JP2007246413A - Composition containing milk-originated basic protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition without having astringent taste peculiar to iron, and exhibiting excellent bone-reinforcing action, bone absorption inhibitory effect, bone formation-accelerating effect and collagen production-accelerating effect. <P>SOLUTION: This composition containing a basic protein originated from milk is formed by dissolving/mixing a basic protein fraction originated from the milk with carbonic acid and/or bicarbonic acid and iron. Since the composition containing the basic protein originated from milk does not have the astringent taste peculiar to the iron, and also is excellent in bone-reinforcing action, bone absorption inhibitory effect, bone formation-accelerating effect and collagen production-accelerating effect, it is useful for the prevention and treatment of two diseases of anemia and bone disease, and also useful as a skin-beautifying agent. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a milk-derived basic protein-containing composition. The milk-derived basic protein-containing composition of the present invention contains iron, but does not exhibit iron-specific astringent taste, and therefore has a feature of preventing or treating anemia, and pharmaceuticals, foods and drinks, and cosmetics for the purpose of iron supplementation. And useful as an active ingredient such as feed. Moreover, since the milk-derived basic protein-containing composition of the present invention has a bone strengthening action, a bone resorption suppressing action, a bone formation promoting action, and a collagen production promoting action, prevention or treatment of bone diseases, bone strengthening, skin beautification, etc. It is useful as an active ingredient for pharmaceuticals, foods and drinks, cosmetics, feeds, etc.

  Anemia caused by iron deficiency is a common problem in countries around the world, and it is reported that about half of adult women in Japan are iron deficient anemia or its reserve group. In addition, iron intake has been decreasing in recent years due to changes in dietary habits and excessive diets. In the 2003 National Nutrition Survey, women of all ages fell below the recommended amount set by the Ministry of Health, Labor and Welfare. Yes. Under these circumstances, it is desirable to supply foods and drinks and pharmaceuticals that have been strengthened with iron. However, adding inorganic iron salts such as iron sulfate and iron citrate, which are generally used for iron strengthening, to foods and drinks, the iron has a unique astringent taste. There is a concern of damaging the gastrointestinal mucosa, and the addition amount has a limit. In addition, organic iron heme iron has a problem of flavor such as metal taste and raw odor, and there are restrictions on addition to food and drink. Furthermore, for the purpose of promoting iron absorption, attempts have been made to add milk casein, amino acids, and casein phosphopeptides (see, for example, Patent Document 1). However, these methods cannot eliminate the astringent taste peculiar to iron, and if the amount of iron added is such that the astringent taste peculiar to iron is not felt, it has an effect on prevention or treatment of iron deficiency anemia. There was a problem of not being.

In view of the problems of such iron materials, iron-lactoferrin complexes that are unique to iron and that are obtained by mixing iron and lactoferrin together with carbonic acid and bicarbonate have been invented by previous research. (For example, refer to Patent Document 2). Similarly, an iron-casein complex obtained by mixing iron and casein (see, for example, Patent Document 3) and an iron-whey protein hydrolyzate obtained by mixing iron and whey protein hydrolyzate Compound complexes (for example, see Patent Document 4) have been invented.
However, there is no known milk-derived basic protein-containing composition obtained by dissolving and mixing milk-derived basic protein fraction, carbonic acid and / or bicarbonate, and iron as in the present invention.

  Iron also plays an important role in bone formation, and is involved in hydroxylation of proline and lysine in bone collagen as a coenzyme of procollagen proline hydroxylase and procollagen lysine hydroxylase.

  In recent years, the number of people suffering from various bone diseases such as osteoporosis, fractures, and back pain has been rapidly increasing, and the establishment of prevention and treatment methods has been urgently required. Bone diseases such as osteoporosis are said to be caused by insufficient intake of calcium, a decrease in calcium absorption ability, postmenopausal hormone imbalance, and the like. In order to prevent such bone diseases, it is extremely important to acquire as much bone mass as possible in childhood and adolescence and increase the maximum bone mass. However, the calcium intake does not meet the target amount in the 2003 National Nutrition Survey, and is one of the nutrients that are difficult to satisfy in the Japanese diet. Against this background, milk-derived basic protein fractions have been invented, which are effective materials for the prevention or treatment of bone diseases, which have an effect of promoting bone formation and suppressing bone resorption (for example, patent documents) 5).

Furthermore, it has also been found that the milk-derived basic protein fraction has an effect of increasing the amount of collagen in the skin (see, for example, Patent Document 6). Collagen loss is one of the causes of skin aging. In addition to attenuation of metabolism due to aging, actions such as sunlight (ultraviolet rays), drying, and oxidation are involved in a complex manner. When the tension-holding mechanism such as skin firmness and elasticity held by collagen is destroyed, the skin becomes wrinkled and sagging and becomes aged. Collagen can also retain moisture in its molecules, thereby helping to keep the skin moist, so when the collagen is destroyed, the skin becomes dry and rough . The milk-derived basic protein fraction is an excellent skin beautifying agent that can prevent skin aging by accelerating the biosynthesis of collagen and has no problem in terms of safety.
However, a milk-derived basic protein-containing composition obtained by dissolving and mixing a milk-derived basic protein fraction, carbonic acid and / or bicarbonate, and iron as in the present invention has a bone strengthening action, bone It is not known to have an absorption suppressing action, an osteogenesis promoting action and a collagen production promoting action.
JP 59-162843 JP-A-7-304798 JP-A-9-77793 Japanese Unexamined Patent Publication No. 2000-50812 JP-A-8-15131 Japanese Patent Laid-Open No. 2003-144095

  Addition of inorganic or organic iron, which is used to prevent or treat iron deficiency anemia, to foods and drinks may cause flavor problems such as the astringent taste peculiar to iron or have a raw odor, or damage to the gastrointestinal mucosa. There was a restriction on the addition to food and drink. Studies so far have reported iron-lactoferrin complex, iron-casein complex, iron-whey protein hydrolyzate complex, etc., but there is no iron-specific astringency than these substances, In addition, it is an object to provide a substance exhibiting excellent bone strengthening action, bone resorption inhibiting action, bone formation promoting action and collagen production promoting action.

As a result of diligent studies to solve the above problems, the inventors of the present invention found that the basic protein fraction derived from milk dissolves and mixes carbonic acid and / or bicarbonate and iron, and has an astringent taste unique to iron. It has been found to form a stable milk-derived basic protein-containing composition without water. Furthermore, the milk-derived basic protein-containing composition in which the milk-derived basic protein-containing fraction is combined with carbonic acid and / or bicarbonate and iron is a raw material milk-derived basic protein fraction or an iron-lactoferrin complex. Body, iron-casein complex, and iron-whey protein hydrolyzate complex significantly improved bone strengthening action, bone resorption suppression action, bone formation promoting action and collagen production promoting action The invention has been completed. Therefore, the present invention has a milk-derived basic protein fraction and a carbonic acid and / or heavy weight that have no astringent taste unique to iron and are excellent in bone strengthening action, bone resorption inhibition action, bone formation promoting action and collagen production promoting action. A milk-derived basic protein-containing composition in which carbonic acid and iron are combined is provided.
In the present invention, after bringing milk or a milk-derived raw material into contact with a cation exchange resin, a fraction obtained by eluting the fraction adsorbed on the resin with an eluent having a salt concentration of 0.1 to 1.8 M is expressed as “milk”. The composition obtained by dissolving and mixing carbonic acid and / or bicarbonate and iron with this “milk-derived basic protein fraction” as a raw material. It is called a “sexual protein-containing composition”.

  The milk-derived basic protein-containing composition in which the milk-derived basic protein fraction of the present invention is combined with carbonic acid and / or bicarbonate and iron is capable of strengthening iron, and at the same time, strengthening bone and suppressing bone resorption. The bone formation promoting action and the collagen production promoting action are significantly superior to the milk-derived basic protein fraction as a raw material. In addition, the bone strengthening action and bone resorption significantly superior to the iron-lactoferrin complex, iron-casein complex, and iron-whey protein hydrolyzate complex already published by the same applicant as the present invention. It exhibits inhibitory action, bone formation promoting action and collagen production promoting action. In this way, since iron and a component having bone strengthening action, bone resorption inhibiting action, bone formation promoting action and collagen production promoting action can be taken at the same time, it has been increasing in recent years and has become a big social problem. It is an extremely excellent material useful for the prevention or treatment of two diseases, anemia and bone disease, and also useful as a skin beautifying agent.

  Since the milk-derived basic protein-containing composition of the present invention uses a milk-derived basic protein fraction that is a crudely purified fraction of milk protein as a raw material, lactoferrin, which is a purified fraction, invented so far, is used as a raw material. Compared to the iron-lactoferrin complex, raw material costs can be reduced. Therefore, according to the present invention, it is possible to supply a composition having no astringency unique to iron at a lower price than before.

  The present invention is also useful in terms of facilitating measurement of the content of the milk-derived basic protein fraction. 95% by weight or more of the conventional milk-derived basic protein fraction is protein, and when it is added in the presence of many other proteins as in normal food and drink, the milk-derived basic protein fraction is contained. In order to accurately measure the amount, it was necessary to use a complicated measurement method. On the other hand, since the present invention contains iron as its component, the iron content can be accurately measured by analysis using, for example, an inductively coupled plasma emission spectrometer (ICP-AES). Therefore, according to the present invention, it is possible to easily measure the content of the milk-derived basic protein fraction even when added to general pharmaceuticals, foods and drinks, cosmetics, feeds, etc. It is extremely useful.

Furthermore, since the present invention uses a milk-derived basic protein fraction containing lactoperoxidase exhibiting strong antibacterial activity as a raw material, the iron-lactoferrin complex, iron-casein complex, iron- Compared to whey protein hydrolyzate complex, etc., it exhibits excellent antibacterial properties and can be stored for a long time.
Furthermore, since lactoperoxidase has a bactericidal action against caries-causing bacteria and periodontal disease bacteria, the milk-derived basic protein-containing composition of the present invention is also useful in oral hygiene such as mouthwash and toothpaste.

  And the milk-derived basic protein-containing composition of the present invention is capable of iron strengthening, and at the same time, is significantly superior to the milk-derived basic protein fraction that is the raw material, bone strengthening action, bone resorption inhibiting action, bone Bone strengthening and bone resorption significantly superior to known iron-lactoferrin complex, iron-casein complex, and iron-whey protein hydrolyzate complex as well as formation promoting action and collagen production promoting action It exhibits inhibitory action, bone formation promoting action and collagen production promoting action.

  As described above, the milk-derived basic protein-containing composition of the present invention has a feature that it does not exhibit iron-specific astringent taste, and has a higher bone strengthening action and bone than the milk-derived basic protein fraction. Since it exhibits an absorption-inhibiting action, an osteogenesis promoting action, and a collagen production promoting action, it is useful for the prevention or treatment of two diseases, anemia and bone disease, and is also useful as a skin beautifying agent. In addition, the industrial utility of milk proteins, especially milk-derived basic protein fractions, is increased by reducing the price of iron materials, improving storage stability, and simplifying the method for quantifying milk-derived basic protein fractions. is there.

The milk-derived basic protein fraction used as a raw material in the present invention has the following properties.
1) According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it consists of several proteins with molecular weights ranging from 1,000 to 100,000.
2) 95% by weight or more is protein and contains a small amount of other fat and ash.
3) Protein mainly consists of lactoferrin and lactoperoxidase.
4) The amino acid composition of the protein contains 15% by weight or more of basic amino acids such as lysine, histidine and arginine.
Such a basic protein fraction used in the present invention is obtained by, for example, bringing a milk material such as skim milk or whey into contact with a cation exchange resin to adsorb the basic protein, and adsorbing the basic protein to the resin. The fraction is eluted with an eluate with a salt concentration of 0.1 to 1.8 M. The eluted fraction is collected, desalted and concentrated by reverse osmosis (RO) membrane, electrodialysis (ED) method, etc., and dried as necessary. Can be obtained.

As a method of obtaining a milk-derived basic protein fraction, milk or a milk-derived raw material is brought into contact with a cation exchanger to adsorb a basic protein, and then the basic protein adsorbed on the cation exchanger is used. A method for obtaining a protein fraction by elution with an eluate having a pH exceeding 5 and an ionic strength exceeding 0.5 (Japanese Patent Laid-Open No. 5-202098), a method using alginate gel (Japanese Patent Laid-Open No. 61-246198), A method for obtaining from whey using inorganic porous particles (Japanese Patent Laid-Open No. 1-86839), a method for obtaining from milk using sulfated ester compounds (Japanese Patent Laid-Open No. 63-255300), etc. are known. In the present invention, the basic protein fraction obtained by such a method can be used. Moreover, the milk or the basic protein fraction derived from milk partially decomposed | disassembled by the proteolytic enzyme can also be used.
The milk-derived basic protein fraction used in the present invention preferably contains 15% by weight or more of basic amino acids in its amino acid composition. If it is less than 15% by weight, the effect of the present invention cannot be exhibited.

  As milk or a milk-derived raw material that serves as a source of the basic protein fraction, milk such as cow's milk, human milk, goat milk, and sheep milk can be used, and these milks can be used directly or reduced. Milk, skim milk, whey and the like can be used.

  The milk-derived basic protein-containing composition of the present invention is obtained by dissolving and mixing a milk-derived basic protein fraction, carbonic acid and / or bicarbonate, and iron.

The milk-derived basic protein fraction, carbonic acid and / or bicarbonate, and iron may be mixed in advance after being made into a solution, or may be added to the solution and mixed in a powder state. For example, a milk-derived basic protein fraction and carbonic acid and / or bicarbonate may be dissolved in water and then mixed with an iron solution, or a milk-derived basic protein fraction and iron may be dissolved in water and then carbonated. And / or may be mixed with a bicarbonate solution, or may be dissolved and mixed by simultaneously adding a milk-derived basic protein fraction, carbonate and / or bicarbonate, and iron to water.
Carbonic acid and / or bicarbonate added in the present invention may be added in the form of an acid, or may be added in the form of a water-soluble salt. Iron is usually added in the form of a water-soluble salt.

  Examples of carbonic acid or bicarbonate include carbonated water, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, calcium carbonate and the like. Examples of the iron compound that can be used in the present invention include trivalent iron compounds such as ferric chloride, ferric nitrate, and ferric sulfate, as well as ferrous nitrate and ferrous sulfate. Examples thereof include divalent iron compounds such as iron and iron citrate. In addition, sodium hydroxide, ammonia, potassium hydroxide, hydrochloric acid, citric acid, lactic acid or the like may be mixed and used as a pH adjuster in these solutions. The pH is usually adjusted to 2-9.

  The prepared milk-derived basic protein-containing composition can be directly incorporated into pharmaceuticals, foods and drinks, cosmetics, feeds, etc., or it can be decomposed with proteolytic enzymes such as pepsin, trypsin, chymotrypsin, pancreatin, etc. It can also be added to foods, drinks, cosmetics and feeds.

  The milk-derived basic protein-containing composition of the present invention can be used alone, but in addition to sugars, lipids, flavors, etc., used in combination with raw materials usually used for pharmaceuticals, foods and drinks, cosmetics, feeds, etc. Is also possible.

  EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples and test examples. However, these are merely examples of the present invention, and the present invention is not limited to these examples.

(Preparation of milk-derived basic protein-containing composition (1))
The milk-derived basic protein fraction was prepared by the following method. Specifically, a column (diameter 5 cm × height 30 cm) packed with 400 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki) was thoroughly washed with deionized water, and then 40 liters of unsterilized skim milk (pH 6.7) ) At a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water, and the basic protein fraction adsorbed on the resin was eluted with 0.02 M carbonate buffer (pH 7.0) containing 0.98 M sodium chloride. The eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and lyophilized to obtain 21 g of a powdered basic protein fraction.
This milk-derived basic protein fraction (4.0 g), sodium bicarbonate (18 g) and ferric chloride hexahydrate (2.6 g) are each dissolved in water, and the milk-derived basic protein fraction (200 ml) and sodium bicarbonate are contained. An aqueous solution containing 220 ml and ferric chloride hexahydrate containing 580 ml was prepared.
The above three types of aqueous solutions were mixed and stirred to produce a milk-derived basic protein-containing composition. This solution did not cause turbidity or precipitation.

(Preparation of milk-derived basic protein-containing composition (2))
As in Example 1, the basic protein fraction recovered from the column packed with sulfonated chitopearl was adjusted to pH 4.0 with 0.2 M glycine-hydrochloric acid buffer solution, and pepsin (pepsin A, Porcine stomach mucosa per gram of protein). (Origin, 4,500 units / mg, SIGMA) 20 mg was added and reacted at 37 ° C. for 2 hours. After the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 10 minutes to obtain a basic protein fraction decomposed with a proteolytic enzyme. Using this, a milk-derived basic protein-containing composition was produced in the same manner as in Example 1. This solution did not cause turbidity or precipitation.

(Preparation of milk-derived basic protein-containing composition degradation product)
The milk-derived basic protein-containing composition solution obtained in Example 1 was adjusted to pH 4.0 with 0.2 M glycine-hydrochloric acid buffer, and pepsin (from pepsin A, Porcine stomach mucosa, 4,500 units / g protein). mg, SIGMA) 20 mg was added and reacted at 37 ° C. for 2 hours. After the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 10 minutes to obtain a milk-derived basic protein-containing composition decomposition product. This solution did not cause turbidity or precipitation.

[Test Example 1]
(Sensory evaluation test)
For the milk-derived basic protein-containing composition of Example 1, the milk-derived basic protein-containing composition of Example 2, and the milk-derived basic protein-containing composition decomposed product of Example 3, the following sensory evaluation tests Went.
Each composition contains 0.05 mol / liter imidazole and 0.15 mol / liter sodium chloride, and is a buffer solution that simulates a liquid food at pH 7.5 (simulated buffer solution) and dissolves to an iron concentration of 3.6 mmol / liter. did. With this solution, 10 male and 10 female panelists were judged whether or not they felt astringent taste, using simulated buffer as a control. Each paneler was blindfolded so as not to give judgment factors based on appearance. In addition, the test for one sample was performed in the order of the control and the sample, and after the evaluation of one sample, the test for evaluating the next sample was performed with a minimum interval of one day. Furthermore, in order to eliminate the daily deviation in sample evaluation, the order of sample evaluation was randomized for each panel. The same sensory evaluation test was also conducted on a simulated buffer solution in which ferric chloride or ferrous sulfate was dissolved so that the iron concentration was 3.6 mmol / liter as another iron salt solution sample. As a result, Table 1 shows the number of panelists who felt astringency.

[Table 1]
───────────────────────
Sample Number of panelists who felt astringent taste ───────────────────────
Example 1 0/20
Example 2 0/20
Example 3 0/20
Ferric chloride 12/20
Ferrous sulfate 20/20
───────────────────────

  According to this, the milk-derived basic protein-containing composition of Example 1, the milk-derived basic protein-containing composition of Example 2, and the milk-derived basic protein-containing composition degradation product of Example 3 are unique to iron. It can be seen that no astringent taste is shown.

[Test Example 2]
(Sensory evaluation test after heating)
Each solution of the milk-derived basic protein-containing composition of Example 1, the milk-derived basic protein-containing composition of Example 2, and the milk-derived basic protein-containing composition decomposed product of Example 3 is used as a simulated buffer. After diluting to a protein concentration of 1 mg / ml, it was sealed in a test tube with a screw cap and heated at 90 ° C. for 10 minutes. At this time, each solution did not cause turbidity or precipitation. After heating, it was naturally cooled to room temperature and stored at room temperature for 1 month. When a sensory evaluation test was performed on this solution in the same manner as in Test Example 1, no panelists who felt astringent taste were recognized.

[Test Example 3]
(Hemoglobin level increase promoting effect)
The milk-derived basic protein-containing composition obtained in Example 1 was examined for an effect of promoting the increase in hemoglobin level by animal experiments. For the milk-derived basic protein-containing composition solution (test group) and ferrous sulfate solution (control group 1) prepared in Example 1, ascorbic acid and sodium ascorbate were added so that the iron concentration was 20 mg / 100 ml. The test sample was dissolved in a physiological phosphate buffer (pH 7.2) containing 6.2 mg / 100 g of vitamin C and heated at 90 ° C. for 10 minutes. A test sample was also prepared by heating a physiological phosphate buffer (pH 7.2) containing 6.2 mg / 100 g of ascorbic acid and sodium ascorbate as vitamin C at 90 ° C. for 10 minutes (control group 2).

  Choose 21-day-old Wistar female rats (Charles River Japan) with a body weight of 45-50 g immediately after weaning, and give an iron-free diet (Oriental Yeast Industry, iron content 0.25 mg / 100 g diet) for 2 weeks. Anemia rats with medium hemoglobin values of 7 g / 100 ml or less were prepared. Rats were administered as a group of 7 animals, and the test sample was administered by oral gavage (sonde) for 6 weeks at 1 ml / day while continuing to feed with iron. Six weeks after administration of the test sample, blood was collected from the tail vein, and the hemoglobin value was measured with an automatic blood cell counter (Toa Medical Electronics). The results are shown in Table 2.

[Table 2]
――――――――――――――――――――――――
Hemoglobin value
(Average ± standard deviation)
(g / 100ml)
――――――――――――――――――――――――
Test group 17.5 ± 1.2 ^a
Control group 1 13.3 ± 1.0 ^b
Control group 2 5.4 ± 0.4 ^c
――――――――――――――――――――――――
Significant difference between samples with different a, b, c labels (p <0.05)

  As described above, the milk-derived basic protein-containing composition of Example 1 showed an effect of increasing the blood hemoglobin level of anemic rats, and was found to be useful for anemia treatment. Furthermore, it became clear that the anemia treatment effect is significantly superior to ferrous sulfate which is inorganic iron.

(Preparation of milk-derived basic protein-containing composition)
A milk-derived basic protein fraction was obtained in the same manner as in Example 1, and 4.0 g of this milk-derived basic protein fraction, 18 g of sodium bicarbonate and 0.48 g of ferric chloride hexahydrate were dissolved in water. 200 ml of an aqueous solution containing a milk-derived basic protein fraction, 220 ml of an aqueous solution containing sodium bicarbonate, and 580 ml of an aqueous solution containing ferric chloride hexahydrate were prepared.
The above three types of aqueous solutions were mixed and stirred to produce a milk-derived basic protein-containing composition. This solution did not cause turbidity or precipitation.

[Test Example 4]
(Bone strengthening action)
The milk-derived basic protein-containing composition obtained in Example 4 was examined for bone strengthening action by animal experiments. The milk-derived basic protein-containing composition used was a powder obtained by desalting and concentrating the solution obtained in Example 4 with an ultrafiltration membrane having a molecular weight of 10,000 cut and then freeze-drying. For animal experiments, 4-week-old SD female rats (Nippon Charles River) were used. After preliminary breeding for 1 week, oophorectomy was performed, and then the animals were reared for 5 weeks on a calcium-deficient diet (Oriental Yeast Industry) for animal experiments. Rats that had their ovaries removed and bred on a calcium-deficient diet for 5 weeks were clearly in osteoporosis. Six rats in each group that have caused this osteoporosis state, a control group (Group A), a milk-derived basic protein-containing composition 0.1% by weight administration group (Group B), a 0.25% by weight administration group (Group C), Divided into 4 test groups of the same 0.5 wt% administration group (Group D), each was raised on the test feed shown in Table 3 for 4 weeks. In addition, it adjusted with casein so that the nitrogen content (17.06%) of each test feed might become the same. For each test feed, 300 mg of calcium, 230 mg of phosphorus and 50 mg of magnesium were blended per 100 g. In addition, iron is adjusted with ferric citrate to be 10 mg per 100 g of each test feed, together with those contained in the milk-derived basic protein-containing composition, so that the final total is 100% by weight. It was prepared with sucrose, which is a major component and contained a sufficient amount.

[Table 3]
───────────────────────────────
Group A Group B Group C Group D Group ───────────────────────────────
Casein 20.0 19.9 19.8 19.6 (wt%)
Corn starch 15.0 15.0 15.0 15.0
Cellulose 5.0 5.0 5.0 5.0
Corn oil 5.0 5.0 5.0 5.0
Vitamin mix 1.0 1.0 1.0 1.0
Mineral mixing (excluding iron) 2.63 2.63 2.63 2.63
Ferric citrate 0.06 0.05 0.03 −
Sucrose 51.01 51.02 50.99 50.97
DL-methionine 0.3 0.3 0.3 0.3
Milk-derived basic protein-containing composition-0.10 0.25 0.50
───────────────────────────────

  Four weeks later, the bilateral femurs of the rats in each test group were removed, and the bone strength was measured with a bone rupture force measuring device (Rheometer Max RX-1600 type, iTechno). The result is shown in FIG. According to this, the femoral fracture stress was significantly higher in the milk-derived basic protein-containing composition administration group (Groups B to D) than in the control group (Group A). The femoral fracture strength showed a significantly higher value as the concentration of the milk-derived basic protein-containing composition in the feed increased.

[Test Example 5]
(Comparison with other bone strengthening agents)
By the method similar to Test Example 4, the milk-derived basic protein-containing composition of the present invention obtained in Example 4 was compared with other bone strengthening agents. For animal experiments, 4-week-old SD female rats (Nippon Charles River) were used. After preliminary breeding for 1 week, oophorectomy was performed, and then the animals were reared for 5 weeks on a calcium-deficient diet (Oriental Yeast Industry) for animal experiments. Rats that had their ovaries removed and bred on a calcium-deficient diet for 5 weeks were clearly in osteoporosis. One group of 6 rats that have induced an osteoporosis state, a control group (Group A), a milk-derived basic protein fraction administration group (Group B), an iron-lactoferrin complex administration group (Group C), and Example 4 The milk-derived basic protein-containing composition-administered group (group D) was divided into 4 test groups, and each of the test feeds shown in Table 4 was bred for 4 weeks. In addition, it adjusted with casein so that the nitrogen content (17.06%) of each test feed might become the same. For each test feed, 300 mg of calcium, 230 mg of phosphorus and 50 mg of magnesium were blended per 100 g. In addition, iron was adjusted with ferric citrate so as to be 10 mg per 100 g of each test feed, and finally adjusted with sucrose containing a sufficient amount as a main component so as to be 100% by weight in total.
The milk-derived basic protein fraction administered to Group B was prepared by the method described in Example 1. Further, the iron-lactoferrin complex administered to Group C was prepared as follows based on the method described in JP-A-7-304798. That is, 4.0 g of lactoferrin (DMV), 18 g of sodium bicarbonate and 0.48 g of ferric chloride hexahydrate were dissolved in water, respectively, 200 ml of an aqueous solution containing lactoferrin, 220 ml of an aqueous solution containing sodium bicarbonate, and ferric chloride 6 A 580 ml aqueous solution containing a hydrate was prepared. The above three types of aqueous solutions were mixed and stirred to produce an iron-lactoferrin complex. Furthermore, this solution was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 10,000 cut, and then the powder obtained by lyophilization was used.

[Table 4]
───────────────────────────────
Group A Group B Group C Group D Group ───────────────────────────────
Casein 20.0 19.5 19.6 19.6
Corn starch 15.0 15.0 15.0 15.0
Cellulose 5.0 5.0 5.0 5.0
Corn oil 5.0 5.0 5.0 5.0
Vitamin mix 1.0 1.0 1.0 1.0
Mineral mixing (excluding iron) 2.63 2.63 2.63 2.63
Ferric citrate 0.060 0.060 − −
Sucrose 51.01 51.01 50.97 50.97
DL-methionine 0.3 0.3 0.3 0.3
Milk-derived basic protein fraction − 0.5 − −
Iron-lactoferrin complex − − 0.5 −
Milk-derived basic protein-containing composition---0.5
───────────────────────────────
(weight%)

  Four weeks later, the bilateral femurs of the rats in each test group were removed, and the bone strength was measured with a bone rupture force measuring device (Rheometer Max RX-1600 type, iTechno). The result is shown in FIG. According to this, the femoral fracture stress showed a significantly higher value in the BD groups compared to the control group (Group A). Moreover, in the milk-derived basic protein-containing composition administration group (Group D) of Example 4, the milk-derived basic protein fraction administration group (Group B) and the iron-lactoferrin complex administration group (Group C) were more significant. Showed a high value.

[Test Example 6]
(Osteoblast proliferation activity)
Regarding the milk-derived basic protein fraction (sample B), the iron-lactoferrin complex (sample C), and the milk-derived basic protein-containing composition of the present invention (sample D) obtained in Example 4, osteoblast proliferation The activity was examined.
The cultured osteoblast-like strain (MC3T3-E1) was plated into a 96-well flat-bottom cell culture plate and cultured for 18 hours in α-MEM medium (Flow Laboratories) containing 0.2% by weight bovine serum. During the culture, 2.5 μl of a solution in which samples B to D were dissolved at a concentration of 0.4 wt% was added to 100 μl of the medium. After culturing, thymidine labeled with tritium was added, and the radioactivity of thymidine incorporated into the cells was measured after 2 hours to determine osteoblast proliferation activity (Edited by Cancer Institute, Department of Cancer Research, University of Tokyo) Cell engineering experiment protocol, pp.319-320, 1993). The results are shown in Table 5. In Table 5, the radioactivity of the group cultured with only the medium (sample A) was defined as 100%, and the osteoblast proliferation activity of the group to which samples BD were added was expressed as a relative value of radioactivity.

[Table 5]
――――――――――――――――――――――――
Tritium thymidine incorporation (%)
(Average ± standard deviation)
――――――――――――――――――――――――
Sample A 100 ± 6.7 ^a
Sample B 211 ± 5.6 ^b
Sample C 205 ± 6.3 ^b
Sample D 239 ± 7.0 ^c
――――――――――――――――――――――――
Significant difference between samples with different a, b, c labels (p <0.05)

  As shown in Table 5, the group to which Samples B to D were added exhibited osteoblast proliferation activity that is twice or more that of the group cultured with only the medium (Sample A). Furthermore, compared with the milk-derived basic protein fraction (sample B) and the iron-lactoferrin complex (sample C), the milk-derived basic protein-containing composition (sample D) of the present invention has significantly higher osteoblasts. Proliferative activity was observed.

[Test Example 7]
(Osteoclast resorption suppression effect)
Regarding the milk-derived basic protein fraction (sample B), the iron-lactoferrin complex (sample C), and the milk-derived basic protein-containing composition of the present invention (sample D) obtained in Example 4, osteoclast bone The absorption inhibitory effect was examined. After removing the femur of 10 day-old mice and removing soft tissues, whole bone marrow cells containing osteoclasts mechanically minced in a medium containing 5% FBS were inoculated onto the ivory pieces, and samples B to 10% of a solution prepared by dissolving D to a protein concentration of 0.3% by weight was added and cultured for 2 days. And the bone resorption pit (pit) made on this ivory piece was stained with hematoxylin, and the osteoclast bone resorption suppression action was examined by counting the number (Seiji Junji et al., Animal culture cell manual by research theme, pp.199-200, 1993). The results are shown in Table 6. In Table 6, the number of pits of the group (sample A) cultured only in the medium was used as a control, and each osteoclast bone resorption suppression action was represented by the number of pits.

[Table 6]
――――――――――――――――――――――――
Number of bone resorption pits
(Average ± standard deviation)
――――――――――――――――――――――――
Sample A 177 ± 11 ^a
Sample B 134 ± 15 ^b
Sample C 151 ± 20 ^b
Sample D 102 ± 14 ^c
――――――――――――――――――――――――
Significant difference between samples with different a, b, c labels (p <0.05)

  As shown in Table 6, the group to which samples B to D were added showed significantly higher osteoclast bone resorption inhibitory effect than when cultured in the medium alone (sample A). Furthermore, compared with the milk-derived basic protein fraction (sample B) and the iron-lactoferrin complex (sample C), the milk-derived basic protein-containing composition (sample D) of the present invention has significantly higher osteoclasts. Bone resorption inhibitory action was observed.

[Test Example 8]
(Promoting collagen synthesis)
Collagen synthesis promoting action of milk-derived basic protein fraction (sample B), iron-lactoferrin complex (sample C), and the milk-derived basic protein-containing composition of the present invention (sample D) obtained in Example 4 Were examined according to the method of Woessner [Woessner, JF, Arch. Biochem. Biophys., Vol. 93, pp. 440-447, 1961]. Using α-MEM medium containing 10% fetal bovine serum, MC3T3-E1 cells, which are osteoblasts, are planted in a 96-well plate so that the number of cells is 2 × 10 ⁴ / ml, and 5% CO ₂ exists at 37 ° C. The cells were cultured for 24 hours to obtain test cultured cells. Then, the medium was replaced with α-MEM medium (Flow Laboratories), each of the above samples was added to a final concentration of 50 μg / ml and cultured at 37 ° C. for 3 days, and the amount of synthesized collagen was measured. The amount of collagen was determined by hydrolyzing the cell disruption solution with 6N hydrochloric acid and quantifying hydroxyproline using p-dimethylaminobenzaldehyde. As a control, sample A was cultured in only α-MEM medium without adding each component sample.

  The result is shown in FIG. Compared to the case of culturing only in the medium (sample A), the amount of hydroxyproline increased in any case, and it was found that samples B to D have an action of promoting collagen synthesis of osteoblasts. Furthermore, compared with the milk-derived basic protein fraction (sample B) and the iron-lactoferrin complex (sample C), the milk-derived basic protein-containing composition (sample D) of the present invention has significantly higher osteoblasts. Was observed to promote collagen synthesis.

(Manufacture of soft drinks)
A plate sterilizer was prepared by mixing 0.12 kg of 50% lactic acid solution, 7.5 kg of maltitol, 0.2 kg of fragrance, and 82.18 kg of water with 10 kg of the milk-derived basic protein-containing composition obtained in the same manner as in Example 1. Was sterilized at 90 ° C. for 15 seconds to produce 100 kg of soft drink. This soft drink contained 40 mg of milk-derived basic protein fraction and 5 mg of iron per 100 ml.

(Manufacture of milk drinks)
The milk-derived basic protein-containing composition solution obtained in the same manner as in Example 1 was added to raw milk so as to be 50 ml per liter, homogenized at a pressure of 120 kg / cm ² , and then at 75 ° C. Milk beverage was produced by heat sterilization for 15 seconds. The milk beverage contained 20 mg of milk-derived basic protein fraction and 2.5 mg of iron per 100 ml.

(Manufacture of yogurt)
Skim milk powder is dissolved in water to a solid content of 12%, sterilized by heating at 90 ° C for 20 minutes, cooled to 25 ° C, and Lactobacillus acidophilus L. acidophilus and Streptococcus thermophilus ( S. thermophilus). When the lactic acid acidity reached 1.0% and the pH reached 4.3, it was cooled to 5 ° C. The starter culture thus prepared was inoculated into fresh milk with a fat content of 3.5% pasteurized by heating at 115 ° C. for 2 seconds, and further contained a milk-derived basic protein-containing composition obtained in the same manner as in Example 1. The solution of the product was dissolved and added at 5% by weight. Fermentation and cooling were performed according to conventional methods to produce yogurt. This yogurt contained 20 mg of milk-derived basic protein fraction and 2.5 mg of iron per 100 g.

(Manufacture of tablets)
The milk-derived basic protein-containing composition solution obtained by the same method as in Example 1 was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 10,000 cut, and then freeze-dried to obtain a powder. To this powder 1.0 kg, calcium carbonate 2.0 kg, maltose 4.0 kg, erythritol 1.6 kg, sorbitol 2.0 kg, flavor 4.0 kg, sweetener 0.05 kg, excipient 0.5 kg, lubricant 0.25 kg were added and mixed, The tablet of the milk-derived basic protein-containing composition of the present invention was produced by tableting. This tablet contained 40 mg of milk-derived basic protein fraction and 5 mg of iron per tablet (500 mg).

(Manufacture of enteral nutrients)
The milk-derived basic protein-containing composition solution obtained by the same method as in Example 1 was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 10,000 cut, and then freeze-dried to obtain a powder. This powder 0.025kg, casein 5.5kg, soy protein 5kg, fish oil 1kg, perilla oil 3kg, dextrin 21.475kg, mineral mixture 6kg, vitamin mixture 1.95kg, emulsifier 2kg, stabilizer 4kg, flavor 0.05kg, 200ml The retort pouch was filled and sterilized with a retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, Nisaka Seisakusho) at 121 ° C for 20 minutes to prepare 50 kg of enteral nutrient. This enteral nutrient contained 40 mg of milk-derived basic protein fraction and 5 mg of iron per 100 g.

(Manufacture of feed for dog breeding)
The milk-derived basic protein-containing composition solution obtained by the same method as in Example 1 was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 10,000 cut, and then freeze-dried to obtain a powder. This powder 0.005kg is mixed with soybean cake 12kg, skim milk powder 14kg, soybean oil 4kg, corn oil 2kg, palm oil 33.195kg, corn starch 14kg, wheat flour 9kg, bran 2kg, vitamin mixture 5kg, cellulose 2.8kg, mineral mixture 2kg. Then, sterilization was performed at 120 ° C. for 4 minutes to prepare 100 kg of feed for breeding dogs. This dog feed contained 4 mg of milk-derived basic protein fraction and 0.5 mg of iron per 100 g.

(Manufacture of lotion)
The milk-derived basic protein-containing composition solution obtained by the same method as in Example 4 was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 10,000 cut, and then freeze-dried to obtain a powder. 0.05 kg of this powder, 3 kg of glycerin, 3 kg of 1,3-butylene glycol, 0.5 kg of polyoxyethylene sorbitan monooleate, 0.15 kg of methyl paraoxybenzoate, 0.1 kg of citric acid, 1 kg of sodium citrate, 0.05 kg of fragrance, purified water 92.15 kg was blended to make a uniform solution, and 100 kg of lotion was prepared.

(Manufacture of toothpaste)
The milk-derived basic protein-containing composition solution obtained by the same method as in Example 4 was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 10,000 cut, and then freeze-dried to obtain a powder. 0.2 kg of this powder, calcium hydrogen phosphate dihydrate 4.5 kg, glycerin 1 kg, sorbitol 2.5 kg, sodium carboxymethylcellulose 0.05 kg, carrageenan 0.3 kg, locust bean gum 0.02 kg, saccharin sodium 0.02 kg, sodium lauryl sulfate 0.12 kg, Vitamin E acetate 0.01 kg, flavoring 0.1 kg, water 1.18 kg were blended to prepare 10 kg of dentifrice.

It is explanatory drawing which showed the femur bone strength enhancement effect of the milk-derived basic protein containing composition (Test Example 4). It is explanatory drawing which showed the femur bone strength enhancement effect of the milk-derived basic protein containing composition (Test Example 5). It is explanatory drawing which showed the collagen synthesis promotion effect | action of the milk-derived basic protein containing composition (Test Example 6).

Explanation of symbols

  a, b, c: Significant difference between samples with different labels (p <0.05).

Claims (7)

After bringing milk or a milk-derived raw material into contact with a cation exchange resin, a milk-derived basic protein fraction obtained by eluting the fraction adsorbed on the resin with an eluate having a salt concentration of 0.1 to 1.8 M, carbonic acid and A milk-derived basic protein-containing composition having the following characteristics (1) to (7), obtained by dissolving and / or mixing bicarbonate and iron.
(1) It contains 0.35-350 mg of iron and 0.38 mg or more of carbonic acid and / or bicarbonate per gram of protein in the milk-derived basic protein-containing composition.
(2) No astringent taste unique to iron.
(3) Having a bone strengthening action.
(4) It has a bone resorption suppressing action.
(5) It has a bone formation promoting action.
(6) It has a collagen production promoting action.
(7) Precipitation does not occur even when an aqueous solution prepared so that the protein concentration is 1 mg / ml is heated at 90 ° C. for 10 minutes.   The milk-derived basic protein-containing composition according to claim 1, wherein the milk-derived basic protein fraction is a fraction containing 15% by weight or more of basic amino acids in the amino acid composition.   The milk-derived basic protein-containing composition according to claim 1, wherein the milk-derived basic protein fraction is degraded with a proteolytic enzyme.   A milk-derived basic protein-containing composition decomposition product obtained by decomposing the milk-derived basic protein-containing composition according to claim 1 or 2 with a proteolytic enzyme.   A pharmaceutical comprising the milk-derived basic protein-containing composition or the milk-derived basic protein-containing composition decomposed product according to any one of claims 1 to 4.   A food and drink and a feed comprising the milk-derived basic protein-containing composition or the milk-derived basic protein-containing composition decomposition product according to any one of claims 1 to 4.   Cosmetics containing the milk-derived basic protein-containing composition or the milk-derived basic protein-containing composition decomposition product according to any one of claims 1 to 4.