WO2015098634A1 - 水不溶性高分子化合物の分解物の連続的製造方法 - Google Patents
水不溶性高分子化合物の分解物の連続的製造方法 Download PDFInfo
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4741—Keratin; Cytokeratin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0036—Galactans; Derivatives thereof
- C08B37/0039—Agar; Agarose, i.e. D-galactose, 3,6-anhydro-D-galactose, methylated, sulfated, e.g. from the red algae Gelidium and Gracilaria; Agaropectin; Derivatives thereof, e.g. Sepharose, i.e. crosslinked agarose
Definitions
- the present invention relates to a method for continuously producing a degradation product of a water-insoluble polymer compound (protein or polysaccharide) by solubilizing and reducing the molecular weight of the water-insoluble polymer compound (protein or polysaccharide).
- jellyfish have become a serious social problem in large numbers around the coast of Japan, causing serious damage to the fishery and gathering at the intakes of factories and power plants, and cost for their removal and disposal. Therefore, it is desirable that jellyfish can be effectively used as a collagen raw material as described above. So far, jellyfish used as a collagen raw material are mainly small jellyfishes that are easily liquefied, and the present situation is that jellyfishes such as bizen jellyfish and large amounts of water-insoluble proteins are not effectively used.
- solubilization and low molecular weight formation of water-insoluble proteins are performed by heat treatment, acid or alkali treatment, proteolytic enzyme (protease) treatment, and the like.
- heat treatment has a problem that it undergoes heat denaturation depending on the type of protein.
- the acid or alkali treatment may destroy the amino acid, and the waste liquid treatment after the treatment is costly and labor intensive.
- Proteolytic enzyme treatment requires setting and control of temperature and pH in order to match the optimum conditions of the enzyme used, and if the enzyme remains in the treated product, it must be deactivated or removed.
- Patent Document 3 a method of hydrolyzing protein using a solid acid catalyst is known (Patent Document 3), and is also used for the production of jellyfish collagen peptide (Patent Document 4).
- Patent Document 4 a method of hydrolyzing protein using a solid acid catalyst.
- lignocellulose decomposition techniques include acid saccharification and enzymatic saccharification, but acid saccharification using sulfuric acid has the advantage of fast reaction, but overdegradation of the product (monosaccharide) becomes a problem. There is a problem of environmental burden in the waste liquid treatment of acid.
- cellulose is a strong polymer that is insoluble in water by hydrogen bonding between polymers in which ⁇ -glucose molecules are polymerized by 1,4-glucoside bonds. Since it has a simple crystal structure, there is a problem that the contact area with the cellulase enzyme is small and the reaction is slow.
- an object of the present invention is to produce a new functional material by easily solubilizing and reducing the molecular weight of a water-insoluble polymer compound such as a water-insoluble protein or a water-insoluble polysaccharide by an easy operation. .
- the present inventors have processed water-insoluble protein with a solid acid catalyst, and surprisingly, the water-insoluble protein degradation product is mainly non-solid acid catalyst. It was found to be obtained in the adsorption fraction. Then, after repeating the operation of recovering the non-adsorbed fraction, the water-insoluble proteolysate was successfully produced in high yield continuously by combining the adsorbed fraction eluted from the solid acid catalyst.
- the present invention has been completed.
- the present invention includes the following inventions.
- a method for producing a degradation product of a water-insoluble polymer compound comprising the following steps: (A) a step of bringing a solid acid catalyst into contact with a water-insoluble polymer compound, followed by heat treatment, and collecting a supernatant; (B) Subsequent to step (A), an aqueous medium is added to the solid acid catalyst, and the mixture is stirred and heated, and then the supernatant is recovered.
- step (C) Following the step (B), the step of washing the solid acid catalyst with an aqueous medium and recovering the washing solution, (D) A step of combining the supernatant liquid collected in step (A), the supernatant liquid collected in step (B), and the washing liquid in step (C) to obtain a fraction that is not adsorbed on the solid acid catalyst, (E) Following the step (D), a step of eluting the components adsorbed from the solid acid catalyst and collecting the eluate to obtain a fraction adsorbed on the solid acid catalyst.
- the water-insoluble polymer compound is a water-insoluble protein or a water-insoluble polysaccharide.
- step (A) and (B) are repeated a plurality of times until the yield of the decomposition product of the water-insoluble polymer compound in the non-adsorbed fraction is 50% or more.
- Any one of the methods Any one of the methods.
- the solid acid catalyst is at least one selected from the group consisting of a cation exchanger, zeolite, and diatomaceous earth.
- a method for producing a decomposition product of a water-insoluble polymer compound is provided.
- the method of the present invention it is possible to continuously produce a degradation product of a water-insoluble polymer compound from a material that could not be effectively used until now at high efficiency and at low cost.
- the method of the present invention can perform a reliable hydrolysis treatment without unnecessary modification of the polymer compound. There is no need for complicated operations or special control.
- FIG. 1 shows an embodiment of a method for recovering a solid acid catalyst non-adsorbed fraction (supernatant) in a continuous production method of a water-insoluble protein degradation product which is an embodiment of the present invention.
- Transition of recovery amount of water-insoluble protein degradation product (bizen jellyfish umbrella collagen peptide) in non-adsorbed fraction of solid acid catalyst (Fig. 3a: reaction time 24 hours, Fig. 3b: reaction time 48 hours) and comparison of absorption amount by reaction time FIG. 3c is shown.
- FIG. 3a reaction time 24 hours
- Fig. 3b reaction time 48 hours
- FIG. 4a shows the molecular weight distribution of the water-insoluble proteolysate (bizen jellyfish umbrella collagen peptide) in the solid acid catalyst non-adsorbed fraction (reaction time 24 hours, upper figure: first to sixth recovery liquids, lower figure: Recovery liquids 7 to 12 times, pullulan standard molecular weight M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- 4b shows the molecular weight distribution of the water-insoluble protein degradation product (bizen jellyfish umbrella collagen peptide) in the solid acid-catalyzed non-adsorbed fraction (reaction time 48 hours, pullulan standard product molecular weights M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- the molecular weight distribution of the water-insoluble protein degradation product (bizen jellyfish umbrella collagen peptide) in the solid acid catalyst adsorption fraction is shown (upper figure: reaction time 24 hours, lower figure: reaction time 48 hours, pullulan standard product molecular weight M1: 112kDa , M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- the transition of the recovery amount of the water-insoluble protein degradation product (sericin peptide) in the solid acid catalyst non-adsorbed fraction is shown.
- control substance M1 5.9kDa (Showa Denko Shodex STANDARD P-82, M2: 3 sugar (raffinose), M3: Disaccharide (sucrose), M4: Monosaccharide (glucose)).
- FIG. 1 shows an outline of the manufacturing process.
- A a step of bringing a solid acid catalyst into contact with a water-insoluble polymer compound, followed by heat treatment, and collecting a supernatant;
- B Subsequent to step (A), an aqueous medium is added to the solid acid catalyst, and the mixture is stirred and heated, and then the supernatant is recovered.
- step (C) Following the step (B), the step of washing the solid acid catalyst with an aqueous medium and recovering the washing solution, (D) A step of combining the supernatant liquid collected in step (A), the supernatant liquid collected in step (B), and the washing liquid in step (C) to obtain a fraction that is not adsorbed on the solid acid catalyst, (E) Following the step (D), a step of eluting the components adsorbed from the solid acid catalyst and collecting the eluate to obtain a fraction adsorbed on the solid acid catalyst.
- the “water-insoluble polymer compound” used in the method of the present invention is preferably a water-insoluble protein or a water-insoluble polysaccharide.
- the “decomposition product of the water-insoluble polymer compound” may be any product in which the water-insoluble polymer compound is solubilized and decomposed, and the average molecular weight and molecular weight distribution are not limited.
- Process (A) In the step (A), after bringing the solid acid catalyst into contact with the water-insoluble polymer compound, heat treatment is performed, and the supernatant is recovered.
- water-insoluble means a state in which it is not solubilized in water.
- the water-insoluble polymer compound includes a water-insoluble protein or a water-insoluble polysaccharide, and may be animal or vegetable, and the type is not particularly limited.
- water-insoluble proteins include collagen, sericin, fibroin, keratin, casein, albumin, globulin, elastin, myosin, actin, whey protein, soy protein, wheat protein, sesame protein, egg protein and the like.
- collagen of jellyfish is preferable, and jellyfish is not limited, but belongs to Scyphozoa, Rhizostomeae. Sakasa jellyfish.
- water-insoluble polysaccharide examples include cellulose, hemicellulose, lignocellulose, inulin, pectin, glucan, carrageenan, agarose, chitin and chitosan.
- the water-insoluble polymer compound raw material is coarsely pulverized using a cutting machine, mincing machine, cutter, etc. as a pretreatment, and further pulverized in order to promote solubilization and molecular weight reduction by contact with the fixed acid catalyst It is preferable to keep it.
- the powderization can be performed by a machine usually used by those skilled in the art, such as a hammer mill, a bead mill, a roller mill, a pin mill, and a blender.
- the water-insoluble polymer compound subjected to the pretreatment as described above may be wetted with an aqueous medium before contact with the solid acid catalyst. Wetting is preferably performed for about 1 to 36 hours, depending on the type of water-insoluble polymer compound.
- the solid acid catalyst used in the present invention is preferably at least one selected from the group consisting of a cation exchanger, zeolite and diatomaceous earth, and more preferably a cation exchanger. These solid acid catalysts can be used alone or in combination of two or more.
- the cation exchanger is preferably a resin having at least one of a sulfone group and a carboxyl group, and the sulfone group and the carboxyl group may be a sulfopropyl group and a carboxymethyl group, respectively.
- the counter ion of the cation exchanger is other than proton, it is preferable to use it after substitution with the proton type.
- the sulfonic acid type cation exchanger is an ion exchanger having a cation exchange ability containing a sulfonic acid group (—SO 3 H), and as a preferred form, a cation exchange resin containing a sulfonic acid group and a sulfonic acid group
- cation exchange membranes containing Preferred cation exchange resins include those containing a hydrophilic vinyl polymer as a base material such as TOYOPEARL SP-650C and SP-550C manufactured by Tosoh Corporation, and perfluorosulfonic acid such as Nafion (registered trademark).
- the polytetrafluoroethylene copolymer containing can be mentioned.
- any zeolite that is generally used as a zeolite catalyst can be used, and is not particularly limited, and examples thereof include Zeorum (registered trademark) manufactured by Tosoh Corporation.
- Any diatomaceous earth can be used as long as it can function as an acid catalyst, and is not particularly limited. Examples thereof include diatomaceous earth (granular) manufactured by Wako Pure Chemical Industries, Ltd.
- the shape of the solid acid catalyst used in the present invention may be either granular or powdery. Further, those having an average particle diameter of about 2 ⁇ m to 2 mm and an ion exchange capacity of about 0.01 to 1 eq / L can be preferably used.
- the solid acid catalyst is preferably a porous body.
- the above-mentioned TOYOPEARL SP-650C and SP-550C manufactured by Tosoh Corporation are porous bodies in which a sulfonic acid group is introduced as an ion exchange group into a filler for gel filtration chromatography.
- the pore size of the porous body varies depending on the molecular weight of the target water-insoluble polymer compound, and is, for example, 0.01 to 0.75 ⁇ m, preferably 0.05 to 0.6 ⁇ m. .
- the amount of the aqueous medium at the time of contacting the water-insoluble polymer compound and the solid acid catalyst is 1 to 50 times by mass ratio with respect to the solid acid catalyst, preferably including the aqueous medium used for wetting the water-insoluble polymer compound, preferably 5 to 15 times.
- the decomposition product of the water-insoluble polymer compound is mainly recovered in the non-adsorbed fraction (supernatant liquid) of the solid acid catalyst, if the amount of the aqueous medium is below the above range, a centrifugal separation operation is required, which is preferable. Absent.
- the aqueous medium used for wetting the water-insoluble polymer compound and contacting the water-insoluble polymer compound with the solid acid catalyst is not particularly limited as long as it does not prevent the reaction between the water-insoluble polymer compound and the solid acid catalyst.
- water ion exchange water, purified water, RO water, tap water, well water, etc.
- phosphate carbonate, acetate, borate, citrate, tris, etc.
- Inorganic salt aqueous solutions such as sodium chloride, potassium chloride, and calcium chloride.
- the salt concentration is preferably a low concentration of about 1 mM to 0.1 M.
- the amount of the water-insoluble polymer compound can be appropriately determined according to the catalytic ability of the solid acid catalyst.
- the mass ratio of the solid acid catalyst is 0.01 to 0.5 times, preferably 0.05 to 0. .2 times.
- the heat treatment after the contact between the solid acid catalyst and the water-insoluble polymer compound is essential for allowing the water-insoluble polymer compound to proceed solubilizing and reducing the molecular weight satisfactorily.
- the heating temperature is 40 to 160 ° C., preferably 60 to 120 ° C., more preferably 80 to 100 ° C.
- the heating time depends on the heating temperature, but is 0.1 to 168 hours, preferably 10 to 72 hours, More preferably, it is 24 to 48 hours.
- the water-insoluble polymer compound is a water-insoluble protein, it is important to reduce the molecular weight only to the decomposition to the peptide, but when treated at a high temperature for a long time, the water-insoluble protein is hydrolyzed to the amino acid. Since there exists a possibility that it may decompose
- the contact between the water-insoluble polymer compound and the solid acid catalyst is preferably carried out by a batch method.
- the batch method is used, the solid acid catalyst and the water-insoluble polymer compound in the container are mixed well and then heated. Heating is preferably performed after sealing the container. The heating conditions are as described above.
- One or more containers may be used in the batch method.
- the supernatant liquid is recovered.
- the supernatant liquid can be collected by methods such as sedimentation separation, flotation separation, filtration, membrane separation, and centrifugation, but sedimentation separation is simple and preferable.
- An example of a method for collecting the supernatant is shown in FIG.
- the solid acid catalyst is heavier than the aqueous medium, so if it is left standing, it usually settles in about 15 minutes, but in actual operation, a drain valve is installed slightly above the bottom of the tank (slightly above the position of the precipitated solid acid catalyst). Then, the supernatant can be recovered by draining from there.
- a supernatant liquid containing a decomposition product of a water-insoluble polymer compound is allowed to pass through, but a filter having a pore size that does not allow a solid acid catalyst to pass through is installed at the bottom of the tank, and the supernatant liquid can be recovered by draining from there. .
- the series of operations in this step are performed a plurality of times.
- the number of times can be appropriately changed according to the type of the water-insoluble polymer compound and the target yield of this step, and can be exemplified by about 4 to 5 times.
- the number of repetitions is such that the yield of the decomposition product of the water-insoluble polymer compound in the non-adsorbed fraction obtained by combining the supernatant of this step, the supernatant of the next step (B) and the washing solution of the next step (C) is 50 % Or more, preferably 65% or more, more preferably 70% or more, and most preferably 80% or more.
- the total amount of the water-insoluble polymer compound to be treated is equal to each other from the viewpoint of solubilization / lowering efficiency of the water-insoluble polymer compound by the solid acid catalyst. It is preferable to carry out separately.
- Process (B) In step (B), following step (A), an aqueous medium is added to the solid acid catalyst, and the mixture is stirred and heated, and then the supernatant is recovered.
- the water-insoluble polymer compound that has not been solubilized in step (A) and is attached to the surface of the solid acid catalyst can be recovered by solubilization and low molecular weight.
- the heat treatment in this step may be performed according to the heat treatment conditions (temperature and time) at the time of contacting the solid acid catalyst and the water-insoluble polymer compound. Moreover, it is preferable to perform the collection operation in this step a plurality of times.
- the number of times can be appropriately changed according to the type of the water-insoluble polymer compound, the yield of the decomposition product of the water-insoluble polymer compound in the step (A), and the final target yield, but about 3 to 6 times are exemplified. it can.
- the number of repetitions is such that the yield of the decomposition product of the water-insoluble polymer compound in the non-adsorbed fraction obtained by combining the supernatant of step (A), the supernatant of step (B) and the washing solution of the next step (C) is 50 % Or more, preferably 65% or more, more preferably 70% or more, and most preferably 80% or more.
- the supernatant liquid may be collected in the same manner as in step (A).
- Process (C) In the step (C), following the step (B), the solid acid catalyst is washed with an aqueous medium, and the washing liquid is recovered. In this step, heat treatment is not performed, and the solid acid catalyst may be washed once with an aqueous medium.
- step (D) In step (D), the supernatant liquid recovered in step (A), the supernatant liquid recovered in step (B), and the cleaning liquid recovered in step (C) are combined to obtain a non-adsorbed fraction on the solid acid catalyst. .
- a water-insoluble polymer compound that is solubilized and poorly differentiated by reaction with the solid acid catalyst and is non-adsorbed on the solid acid catalyst can be obtained in high yield.
- step (E) In step (E), following step (D), the components adsorbed from the solid acid catalyst are eluted, and the eluate is recovered to obtain a fraction adsorbed on the solid acid catalyst.
- aqueous solution containing a salt such as sodium chloride at a high concentration (0.1 M or more), an acid such as hydrochloric acid or trifluoroacetic acid, a base such as triethylamine, or the like can be used.
- a decomposition product of the water-insoluble polymer compound can be obtained in a high yield from the above steps (A) to (D), but this step further allows the reaction with the solid acid catalyst.
- the degradation products of the water-insoluble polymer compound solubilized and reduced in molecular weight a degradation product of the water-insoluble polymer compound adsorbed on the solid acid catalyst can be obtained, and the yield can be further improved.
- the non-adsorbed fraction obtained in the step (D) and the adsorbed fraction obtained in the step (E) may be dried to obtain a final product, or may be mixed and dried to obtain a final product.
- the drying method is not particularly limited, and examples thereof include natural drying, freeze drying, blast drying, hot air drying, vacuum drying, and drying by microwave irradiation.
- RO water was added to the remaining cation exchange resin, made up to 100 ml, and stirred well. After reacting at 80 ° C. for 24 hours, the supernatant was recovered. This operation was further repeated 6 times (collection was completed 6 to 11 times). The remaining cation exchange resin was washed with 30 ml of RO water. The collected supernatant and washings were mixed to obtain a collagen peptide solution having a molecular weight (Mp) of 12 kDa or less that was not adsorbed on the cation exchange resin.
- Mp molecular weight
- FIG. 3a shows the transition of the amount of collagen peptide (non-adsorbed peptide) in the collected supernatant (reaction time: 24 hours).
- RO water was added to the remaining cation exchange resin, made up to 100 ml, and stirred well. After reacting at 80 ° C. for 48 hours, the supernatant was recovered. This operation was repeated two more times (collection completed 6-8 times). The remaining cation exchange resin was washed with 80 ml of RO water. The collected supernatant and washings were mixed to obtain a collagen peptide solution having a molecular weight (Mp) of 12 kDa or less that was not adsorbed on the cation exchange resin.
- Mp molecular weight
- FIG. 3b shows the transition of the amount of collagen peptide (non-adsorbed peptide) in the collected supernatant (reaction time 48 hours).
- (3-2) Measurement of peptide amount The amount of collagen peptide obtained in (2) was measured using a BCA protein assay kit manufactured by PIERCE. BCA protein assay reagent A and BCA protein assay reagent B were mixed at a ratio of 50: 1 and used as BCA reagent. 100 ⁇ l of the sample and 2 ml of BCA reagent were mixed and incubated at 37 ° C. for 15 minutes, and then the absorbance was measured at 562 nm. For the absorbance measurement, a digital colorimeter mini photo 10 manufactured by Sangen Co., Ltd. was used. The amount of peptide was calculated from the standard curve of the ragen peptide prepared in (3-1).
- Table 1 and Table 2 show the measurement results in the 24-hour reaction.
- the amount of collagen peptide (non-adsorbed peptide) in the collected supernatant was 3.65 g, and the yield was 73%. Further, the amount of collagen peptide (adsorbed peptide) in the collected eluate was 0.86 g, and the yield was 17%.
- Table 3 and Table 4 show the measurement results in the 48 hour reaction.
- the amount of collagen peptide (non-adsorbed peptide) in the collected supernatant was 3.58 g, and the yield was 72%.
- the amount of collagen peptide (adsorbed peptide) in the collected eluate was 0.77 g, and the yield was 15%.
- reaction time of 24 hours and 48 hours there is no significant difference in the yield between the reaction time of 24 hours and 48 hours, but the reaction time of 48 hours can be said to be efficient because the number of times of recovery is small.
- Fig. 4 shows the molecular weight distribution of collagen peptide (non-adsorbed peptide) in the supernatant (pullulan standard molecular weight ⁇ M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- a sharp peak was obtained at 5.9 kDa or less in terms of pullulan molecular weight in the first and second recovery, but thereafter a broad peak was obtained on the polymer side (FIG. 4a).
- the peak slightly shifted from the polymer side to the low molecule side (FIG. 4b).
- FIG. 5 shows the molecular weight distribution of collagen peptides (adsorbed peptides) in the eluate (pullulan standard molecular weights M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- the adsorbed peptide had a lower molecular weight than the non-adsorbed peptide, and the peak top was 5.9 kDa or less.
- the molecular weight distribution was almost the same for the 24-hour reaction and the 48-hour reaction without being affected by the reaction time.
- Example 2 Sericin Solubilization / Low Molecularization (1) Solubilization / Low molecularization of sericin by solid acid catalyst After wetting 1g of sericin powder (manufactured by Takahara) with 90ml of RO water overnight, mix with 10ml of cation exchange resin (TOYOPEARL SP-550C). Stir well. After solubilization by reacting at 80 ° C. for 24 hours, cooling was performed, and a supernatant containing sericin peptide was recovered. Next, the sericin powder was wetted overnight in the same manner as described above, then added to the cation exchange resin after collecting the supernatant, diluted to 100 ml with RO water, and stirred well.
- cation exchange resin TOYOPEARL SP-550C
- the mixture was reacted at 80 ° C. for 24 hours, and the supernatant was collected again. This operation was repeated three more times (collection completed 1 to 5 times). A total of 5 g of sericin powder was used in an equal amount each time.
- RO water was added to the remaining cation exchange resin, made up to 100 ml, and stirred well. After reacting at 80 ° C. for 24 hours, the supernatant was recovered. This operation was further repeated 6 times (collection was completed 6 to 11 times).
- the remaining cation exchange resin was washed with 60 ml of RO water. The collected supernatant and washing solution were mixed to obtain a sericin peptide solution having a molecular weight (Mp) of 5.9 kDa or less that was not adsorbed on the cation exchange resin.
- Mp molecular weight
- FIG. 6 shows the transition of the amount of sericin peptide (non-adsorbed peptide) in the collected supernatant.
- Fig. 7 shows the molecular weight distribution of sericin peptide (non-adsorbed peptide) in the supernatant (pullulan standard molecular weight M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- pulseulan standard molecular weight M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa In the first and second recovery, a sharp peak was obtained at 5.9 kDa or less in terms of pullulan molecular weight, but thereafter a broad peak was obtained on the polymer side.
- FIG. 8 shows the molecular weight distribution of sericin peptide (adsorbed peptide) in the eluate (pullulan standard product molecular weights M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- the peak top of the adsorbed peptide was not much different from the first and second collections of the non-adsorbed peptide and the 7th to 12th.
- the keratin powder was wetted overnight as described above, and then the supernatant was added to the recovered cation exchange resin, diluted to 100 ml with RO water, and stirred well. The reaction was carried out at 100 ° C. for 48 hours, and the supernatant was collected again. This operation was repeated three more times (collection completed 1 to 5 times). A total of 5 g of keratin powder was used each time. RO water was added to the remaining cation exchange resin, made up to 100 ml, and stirred well. After reacting at 100 ° C. for 48 hours, the supernatant was recovered. This operation was repeated two more times (collection completed 6-8 times). The remaining cation exchange resin was washed with 80 ml of RO water. The collected supernatant and washings were mixed to obtain a keratin peptide solution having a molecular weight (Mp) of 5.9 kDa or less that was not adsorbed on the cation exchange resin.
- Mp molecular weight
- the amount of keratin peptide (non-adsorbed peptide) in the collected supernatant was 2.65 g, and the yield was 53%.
- the amount of keratin peptide (adsorbed peptide) in the collected eluate was 0.20 g, and the yield was 4%.
- FIG. 9 shows the molecular weight distribution of the keratin peptide (non-adsorbed peptide) in the supernatant (pullulan standard product molecular weight M1: 112 kDa, M2: 47.3 kDa, M3: 22.8 kDa, M4: 11.8 kDa, M5: 5.9 kDa).
- a sharp peak was obtained at a pullulan molecular weight conversion of 5.9 kDa or less.
- FIG. 10 shows the molecular weight distribution of keratin peptides (adsorbed peptides) in the eluate.
- the peak top of the adsorbed peptide was not much different from the non-adsorbed peptide.
- Example 4 Solubilization and low molecular weight of agar (1) Solubilization and low molecular weight of agar with solid acid catalyst 1 g of agar powder (Kanto Chemical) was suspended in 90 ml of RO water and cation exchange resin (TOYOPEARL) SP-550C) was mixed with 10 ml and stirred well. After solubilization by reaction at 100 ° C. for 24 hours, cooling was performed, and a supernatant containing agar oligosaccharides was collected. Next, the agar powder was wetted overnight as described above, and then the supernatant was added to the recovered cation exchange resin, made up to 100 ml with RO water, and stirred well. The reaction was carried out at 100 ° C. for 24 hours, and the supernatant was collected again. This operation was repeated three more times (collection completed 1 to 5 times). A total of 5 g of each agar powder was used.
- TOYOPEARL cation exchange resin
- RO water was added to the remaining cation exchange resin, made up to 100 ml, and stirred well. After reacting at 100 ° C. for 24 hours, the supernatant was recovered. This operation was repeated two more times (collection completed 6-8 times). The remaining cation exchange resin was washed with 80 ml of RO water. The collected supernatant and washings were mixed to obtain an agar oligosaccharide solution that was not adsorbed on the cation exchange resin.
- FIG. 11 shows the transition of the amount of agar oligosaccharide (non-adsorbed oligosaccharide) in the collected supernatant.
- Table 7 shows the measurement results.
- the amount of agar oligosaccharide (non-adsorbed oligosaccharide) in the collected supernatant was 4.15 g, and the yield was 83%.
- FIG. 12 shows the molecular weight distribution of the agar oligosaccharide (non-adsorbed oligosaccharide) in the supernatant (control substance M1: 5.9 kDa (M1: Shodex STANDARD P-82, M2: trisaccharide (raffinose), (M3: disaccharide (sucrose), M4: 1 sugar (glucose))
- M1 Shodex STANDARD P-82
- M2 trisaccharide (raffinose)
- M3 disaccharide
- sucrose disaccharide
- M4 1 sugar (glucose)
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Abstract
Description
(1)以下の工程を含む水不溶性高分子化合物の分解物の製造方法:
(A) 水不溶性高分子化合物に固体酸触媒を接触させた後、加熱処理を行い、上澄み液を回収する工程、
(B)工程(A)に続いて、該固体酸触媒に水性媒体を加えて攪拌し、加熱処理した後、上澄み液を回収する工程、
(C)工程(B)に続いて、該固体酸触媒を水性媒体にて洗浄し、洗浄液を回収する工程、
(D)工程(A)で回収した上澄み液と、工程(B)で回収した上澄み液と、工程(C)の洗浄液を合わせて固体酸触媒に非吸着の画分を得る工程、
(E)工程(D)に続いて、該固体酸触媒から吸着した成分を溶出し、溶出液を回収して固体酸触媒に吸着した画分を得る工程。
(2)水不溶性高分子化合物が、水不溶性タンパク質または水不溶性多糖類である、(1)に記載の方法。
(3)水不溶性高分子化合物を固体酸触媒に接触させる前に水性媒体に浸潤させる工程をさらに含む、(1)または(2)に記載の方法。
(4)水不溶性高分子化合物の量が、固体酸触媒に対して質量比で0.01~0.5倍である、(1)~(3)のいずれかに記載の方法。
(5)前記工程(A)において、水不溶性高分子化合物と固体酸触媒の接触時の水性媒体の量を固体酸触媒に対して質量比で1~50倍となるように調整する、(1)~(4)のいずれかに記載の方法。
(6)前記工程(A)および(B)の加熱処理を、40~160℃で0.1~168時間行う、(1)~(5)のいずれかに記載の方法。
(7)前記工程(A)および(B)の操作を、非吸着画分における水不溶性高分子化合物の分解物の収率が50%以上になるまでそれぞれ複数回繰り返す、(1)~(6)のいずれかに記載の方法。
(8)固体酸触媒が、陽イオン交換体、ゼオライト、および珪藻土から成る群より選ばれる少なくとも1種である、(1)~(7)のいずれかに記載の方法。
本発明は、水不溶性高分子化合物の分解物の製造方法であって、以下の工程を包含する。図1にその製造工程の概略を示す。
(A) 水不溶性高分子化合物に固体酸触媒を接触させた後、加熱処理を行い、上澄み液を回収する工程、
(B)工程(A)に続いて、該固体酸触媒に水性媒体を加えて攪拌し、加熱処理した後、上澄み液を回収する工程、
(C)工程(B)に続いて、該固体酸触媒を水性媒体にて洗浄し、洗浄液を回収する工程、
(D)工程(A)で回収した上澄み液と、工程(B)で回収した上澄み液と、工程(C)の洗浄液を合わせて固体酸触媒に非吸着の画分を得る工程、
(E)工程(D)に続いて、該固体酸触媒から吸着した成分を溶出し、溶出液を回収して固体酸触媒に吸着した画分を得る工程。
工程(A)では、水不溶性高分子化合物に固体酸触媒を接触させた後、加熱処理を行い、上澄み液を回収する。本発明において「水不溶性」とは水に可溶化していない状態をいう。
工程(B)では、工程(A)に続いて、該固体酸触媒に水性媒体を加えて攪拌し、加熱処理した後、上澄み液を回収する。本工程により、工程(A)で可溶化されずに固体酸触媒表面に付着している水不溶性高分子化合物を可溶化および低分子化して回収することができる。本工程の加熱処理は、前記の固体酸触媒と水不溶性高分子化合物の接触時の加熱処理条件(温度および時間)に従って行えばよい。また、本工程の回収操作も複数回行うことが好ましい。回数については水不溶性高分子化合物の種類、工程(A)における水不溶性高分子化合物の分解物の収率、および最終的な目標収率に応じて適宜変更できるが、3~6回程度が例示できる。繰り返し回数は、工程(A)の上澄み液と工程(B)の上澄み液と次の工程(C)の洗浄液を合わせた非吸着画分における水不溶性高分子化合物の分解物の収率が、50%以上、好ましくは65%以上、より好ましくは70%以上、最も好ましくは80%以上となるように決定すればよい。上澄み液の回収は、工程(A)と同様に行えばよい。
工程(C)では、工程(B)に続いて、固体酸触媒を水性媒体にて洗浄し、洗浄液を回収する。本工程では加熱処理は行わず、固体酸触媒を水性媒体にて1回洗浄すればよい。
工程(D)では、工程(A)で回収した上澄み液と、工程(B)で回収した上澄み液と、工程(C)で回収した洗浄液を合わせて固体酸触媒に非吸着の画分を得る。本工程により、固体酸触媒との反応で可溶化・低分化し、かつ固体酸触媒に非吸着の水不溶性高分子化合物を高収率で得ることができる。
工程(E)では、工程(D)に続いて、固体酸触媒から吸着した成分を溶出し、溶出液を回収して固体酸触媒に吸着した画分を得る。溶出には、塩化ナトリウムなどの塩を高濃度(0.1M以上)に含む水溶液または塩酸やトリフルオロ酢酸などの酸、トリエチルアミンなどの塩基などを用いることができる。本発明の方法によれば、上記工程(A)から(D)までで水不溶性高分子化合物の分解物を高収率で得ることができるが、さらに本工程により、固体酸触媒との反応で可溶化・低分子化した水不溶性高分子化合物の分解物のうち、固体酸触媒に吸着した水不溶性高分子化合物の分解物をも得ることができ、さらに収率を向上させることができる。
(1) ビゼンクラゲ傘部不溶性コラーゲンの調製
福岡県柳川市、有明海にて採取したビゼンクラゲを傘部と口腕部に分離した。傘部の内側にある茶色い皮を剥いだ後、水道水で洗浄し、さらに微酸性電解水、オゾン水で洗浄殺菌した。個体を1kg切り取り、1mm~1cmの角状断片に破砕した。ガーゼで濾過をし、不溶性コラーゲンを含む固体を得た。得られた固体を9倍量のRO水で懸濁し、濾過により固体を回収した。この操作をさらに2回繰り返し、洗浄・脱塩をした。回収した固体は凍結乾燥を行い、乾燥後にブレンダーにて粉末化し、不溶性コラーゲン粉末を得た。
(2-1)反応時間24時間
(1)で調製した不溶性コラーゲン粉末1gを90mlのRO水で1晩湿潤させた後、陽イオン交換樹脂(TOYOPEARL SP-550C)10mlと混合し、よく撹拌した。80℃、24時間反応させ可溶化した後、冷却を行い、コラーゲンペプチドを含む上澄み液を回収した。次に、(1)で調製したビゼンクラゲ傘部の不溶性コラーゲン粉末を上記と同様にして1晩湿潤させた後、前記の上澄み液回収後の陽イオン交換樹脂に添加し、RO水で100mlにメスアップした後、よく撹拌した。80℃、24時間反応させ、再び上澄み液を回収した。この操作をさらに3回繰り返した(回収1~5回完了)。ビゼンクラゲ傘部の不溶性コラーゲン粉末は各回等量ずつ合計5g使用した。
(1)で調製した不溶性コラーゲン粉末1gを90mlのRO水で1晩湿潤させた後、陽イオン交換樹脂(TOYOPEARL SP-550C)10mlと混合し、よく撹拌した。80℃、48時間反応させ可溶化した後、冷却を行い、コラーゲンペプチドを含む上澄み液を回収した。次に、(1)で調製したビゼンクラゲ傘部の不溶性コラーゲン粉末を上記と同様にして1晩湿潤させた後、前記の上澄み液回収後の陽イオン交換樹脂に添加し、RO水で100mlにメスアップした後、よく撹拌した。80℃、48時間反応させ、再び上澄み液を回収した。この操作をさらに3回繰り返した(回収1~5回完了)。ビゼンクラゲ傘部の不溶性コラーゲン粉末は各回等量ずつ合計5g使用した。
(3-1)コラーゲンペプチド標準サンプルの調製
ビゼンクラゲ傘部の不溶性コラーゲン粉末1gを100mlのRO水で1晩湿潤させた後、陽イオン交換樹脂(TOYOPEARL SP-550C)100gと混合し、よく撹拌した。80℃、24時間反応させ可溶化した後、冷却を行い、RO水500mlで洗浄した。洗浄後の樹脂を5%トリエチルアミン/10%アセトニトリル100mlで3回抽出した。得られた抽出液を混合した後、ロータリーエバポレーターで濃縮乾固した。乾固した抽出物をRO水で溶解し、凍結乾燥した。得られた乾燥物を3.00、1.50、0.75、0.375、0.188mg/mlとなるように調整し、コラーゲンペプチド定量用の標準検量線に用いた。
(2)で得られたコラーゲンペプチド量の測定は、PIERCE社製 BCA protein assay kitを使用した。BCA protein assay reagent AとBCA protein assay reagent Bを50:1で混合し、これをBCA reagentとした。サンプル100μlとBCA reagent 2mlを混合し、37℃で15分間インキュベートした後、562 nmで吸光度を測定した。吸光度測定には三紳社製デジタル比色計mini photo 10を使用した。(3-1)で作成したラーゲンペプチド標準検量線からペプチド量を算出した。
(2)で得られたコラーゲンペプチドの分子量分布はゲル濾過クロマトグラフィーを用いて測定した。測定条件を以下に示す。
a) HPLC装置:ポンプGL Sciences社製 GL-7410、オートサンプラーGL Sciences 社製GL-7420、カラム恒温槽GL Sciences社製CO631C、UV検出器 島津社製SPD-10AVと日立社製L-4200、RI検出器GL Sciences社製GL-7454、真空デガッサーGastorr社製AG-14を用いた。
b) カラム:東ソー社製TSKgel G3000SWXL(7.8×300mm, 5μm)
c) 移動相:50mMリン酸ナトリウム緩衝液/50mM塩化ナトリウム(pH 7.0)
d) 流速:0.5ml/min
e) 温度:20℃
f) 測定波長:215nm、280nm
g) 分子量測定用標準品:昭和電工社製Shodex STANDARD P-82を0.1%となるように調製し、分子量測定用標準液とした。
(1) 固体酸触媒によるセリシンの可溶化・低分子化
セリシン粉末(高原社製)1gを90mlのRO水で1晩湿潤させた後、陽イオン交換樹脂(TOYOPEARL SP-550C)10mlと混合し、よく撹拌した。80℃、24時間反応させ可溶化した後、冷却を行い、セリシンペプチドを含む上澄み液を回収した。次にセリシン粉末を上記と同様にして1晩湿潤させた後、上記の上澄み液回収後の陽イオン交換樹脂に添加し、RO水で100mlにメスアップした後、よく撹拌した。80℃、24時間反応させ、再び上澄み液を回収した。この操作をさらに3回繰り返した(回収1~5回完了)。セリシン粉末は各回等量ずつ合計5g使用した。残った陽イオン交換樹脂にRO水を加え、100mlにメスアップし、よく撹拌した。80℃、24時間反応させた後、上澄み液を回収した。この操作をさらに6回繰り返した(回収6~11回完了)。残った陽イオン交換樹脂を60mlのRO水で洗浄した。回収した上澄み液と洗浄液を混合し、陽イオン交換樹脂に吸着していない分子量(Mp)5.9kDa以下のセリシンペプチド液を得た。
(2-1)セリシンペプチド標準サンプルの調製
(1)で得られた陽イオン交換樹脂に非吸着のセリシンペプチド液を凍結乾燥した。得られた乾燥物を0.75、0.375、0.188、0.094mg/mlとなるように調整し、セリシンペプチド定量用の標準検量線に用いた。
(1)で得られたセリシンペプチド量の測定は、上記のセリシンペプチド定量用の標準検量線を用いる以外は、実施例1の(3-2)と同様にして行った。
(1)で得られたセリシンペプチドの分子量分布は、実施例1と同様の測定条件に従い、ゲル濾過クロマトグラフィーを用いて測定した。
(1)固体酸触媒によるケラチンの可溶化・低分子化
ケラチン粉末(ナカライテスク)1gを90mlのRO水で1晩湿潤させた後、陽イオン交換樹脂(TOYOPEARL SP-550C)10mlと混合し、よく撹拌した。100℃、48時間反応させ可溶化した後、冷却を行い、ケラチンペプチドを含む上澄み液を回収した。次にケラチン粉末を上記と同様にして1晩湿潤させた後、上記の上澄み液を回収後の陽イオン交換樹脂に添加し、RO水で100mlにメスアップした後、よく撹拌した。100℃、48時間反応させ、再び上澄み液を回収した。この操作をさらに3回繰り返した(回収1~5回完了)。ケラチン粉末各回等量ずつ合計5g使用した。残った陽イオン交換樹脂にRO水を加え、100mlにメスアップし、よく撹拌した。100℃、48時間反応させた後、上澄み液を回収した。この操作をさらに2回繰り返した(回収6~8回完了)。残った陽イオン交換樹脂を80mlのRO水で洗浄した。回収した上澄み液と洗浄液を混合し、陽イオン交換樹脂に吸着していない分子量(Mp)5.9kDa以下のケラチンペプチド液を得た。
(2-1)ケラチンペプチド標準サンプルの調製
(1)で得られたイオン交換樹脂に吸着していたケラチンペプチド液(溶出液)を凍結乾燥した。得られた乾燥物を0.75、0.375、0.188、0.094mg/mlとなるように調整し、ケラチンペプチド定量用の標準検量線に用いた。
(1)で得られたケラチンペプチドのうち、溶出液中のケラチンペプチドは、上記のケラチンペプチド定量用の標準検量線を用いる以外は、実施例1の(3-2)と同様にして行った。また、上澄み液(洗浄液を含む)中のケラチンペプチドは、上澄み液を凍結乾燥し、乾燥重量を測定した。
(1)で得られたケラチンペプチドの分子量分布は、実施例1と同様の測定条件に従い、ゲル濾過クロマトグラフィーを用いて測定した。
(1)固体酸触媒による寒天の可溶化・低分子化
寒天粉末(関東化学)1gを90mlのRO水に懸濁し、陽イオン交換樹脂(TOYOPEARL SP-550C)10mlと混合し、よく撹拌した。100℃、24時間反応させ可溶化した後、冷却を行い、寒天オリゴ糖を含む上澄み液を回収した。次に寒天粉末を上記と同様にして1晩湿潤させた後、上記の上澄み液を回収後の陽イオン交換樹脂に添加し、RO水で100mlにメスアップした後、よく撹拌した。100℃、24時間反応させ、再び上澄み液を回収した。この操作をさらに3回繰り返した(回収1~5回完了)。寒天粉末各回等量ずつ合計5g使用した。
(1)で得られた寒天オリゴ糖のうち、上澄み液(洗浄液を含む)中の寒天オリゴ糖は、上澄み液を凍結乾燥し、乾燥重量を測定した。また、溶出液中の寒天オリゴ糖は、次に示すフェノール硫酸法により算出した。まず、サンプル1.0mlに5%フェノール液を1.0ml加え混合した。次に濃硫酸5.0mlを速やかに直接滴下するように加え混合した。室温にて10分放置し、その後は水浴中で10分冷却した。470nmにて吸光度を測定した。吸光度測定には三紳社製デジタル比色計mini photo 10を使用した。標準品には、50、100、150mg/mlに調製したグルコースを用い、標準検量線に用いた。
(1)で得られた寒天オリゴ糖の分子量分布はゲル濾過クロマトグラフィーを用いて測定した。測定には、カラムに東ソー社製TSKgel G-OLIGO-PW(7.8×300mm)を使用する以外は、実施例1と同様の測定条件に従い測定した。また、対照物質として分子量測定用標準品(昭和電工社製Shodex STANDARD P-82(分子量5.9kDa))、1糖(グルコース)、2糖(スクロース)、3糖(ラフィノース)用いた。
本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書に組み入れるものとする。
Claims (8)
- 以下の工程を含む水不溶性高分子化合物の分解物の製造方法:
(A)水不溶性高分子化合物に固体酸触媒を接触させた後、加熱処理を行い、上澄み液を回収する工程、
(B)工程(A)に続いて、該固体酸触媒に水性媒体を加えて攪拌し、加熱処理した後、上澄み液を回収する工程、
(C)工程(B)に続いて、該固体酸触媒を水性媒体にて洗浄し、洗浄液を回収する工程、
(D)工程(A)で回収した上澄み液と、工程(B)で回収した上澄み液と、工程(C)の洗浄液を合わせて固体酸触媒に非吸着の画分を得る工程、
(E)工程(D)に続いて、該固体酸触媒から吸着した成分を溶出し、溶出液を回収して固体酸触媒に吸着した画分を得る工程。 - 水不溶性高分子化合物が、水不溶性タンパク質または水不溶性多糖類である、請求項1に記載の方法。
- 水不溶性高分子化合物を固体酸触媒に接触させる前に水性媒体に浸潤させる工程をさらに含む、請求項1または2に記載の方法。
- 水不溶性高分子化合物の量が、固体酸触媒に対して質量比で0.01~0.5倍である、請求項1~3のいずれかに記載の方法。
- 前記工程(A)において、水不溶性高分子化合物と固体酸触媒の接触時の水性媒体の量を固体酸触媒に対して質量比で1~50倍となるように調整する、請求項1~4のいずれかに記載の方法。
- 前記工程(A)および(B)の加熱処理を、40~160℃で0.1~168時間行う、請求項1~5のいずれかに記載の方法。
- 前記工程(A)および(B)の操作を、非吸着画分における水不溶性高分子化合物の分解物の収率が50%以上になるまでそれぞれ複数回繰り返す、請求項1~6のいずれかに記載の方法。
- 固体酸触媒が、陽イオン交換体、ゼオライト、および珪藻土から成る群より選ばれる少なくとも1種である、請求項1~7のいずれかに記載の方法。
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