precipitate is treated in the same manner as in the above step (c) to obtain a hydrated chitin slurry in which the calcium ion or magnesium ion content is further reduced.

The method of the present invention further includes a step of mechanically mixing the hydrated chitin slurry as necessary. Thereby, a hydration chitin slurry can be made more uniform. Such mechanical mixing can be performed using, for example, various agitators such as a blender and a rotation / revolution type agitator.

The hydrated chitin slurry obtained by the method of the present invention is reduced to such an extent that the protein content and chitosan content do not affect the human body. For example, crustacean allergen (tropomyosin) that can be contained in chitin derived from salmon can be below the detection limit (for example, 0.3 ppm or less) as measured by ELISA. Therefore, the hydrated chitin slurry obtained by the method of the present invention is greatly improved in quality and safety as compared with the conventional hydrated chitin slurry.

The hydrated chitin slurry obtained by the method of the present invention can be suitably applied to the application fields of chitin and chitosan. That is, it can be applied to the following application fields.
(1) Cosmetics field: Health care, skin care, hair care, oral care, anti-aging cream, cleansing, etc. (humectant, thickener, inflammation suppression, ultraviolet light, hair damage suppression, skin damage suppression, skin regeneration), Repellents, etc.
(2) Food field: Improvement of intestinal metabolism (proliferation of lactic acid bacteria, promotion of production of β-galactosase necessary for lactose digestion), immunity enhancement, improvement of bread bulge, promotion of hyaluronic acid production, prevention and improvement of osteoarthritis, etc. Health food, functional food, etc.
(3) Medical field: Wound healing agent (material), non-woven fabric, artificial skin, surgical suture, cream, pharmaceuticals (medicine, non-woven fabric shape cancer cell proliferation suppression, immune enhancement effect (cancer cell growth suppression and opportunistic infection) Fungus-protective effect against Lisera monocytogenes), growth of lactic acid bacteria, promotion of β-galactose production necessary for lactose digestion, hyaluronic acid production, prevention and improvement of osteoarthritis, improvement of inflammation, healing (stomatitis, gingivitis, alveolar pyorrhea) Etc.), analgesic suppressants, hemostatic agents, bactericides), biomaterials (bones, teeth, etc.),
(4) Plant field: soil improvement material, plant control material, etc.
(5) Biotechnology field: Cell growth substrate, etc.
(6) Molding material field: Fillers for plastic products (thickeners, biodegradable materials), natural material molded products, films, etc.
(7) Thickener and the like.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to these.
Moreover, the evaluation methods of physical properties in the following examples and comparative examples are as follows.

(Weight average molecular weight)
The molecular weight of chitin was determined by the following procedure (viscosity method).
(1) A chitin solution having a plurality of known concentrations is prepared, and the viscosities thereof are measured with a cone plate type rotational viscometer (instrument: “DV-II + Pro” manufactured by Brookfield, temperature: 25 ° C.).
(2) The intrinsic viscosity of the target chitin is calculated from the above viscosity.
(3) A weight average molecular weight is calculated from the calculated intrinsic viscosity according to the following viscosity formula.
[Η] = 2.54 × 10 ⁻² M ^0.45
[Wherein [η]: intrinsic viscosity, M: weight average molecular weight].

[Degree of acetylation]
The degree of acetylation of chitin was determined by the following procedure.
(Preprocessing)
(1) Put 100 ml of 2M hydrochloric acid and 2 g of chitin into a 100 ml beaker, and stir (magnetic stirrer) at 400 rpm for 30 minutes.
(2) The mixture is filtered with a filter paper (“QUALITVE FILTER PAPRE NO.1” manufactured by ADVANTEC), and the filtration residue and 100 ml of methanol are put into a beaker and stirred and washed for 15 minutes. The mixture is then filtered again.
(3) Repeat the above operation (2) four or more times.
(4) After completion of washing with methanol, the obtained residue (chitin) is air-dried for about 1 hour. Thereafter, vacuum drying is performed at 50 ° C. for 12 hours.
(Measurement)
(1) Weigh precisely 0.5000 g of the pretreated chitin.
(2) Add 50 ml of distilled water (measured with a graduated cylinder), add 3 drops of phenolphthalein, and titrate with 0.1N aqueous sodium hydroxide while stirring at 400 rpm. The point at which the liquid color (pink) continues for 30 seconds or more is defined as the end point.
(3) The degree of acetylation is calculated from the following formula from the weight of chitin (a) and the titration amount (b) of 0.1N aqueous sodium hydroxide solution.
Degree of acetylation (%) = (2.03 × b) / [a + (5.5 × b × 10 ⁻⁴ )]

[Protein content]
The protein content of chitin was determined by the following procedure using 18 amino acids (lysine, histidine, phenylalanine, leucine, isoleucine, methionine, valine, threonine, tryptophan, arginine, tyrosine, alanine, glycine, proline, glutamic acid, serine, aspartic acid, cystine. ) Was analyzed (amino acid analysis method).
(1) Hydrolyze chitin (hydrochloric acid hydrolysis; methionine and cystine are hydrolyzed with hydrochloric acid after formic acid oxidation treatment).
(2) The amount of various amino acids is measured with an amino acid analyzer (Hitachi high-speed amino acid analyzer L8800k).
(3) Only tryptophan is measured by high performance liquid chromatography (JLC-500 V, manufactured by JEOL Ltd.).

[Chitin concentration in chitin slurry (water content)]
The chitin concentration (water content) in the chitin slurry was calculated by heating the chitin slurry to 200 ° C. with a differential calorimeter, and performing a weight loss analysis.

[Moisture content in chitin powder]
The water content of the chitin powder was calculated by heating to 150 ° C. with a differential calorimeter and performing a weight loss analysis.

[Chitin powder]
In the examples and comparative examples, the following chitin powder was used.
(1) Regenerated chitin (prepared according to the following Production Example 1) based on chitosan (“Floac C” manufactured by Kyowa Technos Co., Ltd.)
a) Weight average molecular weight: 50,000 to 100,000
b) Degree of acetylation: 98.9% to 96.4%
c) Protein content (measured by the above method): Total amount 0.01% by weight (only glycine is detected)
(2) “Kitin TCL” manufactured by Koyo Chemical Co., Ltd.
a) Weight average molecular weight: several hundred thousand b) Degree of acetylation 97.3%
c) Protein content (measured by the above method): Total amount 0.14% by weight

[Amount of residual gel substance and its dry weight]
(1) The amount of residual gel substance (g) is calculated by measuring the weight of the filter cloth together with the chitin solution in the funnel, and then subtracting the dry weight of the filter cloth from the weight. .
(2) The dry weight (g) of the residual gel-like substance is determined by measuring the amount of the residual gel-like substance, immersing the filter cloth in ion-exchanged water placed in a beaker, taking out the gel-like substance from the filter cloth, Stir the contents of the beaker with a glass rod to dissolve water-soluble substances (calcium chloride, etc.). The solution is filtered, and the filtration residue is dried with a dryer, and then the weight is measured to obtain a dry weight.

[Production Example 1] Preparation of regenerated chitin powder (1) 1% (v / v) acetic acid aqueous solution (5 L; Sigma Aldrich Jumpan Co., Ltd., SAJ first grade, acetic acid) and chitosan (100 g: manufactured by Kyowa Technos Co., Ltd.) “Flownack C”) was dissolved.
(2) A solution obtained by mixing methanol (5 L) with acetic anhydride (528 mL; manufactured by Kanto Chemical Co., Inc.) was mixed with the above solution and allowed to stand overnight for N-acetylation.
(3) After neutralizing with 40% (w / v) NaOH aqueous solution (SAJ first grade sodium hydroxide manufactured by Sigma-Aldrich Jumpan Co., Ltd.), a centrifugal dehydrator (“H-122” manufactured by Kokusan Co., Ltd.) was used. The solution was filtered under conditions of 500 to 1,000 rpm and a filter cloth (PR-20 manufactured by Kokusan).
(4) After the above precipitate was immersed in ion-exchanged water, a 40% NaOH aqueous solution was added to adjust the pH to 11, followed by heating at 80 ° C. for 30 minutes for de-O-acylation.
(5) After neutralization with 10% (v / v) hydrochloric acid (manufactured by Sigma-Aldrich Jumpan Co., Ltd., reagent grade), each is immersed in ion-exchanged water, methanol, and acetone, and filtered under the conditions of (3) above. And washed.
(6) After air drying for 48 hours, vacuum drying was performed at 60 ° C. for 18 hours with a dryer (“EYELA VOS301SD” manufactured by Tokyo Science Instruments Co., Ltd.).
(7) The dried product was pulverized with a blender (Oster Blender ST-1 manufactured by Oster) and then passed through a 60 mesh / inch sieve to prepare regenerated chitin powder. The regenerated chitin powder had an acetylation degree of 97.4% and a protein content of 0.01% by weight.

[Example 1] Preparation of hydrated chitin slurry (1) Calcium chloride dihydrate (8.5 kg; “Calcium chloride (2 water)” manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in methanol (10 L) was dissolved in calcium chloride. A dihydrate saturated methanol solution was prepared. Next, the regenerated chitin powder (70 g) obtained in Production Example 1 above was added to the solution (11 L) in a water bath adjusted to 55 ° C. using a stirrer (“FINE FL-105N”). Then, the mixture was dissolved under the stirring conditions of dials 3 to 7 to prepare a chitin solution.
(2) The chitin solution was filtered using a filter cloth (average pore diameter of about 0.2 mm, average mesh number of about 4 / mm) to remove insoluble matters and the like. The filtration time was about 20 minutes.
(3) Next, the filtered chitin solution and 1 to 2 times the amount of ion-exchanged water were stirred at 10,300 to 15,700 rpm using a blender (Oster Blender ST-1 manufactured by Oster). And then allowed to stand overnight to precipitate chitin.
(4) Thereafter, the precipitate was filtered using a centrifugal dehydrator (“H-122” manufactured by Kokusan Co., Ltd.) under the conditions of 500 to 1,000 rpm and filter cloth (PR-20 manufactured by Kokusan). Next, the dehydrated precipitate is further subjected to centrifugal dehydration while adding the same amount of ion-exchanged water as the amount of water discharged by centrifugal dewatering, and washed until the drainage conductivity reaches about 0.3 to 0.2 mS / m or less. Finally, dehydration was performed to obtain a hydrated chitin slurry.
When the residual amount of methanol in the obtained hydrated chitin slurry was measured by gas chromatography (GC14B manufactured by Shimadzu Corporation), it was less than the detection limit of 5 ppm.
(5) A part of the obtained hydrated chitin slurry was dried overnight at 80 ° C. with a dryer (“DN410H” manufactured by Yamato), and the chitin concentration was measured. Finally, 418 g (average chitin concentration 10.4% by weight) of a hydrated chitin slurry was obtained. The yield was about 62%.

[Example 2]
(1) Regenerated chitin powder (acetylation degree: 98.7%, protein content: 0.01% by weight) prepared in the same manner as in Production Example 1 above is placed in a dryer (EYELA VOS301SD manufactured by Tokyo Science Instruments Co., Ltd.). After drying in vacuo at 85 ° C. for 18 hours, 62.5 g of calcium chloride dihydrate saturated methanol prepared in the same manner as in Example 1 (1) above in a water bath adjusted to 55 ° C. The solution was added to the solution (7 L) and dissolved under the stirring conditions of the dials 2 to 7 using a stirrer (manufactured by IKA, LABORTECHNIK RW20.n) to prepare a chitin solution.
(2) The chitin solution was filtered using a filter cloth (average pore diameter of about 0.2 mm, average mesh number of about 4 / mm) to remove insoluble matters and the like. The filtration time was 6 minutes and 40 seconds.
(3) Next, the filtered chitin solution and 1.25 times its ion-exchanged water were subjected to agitation conditions of 10,300 to 15,700 rpm using a blender (Oster Blender ST-1 manufactured by Oster). Mix and then let stand overnight to precipitate chitin.
(4) Thereafter, the precipitate was dehydrated using a centrifugal dehydrator (“H-122” manufactured by Kokusan Co., Ltd.) under conditions of 500 to 1,000 rpm and using a filter cloth (PR-20 manufactured by Kokusan). Next, the dehydrated precipitate is further subjected to centrifugal dehydration while adding the same amount of ion-exchanged water as the amount of water discharged by centrifugal dewatering, and washed until the drainage conductivity reaches about 0.3 to 0.2 mS / m or less. Finally, dehydration was performed to obtain a hydrated chitin slurry.
(5) A part of the obtained hydrated chitin slurry was subjected to raw material analysis with a suggested thermal analyzer, and the chitin concentration was measured. Finally, 569 g (average chitin concentration: 8.3 wt%) of a hydrated chitin slurry was obtained. The yield was about 76%.
(6) When the degree of acetylation of the residue on the filter cloth was measured in (3), it was 98.3%, and low-quality regenerated chitin with a low degree of acetylation was separated.

Example 3
(1) Regenerated chitin powder (80 g; degree of acetylation 96.4%, protein content 0.01% by weight) prepared in the same manner as in Production Example 1 above, in a water bath adjusted to 55 ° C., Add to calcium chloride dihydrate saturated methanol solution (11 L) prepared in the same manner as in Example 1 (1) above, and use dial 2-7 with stirrer (LABORTECHNIK RW20.n, manufactured by IKA). A chitin solution was prepared by dissolving under stirring conditions.
(2) The chitin solution was filtered using a filter cloth (average pore diameter of about 0.2 mm, average mesh number of about 4 / mm) to remove insoluble matters and the like.
(3) Next, the filtered chitin solution was divided into two, and one side was washed in the same manner as in Example 1 (4) and thereafter, and finally 332 g (average chitin concentration 7.7% by weight) of chitin was obtained. A slurry was obtained. The yield was 64%. At this time, the amount of ion-exchanged water used for cleaning exceeded about 120 L, and the final drainage conductivity was also 0.177 mS / m. Then, when it dried and the acetyl degree was measured, 97.6% of acetylation degree with higher quality than before filtration was obtained. Therefore, it was confirmed that the poor quality chitin having a low degree of acetylation was separated and the purity of the prepared chitin slurry was improved.
(4) Sodium bicarbonate (100 g; manufactured by Sigma-Aldrich, sodium bicarbonate, special grade) was mixed with the other chitin solution (4 L), stirred and then filtered to obtain a precipitate.
(5) The obtained precipitate was mixed with a 1% (w / v) aqueous citric acid solution (4 L; manufactured by Sigma-Aldrich, citric acid, first grade), stirred and then filtered to obtain a precipitate.
(6) The obtained precipitate was washed in the same manner as in Example 1 (4) and thereafter, and finally 400 g (average chitin concentration 6.8% by weight) of chitin slurry was obtained. The yield was 68%. At this time, the ion exchange water for washing used was about 100 L, and the final drainage conductivity was also 0.122 mS / m. Therefore, when washing with sodium bicarbonate and washing with citric acid were performed, washing was performed more efficiently than washing with distilled water alone.

Example 4
Regenerated chitin (1.000 g; degree of acetylation 97.5%, protein content 0.01% by weight) prepared in the same manner as in Production Example 1 was prepared in the same manner as in Example 1 (1) above. The solution was added to a calcium chloride dihydrate saturated methanol solution (137 mL), and dissolved by stirring for 7 hours with a stirrer while keeping the temperature in a water bath adjusted to 55 ° C.
The solution was filtered through a high speed funnel made of PP with a filter cloth (average pore size 130 μm, average mesh number 4 mesh / mm), and the time required for 100 mL and 125 mL filtration (filtration time) was measured.
As a result, 100 mL filtration required 397 seconds, and 125 mL filtration required 1680 seconds.
After filtration, the weight of the gel-like substance remaining on and in the filter cloth and its dry weight were measured. The amount of gel substance was 5.813 g, and the dry weight was 0.178 g.
The chitin solution was diluted and washed to obtain a chitin slurry.

Example 5
A chitin solution was obtained in the same manner as in Example 4 except that regenerated chitin having a high degree of acetylation (degree of acetylation 98.7%, protein content 0.01% by weight) was used. The amount of time and filtration residue was measured.
As a result, it took 300 seconds for 100 mL filtration, and 721 seconds for 125 mL filtration.
The amount of the residual gel substance was 5.674 g, and the dry weight was 0.111 g.
The chitin solution was diluted and washed to obtain a chitin slurry.

Example 6
The regenerated chitin used in Example 4 (acetylation degree 97.5%, protein content 0.01% by weight) was dried at 50 ° C. for 12 hours using a dryer (“EYELA VOS301SD” manufactured by Tokyo Science Instrument Co., Ltd.). After vacuum drying, a chitin solution was obtained in the same manner as in Example 4 above, and this was filtered, and the filtration time and the amount of filtration residue were measured.
As a result, 100 mL filtration required 406 seconds, and 125 mL filtration required 1200 seconds.
The amount of residual gel material could not be measured and the dry weight was 0.222 g.
The chitin solution was diluted and washed to obtain a chitin slurry.

Example 7
The regenerated chitin used in Example 4 (acetylation degree: 97.5%, protein content: 0.01% by weight) using a dryer (“EYELA VOS301SD” manufactured by Tokyo Science Instrument Co., Ltd.) at 100 ° C. for 12 hours Vacuum dried. Thereafter, in the same manner as in Example 4 above, a chitin solution was obtained and filtered, and the filtration time and the amount of filtration residue were measured.
As a result, it took 296 seconds for 100 mL filtration, and 945 seconds for 125 mL filtration.
The amount of residual gel substance was 6.971 g, and the dry weight was 0.337 g.
The chitin solution was diluted and washed to obtain a chitin slurry.

Example 8
(1) 50 ml of a chitin solution prepared in the same manner as in Example 4 above (chitin content 0.4 g, chitin acetylation degree 97.5%, chitin protein content 0.01 wt%) and sodium hydrogen carbonate (1 g; Sigma-Aldrich, sodium hydrogen carbonate, special grade) were mixed, stirred, and filtered to obtain a precipitate. The pH at that time was 4.5.
(2) The obtained precipitate was mixed with a 1% (w / v) aqueous citric acid solution (300 ml; manufactured by Sigma-Aldrich, citric acid, first grade), filtered after stirring.
(3) The obtained precipitate was repeatedly washed with ion-exchanged water and filtered to finally obtain 5.2 g of chitin slurry. The yield was 38%.

[Comparative Example 1]
“Chitin TCL” manufactured by Koyo Chemical Co., Ltd. was pulverized, passed through a 60 mesh / inch sieve, and classified. This chitin (1.000 g) was dissolved in a calcium chloride dihydrate saturated methanol solution (137 mL) in the same manner as in Example 4 and filtered.
In 100 mL filtration, there was a chitin solution that could not be filtered after 5400 seconds. Therefore, the filtration time was 5400 seconds or more.
The amount of residual gel material was 20.367 g, and the dry weight was 0.4734 g.

[Comparative Example 2]
To a calcium chloride dihydrate saturated methanol solution (2 L) prepared in the same manner as in Example 1 (1) above, “chitin TCL” (12.5 g) manufactured by Koyo Chemical Co., Ltd. was added. The mixture was put into a beaker and stirred using a stirrer (IKA Corporation, LABORTECHNIK RW20.n) while keeping the temperature in a water bath adjusted to 60 ° C. to dissolve the chitin.
When the above solution was filtered using a filter cloth (average pore diameter: about 0.2 mm, average mesh number: about 4 pieces / mm), the gel-like substance became filter cloth, that is, the filtration was stopped.
The filter cloth was squeezed out by hand and insoluble matter was filtered. At this time, most of the gel substance passed through the filter cloth and mixed into the chitin solution.
Washing was carried out in the same manner as in Example 1, and finally 128 g (average concentration 2.2 wt%) of chitin slurry was obtained. The yield was 22%.

Claims (7)

(A) A chitin having a protein content of less than 0.14% by weight and an acetylation degree of 96% or more is dissolved in a solvent containing methyl alcohol or ethyl alcohol and a calcium halide salt or a magnesium halide salt. Obtaining a chitin solution;
(B) filtering the chitin solution to remove insoluble matter;
(C) Dilute the filtered chitin solution with water, and if necessary, wash the produced chitin precipitate with water or dialyze it to replace calcium ions or magnesium ions and alcohol contained in the solution with water molecules. And a step of obtaining a hydrated chitin slurry. (D) The method according to claim 1, further comprising a step of adding carbon dioxide gas and / or sodium bicarbonate to the chitin solution or hydrated chitin slurry to separate the remaining alcohol. (E) The method according to claim 1 or 2, further comprising a step of adding an organic acid to the chitin solution or hydrated chitin slurry to separate the remaining calcium ions or magnesium ions. The method according to claim 3, wherein the organic acid is selected from the group consisting of citric acid, succinic acid, succinic anhydride, glutamic acid, ethylenediamine acid and asurbic acid. The method according to claim 1, wherein the chitin used in step (a) is dried at a temperature of 60 ° C or higher. The method according to claim 1, wherein the chitin used in step (a) is regenerated chitin. The method according to claim 1, wherein the chitin used in step (a) is de-O-acylated.