JPH08242773A - Method for producing calcium-casein phosphopeptide preparation, bone mineral reduction-inhibiting food and bone mineral reduction inhibitor - Google Patents

Abstract

PURPOSE: To obtain a calcium-casein phosphopeptide preparation which is useful as a food or medicine for inhibiting bone mineral reduction by bringing a casein phosphopeptide into contact with a cation exchange resin and allowing the resultant metal ion-free casein phosphopeptide to react with a calcium salt. CONSTITUTION: Casein phosphopeptide obtained by hydrolysis of casein (preferably whole casein or a fractionated casein) is brought into contact with a cation exchange resin to remove metal ions from the casein phosphopeptide. The metal ion-free casein phosphopeptide is allowed to react with a calcium salt such as calcium hydroxide or calcium carbonate, preferably at a pH of 4.0-8.0 to introduce calcium ion into the metal ion-free phosphopeptide to give the objective preparation having an N/Ca weight ratio of 1-3 and Ca/P weight ratio of 1-3. This preparation is dosed at a daily dosage of 50-400mg/kg/adult.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a calcium-casein phosphopeptide having improved utilization of calcium and phosphorus, a food for suppressing bone mineral loss, and an agent for suppressing bone mineral loss.

[0002]

2. Description of the Related Art Casein phosphopeptide (hereinafter referred to as CPP
Say. ) Is a peptide obtained by decomposing the milk protein casein with trypsin and showing a calcium absorption promoting action.
The manufacturing method of the above is disclosed in JP-A-58-170440 and JP-A-59-159793. Further, other casein peptides showing similar effects are disclosed in JP-A-4-264100.
Japanese Patent Application Laid-Open No. 6-70726 and the like.

However, CPP is in vitro or in situ.
It has been proved in the experiment of 1. that it has a calcium absorption promoting action, but its effect in vivo is still under discussion. It has been reported that calcium in diet exhibits various absorbability due to the influence of its origin and coexisting dietary components. In addition, the absorbability of calcium salts alone varies, and in vivo
It is difficult to prove the calcium absorption promoting effect of CPP in o.

By the way, in the modern aging society,
In particular, bone loss of unknown cause with aging after the growth period,
As a syndrome which presents clinical features such as easy fracture and low back pain, regressive osteoporosis has attracted attention. However, a sufficiently effective calcium-containing food or drug that can be used for symptomatic treatment of these patients with osteoporosis has not yet been provided.

[0005]

Therefore, an object of the present invention is to provide a method for producing calcium-CPP, a bone mineral loss suppressing food, and a bone mineral loss suppressing agent, which are more likely to exhibit an effect of suppressing bone mineral loss in vivo. To do.

[0006]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that after removing metal ions such as calcium bound during CPP production,
It was found that a calcium-casein phosphopeptide preparation having excellent solubility and stability can be obtained by reacting with calcium again. Furthermore, the obtained calcium-casein phosphopeptide preparation thus obtained has high utilization of phosphorus derived from calcium and CPP, and obtained the finding that it effectively acts on bone mineral formation, and completed the present invention.

In the present invention, the CPP obtained by decomposing casein is brought into contact with a cation exchange resin, and the C
Calcium and phosphorus, including a step of separating a metal ion from PP, and a step of reacting the demetallized ion casein phosphopeptide obtained by the step with a calcium salt to introduce a calcium ion into the demetallated ion casein phosphopeptide The present invention provides a method for producing a calcium-casein phosphopeptide preparation having improved availability.

The present invention also provides a calcium-casein phosphopeptide preparation obtained by the above production method. Furthermore, the present invention provides a bone mineral loss suppressing food containing the calcium-casein phosphopeptide preparation obtained by the above-mentioned production method. Furthermore, the present invention provides a bone mineral loss suppressing agent comprising the calcium-casein phosphopeptide preparation obtained by the above production method.

The method for producing the calcium-casein phosphopeptide preparation of the present invention (hereinafter referred to as calcium-CPP preparation) will be specifically described below. As casein as a raw material, either whole casein or fractionated casein can be used. Here, whole casein means acid casein, sodium casein, calcium casein, and rennet casein prepared from whole milk or skim milk. Further, the fractionated casein was separated from the above-mentioned total casein by α
Casein (fractionated into α _s , κ and λ casein),
It refers to β-casein and γ-casein, and these can be used alone or in combination of two or more. As a method for preparing fractionated casein, for example, a method of separating α casein and β casein from the total casein by a urea fractionation method (J.
Dairy Sci., 35, 272 (1952)), a method for obtaining κ casein from total casein by adding calcium salt (J. Food Sci., 4
3, 397 (1978)), a method of hydrolyzing κ-casein obtained from whole casein to obtain β-casein (Japanese Patent Publication No. 6-501455), and the like are known. Among the separated caseins, in the present invention, it is preferable to use at least one selected from the group consisting of α _s , κ and β casein.

The production of CPP by decomposing casein can be carried out by a conventionally known method. For example, the following method may be mentioned, but the method is not limited to this. First, the casein solution is hydrolyzed with trypsin at pH 8.0 and 37 ° C. for 20 hours. Then the pH of the obtained reaction mixture is adjusted to 4.6 and the precipitate is removed by centrifugation. Calcium chloride solution to the obtained supernatant,
Calcium ion is added to be 20 mM, and further,
Add an equal volume of ethanol. CPP is obtained by collecting the formed precipitate by centrifugation and drying.

In the present invention, the CPP obtained as described above is brought into contact with a cation exchange resin to give the C
Metal ions such as calcium, magnesium and iron are separated and removed from PP. As a method of contacting CPP with the cation exchange resin, a method of removing the cation exchange resin after mixing the aqueous solution of CPP and the cation exchange resin in the container (in this case, the pH is usually 2.0 to 7.0). Another method is to pass an aqueous solution of CPP through a column packed with a cation exchange resin.

Next, the CPP from which the metal ions have been separated and removed by contact with the above-mentioned cation exchange resin (hereinafter referred to as demetallized ion CPP) is reacted with a calcium salt to form the demetallized ion CPP. Introduce calcium ions. The reaction between the demetallized ion CPP and the calcium salt is carried out, for example, by adding and mixing the calcium salt to a liquid prepared by suspending the demetallized ion CPP in ion-exchanged water. At that time, the pH of the reaction solution is preferably 4.0 or more, and more preferably in the range of 4.0 to 8.0. pH
If it is too low, the solubility of the demetallized ion CPP is low and the reactivity decreases. Therefore, it is preferable to adjust the pH of the reaction solution to 4.0 or more by using sodium hydroxide, potassium hydroxide, hydrochloric acid or the like depending on the kind and amount of the calcium salt to be added. Also, as the calcium salt,
For example, calcium hydroxide, calcium carbonate, calcium salts which are usually used as a food additive such as calcium chloride can be mentioned, but considering that the above reaction is preferably carried out at pH 4.0 or more, calcium hydroxide And it is preferable to use calcium carbonate. After introducing calcium ions in this way, a calcium-CPP preparation is obtained by, for example, freeze-drying.

Calcium-C obtained as described above
The calcium content of PP preparations is usually as high as 500-800 mg per 10 g of the preparation. Moreover, the preparation has high stability and excellent solubility. And, such a preparation has high availability of calcium and phosphorus, and effectively acts on bone mineral formation. In particular, the N / Ca ratio of such a preparation is from 1 to
When it is 3, and the Ca / P ratio is 1 to 3 on a weight basis, the availability of calcium and phosphorus is high, and it effectively acts on bone mineral formation. Therefore, by using the calcium-CPP preparation as a functional food or a health food, a person suffering from osteoporosis such as an adult or an aged person, or a person susceptible to osteoporosis, infants, growing children, adolescents, etc. It also shows an inhibitory effect on bone mineral loss and an effect on promoting bone mineral formation. It is also useful as an additive for animal feed.

Further, the above calcium-CPP preparation comprises
It is also useful as a medicine for suppressing bone mineral loss and promoting bone mineral formation for patients with osteoporosis, calcium malabsorption and other metabolic bone diseases. In that case, the said calcium-CPP preparation is formulated and orally administered. Examples of dosage forms are solutions, suspensions, capsules, powders, granules,
Examples include fine particles and tablets. In addition, when formulating, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient,
Additives such as binders, disintegrants and lubricants can be used. The dosage of the preparation is usually in the range of 50 to 400 mg / kg (body weight) (Ca of 3 to 1) in terms of preparation weight per adult per day.
30 mg / kg (body weight) is appropriate.

[0015]

【Example】

Example 1 Production Example 1 of Calcium-CPP Preparation 5.0 kg of lactate casein (New Zealand Dairy Board)
After being dispersed in 95.0 kg of water at 5 ° C, calcium hydroxide was gradually added to adjust the pH to 7.8. Next, after adding 50.0 g of trypsin (manufactured by Novo), pH was adjusted using calcium hydroxide.
The hydrolysis was carried out at 50 ° C. for 4 hours while maintaining the temperature at 7.8.
Then, cool the resulting reaction mixture to 30 ° C and
Lactic acid solution was added to bring the pH to 4.6. The generated precipitate was removed with a separator (α, manufactured by Laval). 91.2 kg of the obtained supernatant was used as an anion exchanger (Fuji Spinning Co., Ltd., Chitop
Earl) 6 L and mixed, and kept at 10 ° C for 20 minutes. After collecting the anion exchanger by filtration and washing the anion exchanger, an aqueous solution of calcium hydroxide was added and the mixture was kept at pH 8.0 for 45 minutes to elute the adsorbed CPP. The eluate was concentrated under reduced pressure, desalted by electrodialysis, and then spray-dried to obtain 830 g of CPP powder.

5.0% of the CPP obtained as described above
Dissolved in water at a concentration of (w / w) to give an aqueous solution of 10.0 kg, and the activated aqueous cation exchange resin (diaion,
2.3 kg (manufactured by Mitsubishi Kasei) was added and mixed. Then, when the pH of the solution reached 2.0, the cation exchange resin was removed by filtration, and the filtrate was freeze-dried. The demetalized CPP thus obtained was added to deionized water at 5.0% (w / w).
While maintaining at room temperature, 11.5 g of calcium hydroxide was added and mixed (pH 6.8) to 2.0 kg of the suspension suspended at the concentration of Calcium C.
108 g of PP preparation were obtained. The composition of the resulting preparation is shown in Table 1.
Shown in

[0017]

[Table 1]

Example 2 Production Example 2 of Calcium-CPP Preparation 4.0 kg of lactate casein ( manufactured by New Zealand Daily Board)
It was mixed with 41 kg of a 6.6 M urea solution and dissolved at room temperature. 16.5 kg of water was added to the obtained solution to adjust the concentration of urea to 4.6 M, and 52 kg of a supernatant was obtained by centrifugation (3000 rpm, 20 minutes). 20 kg of water was added to 52 kg of this supernatant to increase the concentration of urea.
The concentration was set to 3.3 M, and 69 kg of supernatant was obtained by the same centrifugation as above. 65 kg of water was added to 69 kg of this supernatant to give a urea concentration of 1.7
After making the solution of M, the pH was adjusted to 4.7 using 0.1N hydrochloric acid solution. The precipitate obtained by centrifuging this solution is 4.6 M
It was redissolved in 20 kg of urea solution. Thereafter, the concentration of urea was adjusted to 3.3 M by adding water in the same manner as above, and 45 kg of a supernatant was obtained by centrifugation. After adding 45 kg of this supernatant to a urea concentration of 1.7 M, the pH was adjusted to 4.7 with 0.1N hydrochloric acid solution. The precipitate obtained by centrifuging this solution was washed with water, methanol and acetone in this order,
Naturally dried. In this way, 500 g of β-casein was obtained.

400 g of the obtained β-casein was added to 3800 g of distilled water
, And the pH of the solution was adjusted to 8.0 with 1N sodium hydroxide solution. Then trypsin (Novo) 400mg
After the addition of 1N sodium hydroxide solution to pH 8.
While maintaining at 0, hydrolysis was carried out at 37 ° C. for 24 hours. Then, the obtained reaction mixture was adjusted to pH 4.6 with a 1N hydrochloric acid solution, and then the precipitate was removed by centrifugation. To 8200 ml of the obtained supernatant, 170 ml of 1M calcium chloride solution was mixed, and 8370 ml of ethanol was added and mixed. The produced precipitate was separated by centrifugation, washed with 50% ethanol and 97% ethanol successively, and then freeze-dried to obtain βCPP28g.

The βCPP obtained as described above was added to 5.0
The solution was dissolved in water at a concentration of% (w / w) to prepare 500 g of an aqueous solution, and 115.0 g of activated cation exchange resin (Diaion, manufactured by Mitsubishi Kasei Co., Ltd.) was added and mixed to the aqueous solution.
Then, when the pH of the solution reached 2.05, the cation exchange resin was removed by filtration and then freeze-dried to obtain 21.5 g of demetalized CPP. 20.0 g of the demetalized βCPP thus obtained was added to ion-exchanged water at 5.0% (w / w).
While maintaining the temperature at room temperature, 400 g of a suspension suspended at a concentration of 3.0 g was added and mixed with 3.0 g of calcium hydroxide with stirring (p
H6.8), followed by lyophilization to give calcium-CPP
21.0 g of preparation were obtained. The composition of the resulting preparation is shown in Table 2.

[0021]

[Table 2]

(Example 3) Formulation Example Production of tablets 800 parts by weight of the calcium-βCPP preparation prepared in Example 2 and 170 parts by weight of lactose were mixed, and then 100 ml of 50% ethanol was added and kneaded. . The obtained kneaded product was granulated using an extrusion granulator and dried at 70 ° C. for 2 hours with a hot air dryer. Next, 30 parts by weight of sucrose fatty acid ester (DK Ester F20W, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) was mixed with the obtained dry granules, and a tablet having a diameter of 5 mm and 100 mg per tablet was prepared with a tableting machine. The 10 tablets thus obtained had a calcium content of 58 mg and a phosphorus content of 29 mg.

Example 4 Bone Density Reduction Inhibition Test As a model animal for postmenopausal osteoporosis, 58-week-old Wister female rats subjected to ovariectomy were used to inhibit the bone density reduction of the calcium-CPP preparation of the present invention. I investigated the effect. A powder feed for rats containing the calcium-CPP preparation prepared in Example 1 in an amount of 8.3% by weight was prepared. The calcium content in the feed was 0.5% by weight, all calcium being derived from the above calcium-CPP preparation. The phosphorus content in the feed was 0.4% by weight, 62.5% of which was derived from the calcium-CPP preparation. In addition, other than minerals, AIN-76 (Jour
nalof Nutrition, 107, 1340-1348 (1977)).

The above feed was given to 58-week-old female Wistar rats (7) subjected to ovariectomy (OVX) and kept for 16 weeks. During this period, the bone mineral content of the femur was measured by dual energy X-ray absorption. The bone density was measured over time using a bone density measuring device (DCS-600, manufactured by Aloka Co., Ltd.). The result is shown in FIG. In addition, FIG. 1 is a plot of the average value ± standard error of the relative value of the bone density of the femur of each rat in each week when the bone density of the femur of each rat before the start of the test is 100%. is there. As a control, 58-week-old Wister female rats (7) subjected to ovariectomy (OVX) with a diet containing calcium carbonate as a calcium source and egg albumin as a protein source were also treated in the same manner as above. The bone density of the bone was measured over time. The result is shown in FIG.

Furthermore, sham surgery (SHAM) fed with the above-mentioned control feed
As for the rats (7), the bone density of the femur was measured with time in the same manner as above. The result is shown in FIG. From the above results, the bone mineral density of the OVX rat group fed the control diet continued to decrease during the test period, whereas the bone mineral density inhibitory effect was observed in the OVX rat fed the diet containing the calcium-CPP preparation. Which was similar to SHAM rats fed the control diet. In particular, at the 4th, 12th, and 16th weeks of breeding, a statistically significant difference was observed with the OVX rats fed the control diet.

Further, the calcium content and phosphorus content of the femur after 16 weeks of breeding of each rat were measured, and the calcium content and phosphorus content of the OVX rat fed the diet containing the calcium-CPP preparation were the same as those of the control diet. OVX was lower than that of fed SHAM rats but was fed control diet
A statistically significant difference was observed with the rat (Fig. 2
reference).

(Example 5) Acute toxicity test Using 6 7-week-old male and female SD rats, acute oral administration of a 30% distilled water suspension of the calcium-CPP preparation prepared in Example 1 once A toxicity test was conducted. The suspension was forcibly administered at a dose of 20 g / kg (body weight) and observed for 14 days, but all showed normal growth and no deaths occurred.
Moreover, no abnormal findings were observed.

[0028]

EFFECTS OF THE INVENTION According to the production method of the present invention, a calcium-CPP preparation having a high calcium content, a high availability of calcium and phosphorus, and an effective action for bone mineral formation can be obtained. By using this preparation as a functional food or a health food, it exhibits an effect of suppressing bone mineral loss and an effect of promoting bone mineral formation in adults, elderly people, infants and young children, and adolescents. It is also useful as an additive for animal feed. Furthermore, such a preparation is effective for suppressing bone mineral loss in patients with osteoporosis, calcium malabsorption and other metabolic bone diseases,
It is also useful as a drug for promoting bone mineral formation.

[Brief description of drawings]

FIG. 1 is a diagram showing a time-dependent change in bone density of a rat femur in Example 4.

FIG. 2 is a diagram showing the calcium content and phosphorus content of the rat femur in Example 4.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yu Kuwata 1-21-3 Sakaemachi, Higashimurayama, Tokyo Meiji Dairy Co., Ltd.

Claims (9)

[Claims] 1. A step of separating a metal ion from the casein phosphopeptide by bringing a casein phosphopeptide obtained by decomposing casein into contact with a cation exchange resin, and a demetallated casein phosphopeptide obtained by the step. A method for producing a calcium-casein phosphopeptide preparation having improved utilization of calcium and phosphorus, which comprises the step of reacting a calcium ion with a demetallized ion casein phosphopeptide by reacting a calcium salt with a calcium salt. 2. The production method according to claim 1, wherein the reaction between the demetallized casein phosphopeptide and the calcium salt is carried out at pH 4.0 to 8.0. 3. The calcium-casein phosphopeptide preparation according to claim 1 or 2, wherein the N / Ca ratio is 1-3 by weight and the Ca / P ratio is 1-3 by weight. Production method. 4. The casein is whole casein.
The manufacturing method according to any one of to 3. 5. Casein is at least one of the fractionated caseins.
The manufacturing method according to any one of claims 1 to 3, which is a seed. 6. The production method according to claim 1, wherein the calcium salt is calcium hydroxide or calcium carbonate. 7. A calcium-casein phosphopeptide preparation obtainable by the method according to any one of claims 1-6. 8. A bone mineral loss-suppressing food comprising a calcium-casein phosphopeptide preparation obtained by the method according to any one of claims 1 to 6. 9. A bone mineral loss suppressing agent comprising a calcium-casein phosphopeptide preparation obtained by the method according to any one of claims 1 to 6.