JP2001211895A - Method of producing physiologically active peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligopeptide derived from the residues of a domestic animal or a fish, most suitable for a health food having an active oxygen free radical elimination activity and angiotensin-I-converting enzyme inhibition activity, and provide a method for producing a white colored crystal consisting of calcium apatite as a main component from the scales of fishes and bones simply and at a low cost. SOLUTION: This method for producing an oligopeptide having the active oxygen free radical-elimination activity and angiotensin-I-converting enzyme inhibition activity is provided by performing a deliming treatment and/or a defat treatment on the scales of the fishes, the bones of the fishes and domestic animals, collagen derived from the skin of the domestic animals, pig hairs, feather meal, keratin originated from hoofs and horns, and enzymatically hydrolyzing them with an alkali protease derived from the genus Bacillus. Also, the white colored crystal consisting of calcium apatite as the main component is produced by treating the scales of the fishes, the bones of the fishes or the domestic animals with a dilute acid, adjusting the dissolved solution at a pH of >=5 with an alkaline solution, separating the precipitated deposition and washing with water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an oligopeptide derived from livestock or fish residue having an active oxygen free radical scavenging activity and angiotensin I converting enzyme inhibitory activity, a method for producing the same, a fish scale, and a livestock bone and fish bone. The present invention relates to a method for producing white crystals containing calcium apatite as a main component.

[0002]

2. Description of the Related Art Reactive oxygen free radicals cause various diseases,
It has been reported to be involved in everything related to human health, such as cancer and aging. For this reason, radical scavengers such as polyphenols are of great interest.
For example, Japanese Patent Application Laid-Open No. 6-145062 describes an active oxygen free radical scavenger containing a green tea extract containing epigallocatechin gallate useful for suppressing arteriosclerosis and the like, and a carotenoid pigment. However, no food material focusing on the radical scavenging activity of oligopeptides is known.

[0003] Recently, angiotensin I converting enzyme inhibitors have attracted attention for uses other than antihypertensive agents, such as therapeutic agents for heart failure. Conventionally, as angiotensin I converting enzyme inhibitors, many natural products and synthetic products have been reported, including peptides in snake venom. For example, Ile-Pr produced by culturing lactic acid bacteria in milk casein
A method for separating and purifying a tripeptide having an amino acid sequence of o-Pro and Val-Pro-Pro (Japanese Patent Laid-Open No. Hei 6-197786), and Leu-Tyr-Pro obtained by enzymatically decomposing fish meat such as bonito. That the tripeptide having a skeleton has an angiotensin I converting enzyme inhibitory action
39195), and that protease degradants such as milk casein and soy protein have an angiotensin I converting enzyme inhibitory action (Japanese Patent Application Laid-Open No. 8-283173). However, all of these naturally occurring peptides have a strong bitter taste and are problematic in making them food additives, and synthetic peptides have the problem that side effects may occur and great attention must be paid to safety. .

[0004] It has been pointed out that calcium is deficient in Japanese dietary habits. In particular, calcium deficiency in young women has become a problem in relation to osteoporosis. It has been reported that beef bone meal is easily transferred to bone after eating because the ratio of calcium and phosphorus is preserved as bone, and in particular, fired bone powder baked and crushed at a high temperature of 800 ° C or more is unfired. Less bitter than bone meal,
It is said to be suitable for food materials. However, the burning of the protein during the baking produces a peculiar odor and requires equipment for deodorization and the like, so that the cost is higher than products imported from overseas. Therefore, a technique for producing inexpensive and bitter-free calcium apatite is desired.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an oligopeptide derived from livestock or fish residue, which is optimal for health foods having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity, and fish scales and bones To provide a simple and inexpensive method for producing white crystals containing calcium apatite as a main component.

[0006]

Means for Solving the Problems The inventors of the present invention conducted a study on the simple and inexpensive production of value-added substances and raw materials from livestock and fishery processing residues, and found that collagen or keratin derived from fish or livestock. Enzymatic hydrolysis treatment with protease to obtain an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity, and the pH of a dilute acid demineralization solution of fish scale obtained by the pretreatment. Was set to near neutrality to obtain white crystals containing calcium apatite as a main component, and completed the present invention.

That is, the present invention provides an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity, which is characterized in that collagen or keratin derived from fish or livestock is enzymatically hydrolyzed with a protease. Method (claim 1), collagen or keratin derived from fish or livestock, fish scales, bones of fish and livestock, collagen derived from livestock skin, or pig hair,
2. The method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to claim 1, wherein the feather meal is keratin derived from a hoof horn, or a fish or livestock. 3. The active oxygen free radical scavenging according to claim 1 or 2, wherein the collagen derived from the class is an extraction residue obtained by extracting a high molecular weight collagen protein from collagen using a weakly acidic solution, a weakly alkaline solution, or warm water. The method for producing an oligopeptide having an activity and an angiotensin I converting enzyme inhibitory action (Claim 3), and the pretreatment performed prior to the enzymatic hydrolysis treatment with a protease is a decalcification treatment and / or a defatting treatment. Claim 1
4. The method for producing an oligopeptide having an active oxygen free radical-scavenging activity and an angiotensin I converting enzyme inhibitory activity according to any one of claims 3 to 3, and a bacterium belonging to the genus Bacillus having an optimum reaction temperature of 45 to 65 ° C. as a protease. A method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to any one of claims 1 to 4, wherein an alkaline protease derived from the enzyme is used (claim 5). 1-5
An oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity, which can be produced by the method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibiting activity according to any one of claims Item 6) or a functional food containing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to Claim 6 as an active ingredient (Claim 7),
An agent for preventing and / or ameliorating a disease caused by a free radical, comprising an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to claim 6 (claim 8), and claim 6. Oxygen Free Radical Scavenging Activity of Angiotensin I
An agent for preventing and / or improving symptoms of hypertension, comprising an oligopeptide having a converting enzyme inhibitory activity as an active ingredient (Claim 9), or a solution prepared by diluting a fish scale or fish or livestock bone with a dilute acid and dissolving the solution with an alkali. The present invention relates to a method for producing white crystals containing calcium apatite as a main component, wherein a pH of the solution is adjusted to 5 or more by a solution, and a precipitated precipitate is separated and washed with water.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The process for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to the present invention comprises subjecting fish or livestock-derived collagen or keratin to enzymatic hydrolysis with a protease. Although it is characterized in that the collagen derived from fish or livestock in the present invention is not particularly limited as long as it is a substance containing collagen obtained from livestock and fishery processing residues, for example, fish scales, fish and livestock bones And collagen derived from the skin of livestock. These may be fresh, dried or a mixture of different origins. In addition, since collagen protein can be extracted as a macromolecule with weak acid, weak alkali, and warm water, it can be extracted from collagen derived from fish scales, fish and livestock bones, livestock skin, etc. The extraction residue obtained by extracting a high molecular weight collagen protein using a solution or hot water can be used as collagen derived from fish or livestock in the present invention.

The keratin derived from fish or livestock in the present invention is not particularly limited as long as it is a substance containing keratin obtained from livestock and fishery processing residues.
Keratin derived from poultry hair, feather meal, hoof horn and the like can be mentioned. These may be fresh, dried or a mixture of different origins.

In the present invention, examples of the pretreatment performed prior to the enzymatic hydrolysis treatment with a protease include a decalcification treatment and a defatting treatment, and such pretreatment includes a fish scale, a bone and a skin containing collagen protein. And pig hairs containing keratin proteins, feather meals and hoofs are usually different. For example, a fish scale contains about 50% of an inorganic component mainly composed of calcium apatite, but has a lipid content of 1%.
% Or less, it is desirable to dissolve the inorganic components with a dilute acid solution, that is, demineralize the skin. The skin has a low inorganic component content but a high lipid content. Since it also contains lipids, it is desirable to perform both degreasing and decalcification. Further, pig hair, feather meal, and hoof horn containing keratin protein have a high protein content but a strong structure. Therefore, it is desirable to perform heat treatment and pulverization.

In the present invention, the enzymatic hydrolysis treatment with a protease is usually carried out using a solid obtained after performing a pretreatment suitable for the type of the above-mentioned livestock and marine processing residue as a substrate. The protease used for the treatment is not particularly limited as long as the oligopeptide having the active oxygen free radical scavenging activity and the angiotensin I converting enzyme inhibitory activity of the present invention can be obtained. Is 45-65 ° C
It is preferable to use an alkaline protease derived from Bacillus bacterium, and it is preferable to use a different protease between a protease for hydrolyzing a collagen protein-containing substance and a protease for hydrolyzing a keratin protein-containing substance. In the enzymatic hydrolysis treatment, utilizing the property that the enzyme is adsorbed on the insoluble substrate, the reaction is performed by adding only the substrate after the reaction, and then allowed to stand.The reaction supernatant containing the solubilized peptide is separated, and then water is added. The enzyme can also be used repeatedly by a method of adding and restarting stirring.

As the protease for hydrolyzing the collagen protein-containing substance, for example, alkaline protease "Proleather" derived from a bacterium belonging to the genus Bacillus (manufactured by Amano Pharmaceutical Co., Ltd.) and alkaline protease "Sumiteam MP" derived from the genus Aspergillus (Shin Nippon Chemical Co., Ltd.) Aspergillus-derived acidic protease "Sumiteam AP" (manufactured by Shin Nippon Chemical Co., Ltd.), Aspergillus-derived acid protease "Protin FA" (Daiwa Kasei Co., Ltd.), Aspergillus-derived acid protease "Denapsin 10P" ( Nagase Seikagaku Kogyo Co., Ltd.). The alkaline protease "Proleather" derived from the bacterium belonging to the genus Bacillus, which has an optimal reaction temperature of 60 ° C. and an optimal reaction pH of 8.0 to 8.3, can be used in the degradation rate and the like. preferable.

As proteases for hydrolyzing the keratin protein-containing substance, alkaline protease "Zavinasase" derived from a bacterium belonging to the genus Bacillus (Savinase; Novo) and alkaline protease "esperase" derived from a bacterium belonging to the genus Bacillus (Esperase; Novo) are used. Co., Ltd.), alkaline protease "Alcalase" derived from Bacillus bacteria (Alcala
se; manufactured by Novo), but the alkaline protease “zabinase” derived from the bacterium belonging to the genus Bacillus, which has an optimum reaction temperature of 50 ° C. and an optimum reaction pH of 8.3, is preferable in terms of the degradation rate and the like.

In the present invention, the oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity is a process for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to the present invention. Any one may be used as long as the average degree of polymerization is 2 to 2
Specific examples of the mixture of peptides having the oligopeptide of 5 as a main component can be given. Angiotensin I-converting enzyme inhibitory activity is considered that a peptide having a specific amino acid sequence among the mixed peptides is considered to have a high activity, but the active oxygen free radical scavenging activity is determined because all peptides have an activity. When the peptide thus obtained is used as a food material or the like, it is not necessary to purify only a specific peptide, nor is it necessary to purify only an oligopeptide. Therefore, the oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity of the present invention also includes an oligopeptide-containing composition having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity.

The functional food containing the oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity of the present invention as an active ingredient includes breads, noodles, beverages, side dishes, yogurt, tofu and the like. There is no particular limitation on foods such as solid foods, liquid foods, and semi-solid foods. The preventive and / or symptom-ameliorating agent for a free radical-induced disease or a hypertension disease, comprising an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity of the present invention as an active ingredient, A known excipient or the like is added to the oligopeptide, and prevention and / or
Alternatively, those prepared as a symptom improving agent can be exemplified.

According to the method of the present invention for producing white crystals containing calcium apatite as a main component, the scale of fish scales or the bones of fish or livestock is treated with a dilute acid, the dissolved solution is adjusted to pH 5 or more with an alkaline solution, and the precipitated precipitates. Separation, characterized by washing with water, such white crystals mainly composed of calcium apatite, in the decalcification step in the above pretreatment, calcium obtained by dilute acid treatment from fish scales or defatted bone The pH of the solution in which apatite is dissolved in dilute acid is raised to 5 or more using an alkaline solution,
It can be obtained by re-precipitating once dissolved calcium apatite as white crystals. The white crystals are precipitated by raising the pH to 5 or more, but it is preferable to raise the pH to 7 or more in order to increase the recovery of calcium and phosphorus. The method for producing white crystals containing calcium apatite as a main component according to the present invention has a feature that it is not necessary to calcine proteins, and it is a low-cost and simple method and does not generate odor.

[0017]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Fresh fish scales were immersed in a 0.6N hydrochloric acid solution 10 times the dry weight for 24 hours, and then filtered to obtain fish scale solids mainly containing collagen after decalcification. After diluting this solid content with water so as to have a dry weight of 62.5 g / l, the solution was put into a stirred reaction tank, and the temperature was set to 60 ° C. and the pH was set to 8.0, and then the protein concentration was 0.0625 g / l.
Was added, and an alkaline protease "Proleather" (manufactured by Amano Pharmaceutical Co., Ltd.) derived from Bacillus sp. By this enzyme treatment, 95% or more of the solid content was decomposed and solubilized. The enzyme-treated solution from which solids had been removed was freeze-dried to obtain a dried enzymatically hydrolyzed oligopeptide. Determined by the trinitrobenzenesulfonic acid method,
The average degree of polymerization of amino acids in the dried oligopeptide is 2
From 5. When the active oxygen free radical scavenging activity of the dried oligopeptide was measured by a luminol-dependent chemiluminescence method, the IPOX ₅₀ that suppressed the amount of generated radicals by 50% was 1 to 3 mg / ml. It had erasing activity. When the inhibitory effect on angiotensin I converting enzyme was examined, the concentration IC ₅₀ required to suppress the activity of angiotensin I converting enzyme to 50% was about 5 mg / ml, and sardines reported to have the same activity were reported. Had the same level of activity as

Example 2 The descaled fish scale solids obtained in Example 1 and containing collagen as a main component were stirred occasionally at 125 g / l at a temperature of 60 ° C. under acidic conditions of pH 3 in a glass beaker. The acid-soluble protein was extracted for 1 hour while heating. An extraction residue and a protein extract are separated by filtration, and the extraction residue is used as a substrate. The alkaline protease "Proleather" derived from Bacillus bacteria having a substrate concentration of 62.5 g / l and a protein concentration of 0.0625 g / l (Amano Pharmaceutical Co., Ltd.) Company), temperature 60
An oligopeptide having an average degree of polymerization of 2 to 5 was obtained by enzymatic decomposition for 1 hour at a temperature of 8.0 ° C. and pH 8.0. The active oxygen free radical scavenging activity of this oligopeptide is IP
About 2mg / ml in OX _50, angiotensin I converting enzyme activity was about 5 mg / ml in IC _50. From these results, it was found that the same oligopeptide as the fish scale sample directly enzymatically degraded without performing the protein extraction operation was obtained.

Example 3 Fresh pork skin was used in a rotary evaporator at a concentration of 150 g / l using acetone as a solvent in a rotary evaporator under room temperature conditions.
Degreasing while rotating slowly for hours. After removing acetone by filtration, acetone was completely removed using a rotary evaporator. The resulting defatted pork skin is coarsely ground with a bone chopper, and the acid-soluble protein is extracted while rotating slowly at 125 g / l at a temperature of 60 ° C. for 1 hour under acidic conditions of pH 3 in a rotary evaporator, followed by filtration. The extraction residue and the protein extract were separated, and the extraction residue was used as a substrate, and the substrate concentration was 62.5 g /
1. The average degree of polymerization of Bacillus bacterium-derived alkaline protease "Proleather" (manufactured by Amano Pharmaceutical Co., Ltd.) with a protein concentration of 0.78 g / l, at 60 ° C. and pH 8.0 for 1 hour. Two to four oligopeptides were obtained. Active oxygen free radical scavenging activity of the oligopeptides of about 2-5 mg / ml in IPOX _50, with angiotensin I converting enzyme activity the IC ₅₀ of about 5～9Mg /
ml. The protein extract was freeze-dried to obtain a dried product, and the molecular weight of the contained protein was determined by SDS-
As a result of examination by polyacrylamide electrophoresis, it was mainly a high molecular weight protein of 100,000 Da or more. Further, as a result of examining the ratio of bound water and free water by a differential scanning calorimeter to a solution prepared so as to have a total organic carbon concentration of about 70 g / l, all water was bound water,
The obtained protein was excellent in water retention.

Example 4 Manufactured as a fertilizer or feed by a rendering company.
Meatmeal containing collagen, fishmeal, beef bone, and feather meal containing keratin were subjected to enzyme treatment without heat treatment and degreasing treatment. The conditions of collagen-based enzyme treatment were as follows: Bacillus-derived alkaline protease “Proleather” (manufactured by Amano Pharmaceutical Co., Ltd.) having a substrate concentration of 62.5 g / l and a protein concentration of 0.78 g / l, a temperature of 60 ° C., and a pH of 8.0. By enzymatic decomposition for 3 hours, the keratin-based enzyme treatment conditions are as follows:
Substrate concentration 62.5 g / l, protein concentration 0.1
6 to 0.31 g / l of an alkaline protease derived from a bacterium belonging to the genus Bacillus “Savinase” (manufactured by Novo), which is enzymatically degraded for 1 hour at a temperature of 50 ° C. and a pH of 8.3 to produce various enzymatically degraded oligopeptides. Obtained. The average degree of polymerization of the obtained oligopeptides is 2 to 4, and the active oxygen free radical scavenging activity is 0.35 in IPOX ₅₀ .
~ 3mg / ml (pig skin; 1-3mg / ml, fish scale;
3 mg / ml, feather meal; 1.25 mg / ml,
Beef bone: 0.8 mg / ml, hoof angle: 0.35 mg / m
l), angiotensin I converting enzyme activity 1 with an IC ₅₀
It was 3 mg / ml.

Example 5 A white precipitate was deposited by raising the pH of the demineralized solution obtained in Example 1 to 7 or more with a 1N aqueous sodium hydroxide solution. After the obtained precipitate was filtered, the precipitate was washed with the same amount of distilled water as the hydrochloric acid solution. FIG. 1 shows the FT-IR spectrum of the white crystal precipitated at the above pH 7, and FIG. 2 shows the FT-IR spectrum of calcium apatite (reagent special grade apatite HAP, single crystal, manufactured by Wako Pure Chemical Industries) as a standard reagent. FIG. 3 shows the spectrum of F of calcium phosphate (reagent special grade tricalcium phosphate manufactured by Wako Pure Chemical Industries, Ltd.) as a standard reagent.
Each T-IR spectrum is shown. As can be seen from FIGS. 1 to 3, the spectrum of the white crystal has an OH stretching vibration having a characteristic of calcium apatite of 3,400 to 3,400.
3,500cm observed at about ^-1, since the spectrum of 400～1,800Cm ^-1, further enters the fingerprint area is similar to calcium apatite rather than calcium phosphate, obtained in white crystals of calcium apatite It is believed that there is. By the way, although calcium ions and phosphate ions were present in the demineralized solution, it was shown that calcium apatite instead of calcium phosphate was precipitated by raising the pH to 7.

[0022]

According to the present invention, livestock or fish optimal for health foods having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity, which are high value-added materials from wastes and the like which are difficult to treat. White crystals containing calcium apatite as a main component can be easily and inexpensively produced from oligopeptides derived from residues and fish scales and bones, which is extremely effective industrially.

[Brief description of the drawings]

FIG. 1 shows the F of white crystals of calcium apatite of the present invention.
It is a figure which shows a T-IR spectrum.

FIG. 2. FT- of calcium apatite as a standard reagent
It is a figure which shows an IR spectrum.

FIG. 3. FT-IR of calcium phosphate as a standard reagent
It is a figure showing a spectrum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C01B 25/32 C07K 4/12 C07K 4/12 A61K 37/02 F-term (Reference) 4B018 MD04 MD20 MD22 MD69 MD73 MD74 ME04 ME06 MF10 MF12 4B064 AG01 AG21 BG01 BG09 CA21 CC03 CC06 CD20 CD21 DA01 DA06 4C084 AA02 AA06 AA07 BA43 CA59 DC40 MA52 ZA422 ZC202 ZC412 4H045 AA10 AA20 AA30 CA40 DA57 EA23 FA16 FA70

Claims (10)

[Claims] 1. A method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity, wherein collagen or keratin derived from fish or livestock is enzymatically hydrolyzed with a protease. 2. The collagen or keratin derived from fish or livestock is a fish scale, a bone derived from fish and livestock, a collagen derived from livestock skin, or a keratin derived from pig hair, feather meal, or hoof horn. The method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to claim 1. 3. The collagen derived from fish or livestock is an extraction residue obtained by extracting a high molecular weight collagen protein from collagen using a weakly acidic solution, a weakly alkaline solution, or warm water. 2. The method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to 2. 4. The scavenging treatment for active oxygen free radicals according to claim 1, wherein the pretreatment carried out prior to the enzymatic hydrolysis treatment with a protease is a decalcification treatment and / or a degreasing treatment. A method for producing an oligopeptide having an activity and an angiotensin I converting enzyme inhibitory action. 5. The protease has an optimum reaction temperature of 4
The method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibiting activity according to any one of claims 1 to 4, wherein an alkaline protease derived from Bacillus bacterium at 5 to 65 ° C is used. 6. An active oxygen free radical scavenging activity and angiotensin I which can be produced by the method for producing an oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to any one of claims 1 to 5. An oligopeptide having a converting enzyme inhibitory action. 7. A functional food containing the oligopeptide having an active oxygen free radical scavenging activity and an angiotensin I converting enzyme inhibitory activity according to claim 6 as an active ingredient. 8. A preventive and / or symptom-ameliorating agent for a free radical-induced disease, comprising an oligopeptide having the active oxygen free radical scavenging activity and the angiotensin I converting enzyme inhibitory activity according to claim 6 as an active ingredient. 9. A preventive and / or symptom-ameliorating agent for hypertension comprising an oligopeptide having an active oxygen free radical-scavenging activity and an angiotensin I converting enzyme inhibitory activity according to claim 6 as an active ingredient. 10. The scale of fish scales or bones of fish or livestock is treated with a dilute acid, and the dissolved solution is adjusted to pH 5 with an alkaline solution.
A method for producing a white crystal containing calcium apatite as a main component, wherein the precipitated precipitate is separated and washed with water.