JP3501495B2 - Method for producing composition containing bovine insulin-like growth factor-1 - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an insulin-like growth factor-1 containing composition. This composition containing insulin-like growth factor-1 has a bone-reinforcing effect and is useful for the prevention and treatment of osteoporosis.

[0002]

2. Description of the Related Art In recent years, the number of patients with various bone diseases such as osteoporosis, bone fracture and back pain has been increasing with the aging of the population. It is said that this is due to insufficient intake of calcium, decreased ability to absorb calcium, and hormone imbalance after menopause. In order to prevent various bone diseases such as osteoporosis and bone fracture due to aging, it is said that it is effective to increase the bone mass as much as possible to increase the peak bone mass. And increasing the maximum bone mass is nothing but strengthening the bone.

Under such circumstances, calcium salts such as calcium carbonate, calcium lactate and calcium phosphate and natural calcium agents such as beef bone powder, egg shell and fish bone powder are used for the purpose of supplementing calcium. However, some of these calcium forms an insoluble salt in the digestive tract and is not sufficiently absorbed in the body. In addition, vitamin D ₃ and calcitonin preparations are used as drugs for treating osteoporosis and bone strengthening, but when these drugs are used, side effects such as tinnitus, headache, and loss of appetite may occur. Therefore, due to the nature of the disease called osteoporosis, it is desired to develop a food that can be taken orally for a long period of time and that can be expected to have a preventive or therapeutic effect.

On the other hand, insulin-like growth factor-1 (hereinafter,
(Abbreviated as IGF-1) is a polypeptide having a molecular weight of about 7,800 that belongs to the somatomedin class, and plays an important role in bone metabolism such as activating osteoblasts, increasing bone mass and strengthening bone. It is known to be a factor to fulfill. In addition, it was recently revealed that the receptor for IGF-1 is present in the digestive tract, and it has been suggested that orally ingested IGF-1 acts through the receptor in the digestive tract [hormone and clinical , Volume 39, 31-
37, 1991]. It has been confirmed that this IGF-1 is present in human milk [J.
Clin. Endocrinol. Metab. , Fifth
8, 955-959, 1984], and other than that, it is confirmed to be present in all organs including serum and liver [Proc. Natl. Acad. Sci. US
A, Vol. 81, pp. 935-939, 1984].

As a method for preparing this IGF-1, a method for isolation from serum or the like [J. Biol. Ch
em. 261, pp. 569-575, 1986]
Or a method of producing by genetic recombination [JP-A-63-
No. 269984] is known. However, considering the addition of IGF-1 to foods for the purpose of preventing or treating osteoporosis, those isolated from serum or those produced by gene recombination have problems in terms of cost and safety. .

By the way, it has been confirmed that IGF-1 also exists in milk, and it has been reported that the chemical structure of bovine IGF-1 is exactly the same as that of human IGF-1 [B.
iochm. J. 251, pp. 95-103, 19
1988]. This indicates that bovine IGF-1 has the same action as human insulin-like growth factor-1, and if bovine IGF-1 present in milk is added, it is completely safe to add it to food. It can be said that there is no.

As a method for preparing bovine IGF-1 from milk, a method is known in which bovine colostrum is acid-extracted and then treated by a combination of cation exchange chromatography, gel filtration, reverse phase HPLC and the like. [Bioch
em. J. 233, pp. 207-213, 1986.
Year〕. However, in practice, this method has complicated steps,
In particular, most of the milk protein is acid-precipitated by acid extraction of bovine colostrum, and the acid-precipitated milk protein cannot be effectively used, so it cannot be said to be an industrially preferable method. Further, a method for isolating a peptide lacking the N-terminal 5 residues of bovine IGF-1 from milk is known [Japanese Patent Publication No. 63-501567]. However, this method is also based on acid precipitation and cation exchange chromatography, and it cannot be said to be industrially preferable.

The present inventors have previously proposed a method of preparing a composition containing IGF-1 by heating milk or a raw material derived from milk [Japanese Patent Application No.
97273]. However, the IGF-1 content of this IGF-1 containing composition is about 10 to 25 μg / g, and development of a method for preparing a composition having a higher IGF-1 content is desired. In addition, this method also has a problem that the milk protein precipitated by heating cannot be effectively used.

[0009]

In view of the above-mentioned problems, the inventors of the present invention have selected bovine IG from milk or raw materials derived from milk.
As a result of intensive research on a method for separating F-1, bovine IGF-
The inventors have found that a composition having a high content of 1 can be obtained, and have completed the present invention. Therefore, the present invention relates to human IGF
It is an object of the present invention to provide a method for efficiently separating a composition containing bovine IGF-1 having the same chemical structure as that of -1 and having a high bone strengthening effect and preventing or treating osteoporosis from a high degree from milk. .

[0010]

In the present invention, bovine IGF is used.
In producing a composition highly containing -1, milk or a raw material derived from milk is contacted with a cation exchanger to adsorb the bovine IGF-1 fraction, and then the fraction is diluted with an appropriate eluent. Elute and collect.

The milk or the raw material derived from milk used in the present invention is, for example, skim milk, cheese whey, acid whey, colostrum and the like. In addition, whey protein concentrate (WP)
C), whey protein isolate (WPI), whole milk powder, skim milk powder, whey powder, etc. may be reduced. Also,
It is advisable to preheat these materials before contacting them with the cation exchanger. That is, it is possible to reduce the amount of impurities adsorbed on the cation exchanger by heating the raw material and denaturing major milk proteins such as casein, α-lactalbumin, β-lactoglobulin present in milk. it can. As a result, the bovine IGF-1 content in the composition will be improved. Although bovine IGF-1 can be sufficiently recovered without this heat treatment, contamination with casein and whey protein increases, resulting in IGF- in the composition.
1 Decrease the content. When performing this heat treatment, the heating temperature may be set according to the following formula.

T ≧ −5 (pH) +100 (where T is the temperature in degrees Celsius, and pH is 2 ≦ pH ≦ 7)

The pH of the raw material to be brought into contact with the cation exchanger is not particularly limited, but most milk proteins contaminated when the pH is too low are adsorbed on the cation exchanger, and as a result, the bovine in the composition is IGF-1 content is reduced. Conversely, p
If H is too high, the amount of bovine IGF-1 adsorbed on the cation exchanger decreases, which is not preferable. Therefore, it is advisable to carry out the cation exchange treatment in a neutral range that is about the same as the pH of normal milk.

The salt concentration of the raw material to be brought into contact with the cation exchanger is also not particularly limited, but as in the case of the ion exchange treatment which is usually performed, when the salt concentration of the raw material is high, the adsorption to the cation exchanger becomes poor. , The bovine IGF-1 recovery rate from the raw material is reduced. Therefore, it is preferable to adjust the salt concentration to the same level as that of normal milk or lower.

Further, when the milk or the raw material derived from the milk is brought into contact with the cation exchanger, it is preferable that treatment with a clarifier or the like is performed in advance to remove fine precipitates contained in the raw material.

In the present invention, there is no particular limitation on the method of contacting the milk or the raw material derived from milk and the cation exchanger, and a conventional method using a packed bed type column, a method using a rotary type column, Alternatively, cation exchange can be performed according to a method such as a batch method. The contact time between milk or the raw material derived from milk and the cation exchanger is not particularly limited, and it is better to contact for a long time. It is desirable to set the time to 24 hours. Further, the temperature of the raw material to be brought into contact with the cation exchanger is not particularly limited, but is preferably 4 ° C to 40 ° C.
When the temperature is 40 ° C. or higher, deterioration of the raw material becomes remarkable.

In the present invention, the ratio of the cation exchanger and the raw material is such that cation exchanger / raw material = 1/10 (w / w) to 1.
/ 3,000 (w / w), preferably cation exchanger / raw material = 1/16 (w / w) to 1/1000 (w /
w). The value of cation exchanger / raw material is 1/10 (w
/ W), the cost of the cation exchanger becomes relatively high. Also, the value of cation exchanger / raw material is 1 /
When it is less than 3,000, the bovine IGF-1 recovery rate becomes extremely poor.

The cation exchanger that can be used in the present invention is C having a carboxymethyl group as an exchange group.
M-Cellulofine, CM-Cellulose, Microprep CM Strong Cation Exchange Support,
CM-Sepharose, CM-Sephadex, C-spherosyl, etc. and sulfonated chitopearl having sulfonic acid group as an exchange group, SP-Toyopearl, S-Sepharose, SP-Sephadex, Indion S3, S-
Spherosyl, Microprep S strong cation exchange support and the like can be exemplified, but in order to obtain a composition having a higher bovine IGF-1 content, a strong acid cation exchanger having a sulfonic acid group as an exchange group is used. Is desirable.

In the present invention, milk or a raw material derived from milk is brought into contact with a cation exchanger to adsorb bovine IGF-1 to the cation exchanger, and then a solution having a salt concentration of less than 0.1 M is first prepared. Alternatively, wash the cation exchanger with deionized water or the like. By performing this washing, a part of the contaminating milk protein can be removed from the cation exchanger, and as a result, the bovine IGF-1 content in the composition can be improved,
It is preferable to perform this treatment. Then, bovine IGF-1 is eluted from the cation exchanger. This elution method may also be carried out according to a commonly used elution method, but the salt concentration of the eluate used for elution is 0.1 M to 0.3 M.
It is necessary to use the thing of the range of. Bovine IGF from cation exchanger when the salt concentration of the eluate is less than 0.1M
-1 cannot be sufficiently eluted. Also, when the salt concentration of the eluate exceeds 0.3 M, bovine IG adsorbed on the cation exchanger.
Proteins other than F-1 will also be eluted together,
As a result, it reduces the bovine IGF-1 content in the composition.

It has been experimentally obtained that the pH of this eluate is not less than 5 and less than 8. Therefore, as the eluate, Tris-hydrochloric acid buffer solution, phosphate buffer solution, carbonate buffer solution, etc. can be used. , In commonly used buffer,
It is preferable to use a solution in which a neutral salt such as sodium chloride, potassium chloride or ammonium acetate is dissolved. Further, as the eluent, a neutral salt solution having a salt concentration of 0.1 M or more and 0.3 M or less and having no buffering capacity can be used, which is preferable in terms of improving operability and reducing cost.

Next, the bovine IGF thus obtained
For the eluate containing the -1 fraction, a commonly used method, for example, an ion exchange resin, a reverse osmosis membrane, an ultrafiltration membrane,
Desalination and concentration can be performed by a method using a dialysis membrane, an electrodialysis membrane, a gel filtration carrier, or a combination of these methods. The desalting and concentration methods include ultrafiltration. And a method combining diafiltration is preferable because concentration and desalting can be performed at the same time. The ultrafiltration membrane that can be used at this time may be any ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less. This bovine IGF-
Although the 1-containing composition concentrate can be used as it is,
If necessary, a dry powder of the bovine IGF-1 containing composition can be obtained by a method such as spray drying or freeze drying. Furthermore, since bovine IGF-1 has a relatively heat-stable property, it is possible to add a heat sterilization step which is usually performed.

The bovine IGF-1 content in the bovine IGF-1 containing composition obtained by the method of the present invention was measured by an immunological assay using an anti-IGF-1 antibody. The recovery rate was about 40% on average. The recovery rate of bovine IGF-1 recovered from colostrum by a combination of acid extraction and cation exchange chromatography is shown in the literature [Biochem. J. 233, 207-21
Page 3, 1986], 25%, and the method of the present invention exceeded that.

Further, according to the method of the present invention, it is possible to reuse the milk component which is not adsorbed on the cation exchanger and the process is not complicated. Therefore, acid extraction and cation exchange chromatography are combined. It is more practical than the method.

The bovine IGF-1 containing composition contains components such as casein and whey protein, and especially lactoferrin, lactoperoxidase,
Although proteins such as secretary components have physiological activity, since these proteins do not affect the physiological action of bovine IGF-1, there is no substantial problem even if these proteins are contained. When inconvenient, these proteins can be inactivated by treatment such as heating. In addition, lactoperoxidase can be separated by a method such as rechromatography, or can be inactivated in an acidic state.

Bovine IGF obtained by the method of the present invention
Since the -1 containing composition has a bone-reinforcing action, food and drink,
It can be added to medicines, feeds and the like to impart effects such as prevention and treatment of osteoporosis. Also, this bovine IGF-
These effects are further improved by adding calcium having good absorbability such as calcium chloride, calcium carbonate, calcium lactate, egg shell, or milk-derived calcium to foods and drinks, medicines, feeds, etc. containing the 1-containing composition. It is possible to increase. As for the bovine IGF-1 containing composition, no acute toxicity was observed as a result of an animal test in rats. Next, the present invention will be specifically described with reference to examples.

[0026]

Example 1 50 l of cheese whey (pH 6.0) sterilized by heating at 150 ° C. for 5 seconds was packed in a column filled with 500 g of sulfonated chitopearl (manufactured by Fuji Spinning Co., Ltd.) thoroughly washed with deionized water, and a flow rate of 25 ml / min. It was passed at. After passing the liquid
After thoroughly washing the sulfonated chitopearl with deionized water,
Bovine IGF-1 adsorbed with 0.02 M carbonate buffer (pH 7.0) containing 0.28 M sodium chloride was eluted.
Then, the eluate was desalted and concentrated with an ultrafiltration membrane having a cut-off molecular weight of 10 kDa, and then freeze-dried to obtain powdered bovine IG.
450 mg of the F-1 containing composition was obtained. This bovine IGF-
When the content of bovine IGF-1 contained in the 1-containing composition was measured by radioimmunoassay (RIA), it was 160
It was found to be μg / g.

[0027]

Example 2 Unsterilized skim milk (pH 6.5) 1,000
1 to a column packed with 1 kg of SP Toyo Pearl (manufactured by Tosoh Corporation) thoroughly washed with deionized water, and a flow rate of 30 m
The solution was passed at 1 / min. After passing the solution, SP Toyopearl was thoroughly washed with deionized water, and then bovine IGF-1 adsorbed with 0.05M carbonate buffer (pH 7.5) containing 0.15M sodium chloride was eluted. Then, this eluate was subjected to ultrafiltration and diafiltration with a membrane having a cut-off molecular weight of 8 kDa to desalt and concentrate, and then freeze-dried to obtain 50 g of a powdery IGF-1 containing composition. This bovine IGF
When the content of bovine IGF-1 contained in the -1 containing composition was measured by radioimmunoassay (RIA), it was found to be 65.
It was found to be μg / g.

[0028]

Example 3 A whey protein solution (pH 6.8) (40 l) was prepared by sufficiently dissolving whey protein concentrate (WPC) in distilled water to a concentration of 10% by weight. This whey protein solution was passed through a column filled with 400 g of CM-cellulofine (manufactured by Seikagaku Corporation) thoroughly washed with deionized water at a flow rate of 20 ml / min. After passing the solution, 0.03M phosphate buffer containing 0.02M sodium chloride (pH 7.4)
After thoroughly washing CM-cellulofine with 0.20M
0.10M citrate buffer containing sodium chloride (pH
The bovine IGF-1 adsorbed in 6.2) was eluted. Then, the eluate was desalted and concentrated by an electrodialysis (ED) method, and then freeze-dried to obtain 1.3 g of a powdery bovine IGF-1 containing composition. When the content of bovine IGF-1 contained in this bovine IGF-1 containing composition was measured by radioimmunoassay (RIA), it was found to be 35 μg / g.

[0029]

Example 4 Whey protein isolate (WPI) was sufficiently dissolved in distilled water to a concentration of 10% by weight to prepare 80 l of whey protein solution (pH 6.5). This whey protein solution was passed through a column filled with 800 g of SP-Sepharose (Pharmacia) thoroughly washed with deionized water at a flow rate of 24 ml / min. After the passage, the SP-Sepharose was thoroughly washed with a 0.05M carbonate buffer (pH 7.6) containing 0.01M sodium chloride, and then a 0.20M citrate buffer (pH 5.
The bovine IGF-1 adsorbed in 7) was eluted. Then, this eluate was desalted by ion exchange chromatography and then spray-dried to obtain 29.6 g of a powdery bovine IGF-1 containing composition. The content of bovine IGF-1 contained in this bovine IGF-1 containing composition was measured by radioimmunoassay (RIA) and found to be 51.2 μg / g.

[0030]

Example 5 Acid casein whey 3t heat-sterilized at 121 ° C. for 30 seconds was adjusted to pH 6.0 with sodium bicarbonate and then thoroughly washed with deionized water to fill with 30 kg of SP-Sephadex (Pharmacia). In the column
Liquid was passed at a flow rate of 10 l / min. After passing the liquid, SP with deionized water
-After thoroughly washing Sephadex, bovine IGF-1 adsorbed with 0.05 M carbonate buffer (pH 7.0) containing 0.25 M sodium chloride was eluted. The eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and then spray-dried to obtain 176 g of a powdery bovine IGF-1 containing composition.
Bovine IGF contained in the composition containing bovine IGF-1
The content of -1 was measured by radioimmunoassay (RIA) and found to be 106 μg / g.

[0031]

Example 6 A whey protein solution (pH 6.8) was prepared by sufficiently dissolving whey protein concentrate (WPC) in distilled water to a concentration of 10% by weight, and then the solution was kept at 90 ° C. for 1 hour.
A column filled with 500 g of Indion S3 (manufactured by Organo) thoroughly washed with deionized water was heated with 0 l and centrifuged at 17,000 x G for 10 l of the supernatant.
Liquid was passed at a flow rate of 18 ml / min. After passing the solution, thoroughly wash Indion S3 with 0.07 M Tris-HCl buffer,
Bovine IGF-1 adsorbed with a 0.3 M sodium chloride solution (pH 7.3) was eluted. Then, this eluate was desalted by gel filtration chromatography and then freeze-dried to obtain 1.4 g of a powdery bovine IGF-1 containing composition. Bovine IGF-1 contained in this bovine IGF-1 containing composition
Content was measured by radioimmunoassay (RIA) and found to be 42 μg / g.

[0032]

Example 7 Colostrum (pH at 150 ° C. for 5 seconds)
6.8) 2 l was passed through a column filled with 100 g of S-spherocyl (manufactured by IBF) thoroughly washed with deionized water at a flow rate of 20 ml / min. After passing the solution, S- with deionized water
After sufficiently washing spherocyl, bovine IGF-1 adsorbed with a 0.3 M sodium chloride solution (pH 7.3) was eluted. Then, this eluate was subjected to ultrafiltration and diafiltration with a membrane having a cut-off molecular weight of 10 kDa to desalinize and concentrate, and then freeze-dried to obtain powdered bovine IGF-1.
1.5 g of the composition containing was obtained. The content of bovine IGF-1 contained in this bovine IGF-1 containing composition was measured by radioimmunoassay (RIA) to be 184 μg / g.
Was found.

[0033]

Example 8 A column packed with 100 kg of skim milk (pH 6.5) sterilized by heating at 121 ° C. for 30 seconds and 0.5 kg of Microrep S Strong Cation Exchange Support (manufactured by Bio-Rad) thoroughly washed with deionized water. The solution was passed through at a flow rate of 35 ml / min. After passing through the liquid, 0.05
After thoroughly washing the Microrep S Strong Cation Exchange Support with M carbonate buffer, 0.10 M Tris-HCl buffer containing 0.20 M sodium chloride (p
The bovine IGF-1 adsorbed at H7.4) was eluted. The eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and then spray-dried to obtain 1.3 g of a powdery bovine IGF-1 containing composition. When the content of bovine IGF-1 contained in this bovine IGF-1 containing composition was measured by radioimmunoassay (RIA), it was found to be 114 μg / g.

[0034]

[Example 9] Unpasteurized cheese whey (pH 6.5) 20
1 was thoroughly washed with deionized water to obtain C-spherosyl (I
BF) 300g column, flow rate 25ml
The solution was passed at a rate of / minute. After passing the solution, C-spherosyl was thoroughly washed with deionized water, and further, 0.07M phosphate buffer solution (p
After thorough washing with H7.2), bovine IGF-1 adsorbed with 0.05 M citrate buffer (pH 6.5) containing 0.25 M sodium chloride was eluted. Then, the eluate was desalted with a nanofiltration membrane, concentrated, and then freeze-dried to give a powdered bovine IGF-1 containing composition (890 mg).
Got The content of bovine IGF-1 contained in this bovine IGF-1 containing composition was determined by radioimmunoassay (RIA).
It was found to be 43 μg / g.

[0035]

Test Example 1 Bovine IGF-1 obtained in Examples 1 to 9
The osteoblast proliferation effect of the contained composition was examined. The cultured osteoblast-like cell line (MC3T3-E1) was seeded on a 96-well flat-bottom cell culture plate, and α-MEM medium (Flow Laboratories) containing 0.3% by weight of bovine serum was used.
Culture) for 18 hours. In this culture, 2 μl of a solution prepared by dissolving the bovine IGF-1 containing composition in a concentration of 0.5% by weight was added to 100 μl of the medium. After culturing, thymidine labeled with tritium was added, and after 2 hours, the radioactivity of thymidine incorporated into cells was measured to determine the proliferative activity of osteoblasts.
The result is shown in FIG. In FIG. 1, bovine I was added to the medium.
The radioactivity of the group to which the GF-1 containing composition was not added was 1
The cell proliferation activity of the group to which the composition containing bovine IGF-1 was added was set to 00%, and the cell proliferation activity was shown from the radioactivity. According to this, the group to which the bovine IGF-1 containing composition obtained in Examples 1 to 9 was added was 1.8 to 2.7 times that of the group to which the bovine IGF-1 containing composition was not added. And showed proliferative activity of osteoblasts.

[0036]

Test Example 2 The bovine IGF-1 containing composition obtained in Example 5 was tested for bone strengthening activity by animal experiments. Four-week-old SD female rats were used as experimental animals, and seven rats were included in one test group. The osteoporosis model rat was preliminarily bred for 1 week, then subjected to oophorectomy, and further bred for 5 weeks on a calcium-deficient diet for the experiment. In addition, a sham rat, which was subjected to only sham operation and whose ovaries were not removed, was also used in the experiment. Then, osteoporosis model rats were used as a control group (group A) and bovine I.
GF-1-containing composition administration group (B) and bovine IGF-1
The composition-containing composition and the calcium-administered group (C) were divided into 3 groups, and the test feeds shown in Table 1 were each raised for 3 weeks.

[0037]

[Table 1] ────────────────────────────────────                               (A) group (B) group (C) group ────────────────────────────────────   Sucrose 51.05 (%) 51.46 (%) 50.62 (%)   Casein 20.0 18.0 18.0   Cornstarch 15.0 15.0 15.0   Cellulose 5.0 5.0 5.0   Corn oil 5.0 5.0 5.0   Vitamin mixture 1.0 1.0 1.0   Mineral mix 2.65 2.43 3.27   DL-methionine 0.3 0.3 0.3   Bovine IGF-1 containing composition-1.81 1.81 ────────────────────────────────────

The bovine IGF-1 containing composition (1.81% by weight) was replaced with a nitrogen amount corresponding to 2% by weight of casein.
Was added. In addition, regarding the amount of calcium, phosphorus and magnesium in the feed, per 100 g of feed, 3
It was set to 00 mg, 230 mg, and 50 mg. further,
As for the group (C), the amount of calcium and the amount of phosphorus were 520 mg and 400 mg per 100 g of the feed.

After 3 weeks, the bilateral femurs of the rats in each group were excised and the bone strength was measured with a fracture characteristic device. The result is shown in FIG. According to this, the femoral fracture stress is
The value was statistically significantly higher in the bovine IGF-1 containing composition administration group (B) than in the control group (A). Furthermore, the bovine IGF-1 containing composition + calcium administration group (C)
The value was statistically significantly higher than that of the bovine IGF-1 containing composition-administered group (B).

Next, bovine IGF produced by the method of the present invention
Reference examples of foods and drinks to which the -1 containing composition is added are shown below.

[0041]

Reference Example 1 Bovine IGF having the composition shown in Table 2 according to a conventional method
A fruit juice beverage containing the -1 containing composition was produced.

[0042]

[Table 2] ────────────────────────────────────         Mixed isomerized sugar 15.0 (wt%)         Fruit juice 10.0         Citric acid 0.5         Bovine IGF-1 containing composition 0.5         Perfume 0.1         Calcium 0.1         Water 73.5 ────────────────────────────────────

[0043]

Reference Example 2 Bovine IGF having the composition shown in Table 3 according to a conventional method
A calcium agent containing a -1 containing composition was produced.

[0044]

[Table 3] ────────────────────────────────────         Hydrous crystalline glucose 73.5 (wt%)         Bovine IGF-1 containing composition 20.0         Calcium 5.0         Sugar ester 1.0         Fragrance 0.5 ────────────────────────────────────

[0045]

INDUSTRIAL APPLICABILITY According to the method of the present invention, bovine I containing a high amount of bovine IGF-1 from milk or a raw material derived from milk.
It becomes possible to provide a GF-1 composition. Since this bovine IGF-1 containing composition has a bone-reinforcing effect, it is useful for preventing or treating various bone joint diseases, particularly osteoporosis. In addition, this bovine IGF is used during human growth.
The maximum bone mass can be increased by ingesting the -1 composition. Therefore, this bovine IGF-1 containing composition is useful as a material for foods and drinks, medicines, feeds and the like.

[Brief description of drawings]

FIG. 1 shows bovine IGF-1 obtained in Examples 1 to 9.
It is what showed the osteoblast proliferation promotion effect of a containing composition.

FIG. 2 shows the bone-reinforcing action of the bovine IGF-1 containing composition obtained in Example 5.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masatoshi Yahiro 2-8-13 Kumegawa-cho, Higashimurayama-shi, Tokyo (56) Reference JP-A 63-109794 (JP, A) International Publication 92/012993 (WO, A1) (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 1/14-1/36 C07K 14/65 C12P 21/00-21/02 BIOSIS (DIALOG)

Claims (5)

(57) [Claims] 1. A bovine insulin-like growth factor, comprising contacting cow's milk or a raw material derived from cow's milk with a cation exchanger to adsorb bovine insulin-like growth factor-1, followed by elution and recovery. 1. A method for producing a containing composition. 2. The salt concentration of the eluate used for elution is 0.1M
The method according to claim 1, wherein the pH is 0.3 M or less and the pH is 5.6 or more and less than 8. 3. The production method according to claim 1, wherein preheated milk or a raw material derived from milk is used. 4. The method according to claim 1, wherein the cation exchanger is a strongly acidic cation exchanger having a sulfonic acid group as an exchange group. 5. The method according to claim 1, wherein the eluate containing bovine insulin-like growth factor-1 is desalted and concentrated with an ultrafiltration membrane having a cut-off molecular weight of 10 kDa or less.