WO1995026984A1 - Process for producing composition containing bovine insulin-like growth factor 1 - Google Patents

Process for producing composition containing bovine insulin-like growth factor 1 Download PDF

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Publication number
WO1995026984A1
WO1995026984A1 PCT/JP1995/000620 JP9500620W WO9526984A1 WO 1995026984 A1 WO1995026984 A1 WO 1995026984A1 JP 9500620 W JP9500620 W JP 9500620W WO 9526984 A1 WO9526984 A1 WO 9526984A1
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Prior art keywords
igf
milk
composition containing
growth factor
composition
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PCT/JP1995/000620
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French (fr)
Japanese (ja)
Inventor
Ken Kato
Hiroaki Matsuyama
Yukihiro Takada
Yoshihiro Kawasaki
Toshiaki Uchida
Masatoshi Yahiro
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Snow Brand Milk Products Co., Ltd.
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Priority to NL9520002A priority Critical patent/NL194730C/en
Publication of WO1995026984A1 publication Critical patent/WO1995026984A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for producing a composition containing insulin-like growth factor-11.
  • This insulin-like growth factor-11-containing composition has a bone strengthening effect and is useful for the prevention and treatment of osteoporosis.
  • calcium salts such as calcium carbonate, calcium lactate, and calcium phosphate
  • natural calcium agents such as bovine bone meal, eggshell, and fish bone powder
  • some of these calcium forms insoluble salts in the digestive tract and are not well absorbed by the body.
  • Vita Mi emissions D 3 and calcium Bok Nin formulation is used, the use of these medicines, tinnitus, headache, such as anorexia May have side effects. Therefore, due to the nature of the disease osteoporosis, long-term oral There is a demand for the development of foods that can be used and can be expected to have a preventive or therapeutic effect.
  • IGF-1 insulin-like growth factor-11
  • IGF-1 insulin-like growth factor-11
  • somatomedin a polypeptide having a molecular weight of about 7,800 belonging to the class of somatomedin, and activates osteoblasts. It is known to be a factor that plays an important role in bone metabolism, such as increasing bone mass and strengthening bone.
  • IGF-1 receptor is present in the gastrointestinal tract, suggesting that orally ingested IGF-1 acts via a gastrointestinal receptor [hormones and Clinical, Vol. 39, pp. 31-37, 1991].
  • This IGF-1 has been confirmed to be present in human milk [J. Clin. Endocrinol. Metab., Vol. 58, pp. 955-959, 1 1984), but it was also confirmed to be present in all organs including serum and liver [Pr0N atl. Acad. Sci. USA, Vol. 81, 9] Pp. 35-93, 1984).
  • IGF-1 is also present in milk, and it has been reported that the chemical structure of IGF-1 is exactly the same as that of human IGF-1 [Biochem. J , Vol. 251, pp. 95-103, 198 8 years]. This indicates that espresso IGF-1 has the same effect as human insulin-like growth factor-11, and if espresso IGF-1 is present in milk, it is added to food. It is safe to say that there is no problem at all.
  • the present inventors have previously proposed a method for preparing a composition containing IGF-1 by heat-treating milk or a raw material derived from milk [Japanese Patent Application No. 5-97272]. .
  • the IGF-1 content of the IGF-1 containing composition is about 10 to 25 // gg, and development of a method for preparing a composition having a higher IGF-1 content is desired.
  • this method also has a problem that milk protein precipitated by heating cannot be used effectively.
  • the present inventors have conducted intensive studies on a method of isolating IGF-1 from milk or a milk-derived material.
  • the present inventors have found that a composition having a high content of 1 can be obtained, and have completed the present invention. Therefore, Akira has a chemical structure identical to that of human IGF-1 and is useful for bone strengthening and prevention and treatment of osteoporosis.
  • the task is to provide Disclosure of the invention
  • the milk or raw material derived from milk used in the present invention includes, for example, skim milk, cheese whey, acid whey, colostrum, and the like, whey protein concentrate (WPC), whey protein isolate (WPI), Whole milk powder, skim milk powder, whey powder, etc. may be reduced. Further, it is preferable to heat these raw materials before they are brought into contact with the cation exchanger. In other words, the amount of impurities adsorbed on the cation exchanger is reduced by heating the raw materials and denaturing major milk proteins such as casein, ⁇ -lactalbumin, and ⁇ -lautoglopurin present in milk. Can be done. As a result, the content of IGF-1 in the composition is improved.
  • the heating temperature may be set according to the following equation.
  • the salt concentration of the raw material there is no particular limitation on the salt concentration of the raw material to be brought into contact with the cation exchanger.
  • the salt concentration of the raw material is high, adsorption to the cation exchanger becomes poor, and The IGF-1 recovery rate decreases. Therefore, it is preferable to adjust the salt concentration to the same level as or less than that of ordinary milk.
  • the milk or the milk-derived material is brought into contact with the cation exchanger, it is preferable to carry out a treatment with a clarifier or the like in advance to remove fine precipitates and the like contained in the material.
  • the method of contacting milk or a raw material derived from milk with a cation exchanger there is no particular limitation on the method of contacting milk or a raw material derived from milk with a cation exchanger, and a method using a conventional packed bed type column, a method using a rotary column, or Cation exchange can be performed according to a method such as a batch method.
  • the contact time between the milk or the milk-derived material and the cation exchanger there is no particular limitation on the contact time between the milk or the milk-derived material and the cation exchanger, and it is better to keep the contact for a long time, but if the contact is carried out for an excessively long time, the raw material deteriorates. It is desirable to set the time from minutes to 24 hours.
  • the temperature of the raw material to be brought into contact with the cation exchanger is not particularly limited, but is preferably 4 to 40 ° C. When the temperature exceeds 40 ° C, the raw material deteriorates significantly.
  • the value of the cation exchanger Z raw material is larger than 1 Z10 (w / w)
  • the cost of the cation exchanger becomes relatively high.
  • the value of the cation exchanger raw material is smaller than 13,000, the recovery rate of PG IGF-1 becomes extremely poor.
  • Examples of the cation exchanger that can be used in the present invention include CM—cellulofine, CM—cellulose, microprep CM strong cation exchange support, and CM—Sepharose having a carboxymethyl group as an exchange group.
  • CM-Sephadex, C-Sulferosyl, etc. Sulfonated chitopearl having sulfonate group as an exchange group, SP-Toyo-Pearl, S-Sepharose, SP-Sephadex, Indion S3, S-Suefrosil, Microbrep Examples include S Strong Cation Extraction Sabot.
  • a solution having a salt concentration of less than 0.1 M wash the ion exchanger with deionized water.
  • a part of the contaminating milk protein can be removed from the cation exchanger, and as a result, the content of PG-IGF-1 in the composition can be improved. It is preferable to do it.
  • This elution method may be performed according to a commonly used elution method, but the salt concentration of the eluate used for elution must be in the range of 0.1 M to 0.3 M.
  • the concentration of IGF- 1 Cannot be eluted sufficiently.
  • the salt concentration of the eluate exceeds 0.3 M, proteins other than IGF-1 adsorbed on the cation exchanger will be eluted together, and as a result, Reduces IGF-1 content.
  • the pH of this eluate is preferably 5 or more and less than 8, and therefore, eluents such as Tris-HCl buffer, phosphate buffer, carbonate buffer, etc.
  • a neutral salt such as sodium chloride, potassium chloride, or ammonium acetate
  • a neutral salt solution having a salt concentration of 0.1 M or more and 0.3 M or less and having no buffer capacity can be used as the eluate, which is preferable in terms of improving operability and reducing costs.
  • the eluate containing the thus-obtained IGF-1 fraction is subjected to a conventional method, for example, ion exchange resin, reverse osmosis membrane, ultrafiltration membrane, dialysis membrane.
  • Desalting and concentration can be performed by a method using an electrodialysis membrane, a gel filtration carrier, or the like, or by a method combining these methods.
  • a combination of filtration and diamond filtration is preferred, since concentration and desalting can be performed simultaneously.
  • the ultrafiltration membrane that can be used at this time may be any ultrafiltration membrane as long as the molecular weight cut off is 10 kDa or less.
  • the concentrate of the composition containing the IGF-1 can be used as it is, but if necessary, a dry powder of the composition containing the IGF-1 can be obtained by a method such as spray drying or freeze drying.
  • a heat sterilization step since PIG IGF-1 has a relatively heat-stable property, it is possible to add a heat sterilization step as is usually performed.
  • the IGF-1 content of the IGF-1 containing composition obtained by the method of the present invention was measured by an immunoassay using an anti-IGF-1 antibody.
  • the recovery of IGF-1 from the raw material was about 40% on average.
  • the recovery rate of PIG IGF-1 recovered from colostrum by a combination of acid extraction and positive ion exchange chromatography was described in literature [Biochem. J., Vol. 233, pp. 207-213, 1 986], 25%, and the method of the present invention exceeded it.
  • the composition containing casein and whey protein is contained in the composition containing the casein IGF-1.
  • proteins such as lactoferrin, lactopaboxidase, and secretory components are included. Although these proteins have physiological activity, these proteins do not affect the physiological action of the IGF-1 at all, so there is no substantial problem with the inclusion of these proteins. If inconvenient, these proteins can be inactivated by treatment such as heating.
  • lactopoxidase can be separated by a method such as rechromatography or inactivated by making it acidic.
  • the pharmaceutical composition containing IGF-1 obtained by the method of the present invention has a bone-strengthening effect, and thus can be added to foods and drinks, pharmaceuticals, feeds, and the like to impart effects such as prevention and treatment of osteoporosis.
  • calcium, calcium carbonate, calcium lactate, eggshell, or calcium with good absorbability, such as milk-derived calcium is added to foods, drinks, medicines, feeds, etc., containing this composition containing IGF-1.
  • these effects can be further enhanced.
  • no acute toxicity was observed for the composition containing IGF-1.
  • FIG. 1 shows the osteoblast proliferation-promoting effect of the composition containing PIG IGF-1 obtained in Examples 1 to 9 below.
  • FIG. 2 shows the bone-strengthening action of the composition containing PIG IGF-1 obtained in Example 5 below.
  • the eluate was desalted and concentrated using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa, and then lyophilized to obtain 450 mg of a powdery IGF-1 containing composition.
  • a radioimmunoassay RIA
  • 1,000 sterile non-sterilized skim milk (pH 6.5) is passed through a column packed with 1 kg of SP Toyo Pearl (manufactured by Tosoh I), thoroughly washed with deionized water, at a flow rate of 30 ml / min. Liquid. After passing the solution through, thoroughly wash the SP Toyo Par with deionized water, and then add 0.15M sodium chloride and 0.05M carbonate The adsorbed IGF-1 was eluted with a buffer (pH 7.5).
  • the eluate is subjected to ultrafiltration and diafiltration using a membrane having a molecular weight cut-off of 8 kDa, desalted and concentrated, and then freeze-dried to obtain a powdery composition containing IGF-1 50. g was obtained.
  • a radioimmunoassay RIA
  • Whey protein concentrate to be a 10% strength by weight was prepared Hoe one protein solution (P H 6. 8) 40 1 fully dissolved in distilled water.
  • This whey protein solution was passed through a column packed with 400 g of CM-cellulofine (manufactured by Seikagaku Corporation) sufficiently washed with deionized water at a flow rate of 2 Om 1 Z min. After passing the solution through, thoroughly wash the CM-cell fin with 0.03 M phosphate buffer (pH 7.4) containing 0.02 M sodium chloride, and then add 0.20 M sodium chloride.
  • the adsorbed IGF-1 was eluted with a 0.10 M citrate buffer (pH 6.2).
  • the eluate was desalted and concentrated by an electrodialysis (ED) method, and then freeze-dried to obtain 1.3 g of a powdery IGF-11-containing composition.
  • the content of the IGF-1 contained in the composition containing the IGF-1 was measured by a radioimmunoassay (RIA) and found to be 35 gZg.
  • RIA radioimmunoassay
  • the whey protein isolate (WPI) was sufficiently dissolved in distilled water to a concentration of 10% by weight to prepare a whey protein solution (pH 6.5) 801.
  • This whey protein solution was passed through a column packed with 800 g of SP-Sepharose (manufactured by Pharmacia) thoroughly washed with deionized water at a flow rate of 24 m 1 Z minute. After passing through, 0.01 M containing sodium chloride 0.05 M carbonate After the SP-Sepharose was thoroughly washed with buffer (pH 7.6), it was adsorbed with 0.20 M citrate buffer (pH 5.7) containing 0.1 M sodium chloride. IGF-1 was eluted.
  • the eluate was desalted by ion exchange chromatography, and then spray-dried to obtain 29.6 g of a powdery IGF-1 containing composition.
  • the content of the IGF-1 contained in the composition containing the IGF-1 was measured by radioimmunoassay (RIA) and found to be 51.2 gZg.
  • the eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and then spray-dried to obtain 176 g of a powdery IGF-1 containing composition in powder form.
  • RO reverse osmosis
  • RIA radioimmunoassay
  • a whey protein concentrate (WP C) is sufficiently dissolved in distilled water to a concentration of 10% by weight to prepare a whey protein solution (PH 6.8), and this solution is heated at 90 ° C for 10 minutes.
  • the supernatant obtained by centrifugation at 17,000 XG was applied to a column packed with 500 g of Indeion S 3 (manufactured by Organo) thoroughly washed with deionized water at a flow rate of 18 m 1 / In minutes. After the infusion, wash Indion S 3 thoroughly with 0.07 M Tris-HCl buffer. After that, the adsorbed IGF-1 was eluted with a 0.3 M sodium chloride solution (pH 7.3).
  • the eluate was desalted by gel filtration chromatography, and then lyophilized to obtain 1.4 g of a powdery IGF-1 containing composition.
  • RIA radioimmunoassay
  • the eluate is subjected to ultrafiltration and membrane filtration with a membrane having a molecular weight cut-off of 10 kDa, desalted and concentrated, and then lyophilized to form a powdered IGF-1 containing composition 1 .5 g were obtained.
  • a radioimmunoassay RIA
  • the discharged liquid was desalted and concentrated by a reverse osmosis (R0) membrane, and then spray-dried to obtain 1.3 g of a powdery IGF-1 containing composition.
  • the content of the IGF-1 contained in the composition containing the IGF-1 was measured by radioimmunoassay (RIA) and found to be 114 / g / g.
  • Unsterilized cheese whey (pH 6.5) 201 was passed through a column packed with 300 g of C-sfluorosyl (manufactured by IBF) thoroughly washed with deionized water at a flow rate of 25 ml. After passing through, wash C-sulferosyl thoroughly with deionized water, and further thoroughly with 0.07 M phosphate buffer (pH 7.2), then contain 0.25 M sodium chloride The adsorbed IGF-1 was eluted with a 0.05 M citrate buffer (pH 6.5). The eluate was desalted and concentrated using a nanofiltration membrane, and then lyophilized to obtain 890 mg of a powdery IGF-1 containing composition. When the content of the Pacific IGF-1 contained in the composition containing the Pacific IGF-11 was measured by radioimmunoassay (RIA), it was found to be 43 gZg. Test example 1
  • a cultured osteoblast-like cell line (MC 3T3— ⁇ ⁇ ) was inoculated into a 96-well flat bottom cell culture plate, and ⁇ -MEM medium containing 0.3% by weight of serum (F10 w Laboratories) was added. For 18 hours. At the time of this culturing, a solution 21 containing a composition containing the IGF-1 at a concentration of 0.5% by weight was added to 100 zl of the medium. After culture, thymidine labeled with tritium was added, and 2 hours later, the radioactivity of thymidine incorporated into the cells was measured to determine the osteoblast proliferation activity.
  • Figure 1 shows the results. In FIG.
  • the radioactivity of the group to which the medium containing no IGF-1 was added was 100%, and the cell proliferation of the group to which the medium containing the IGF-1 was added was determined from the radioactivity. Showed activity. According to this, the group to which the IGF-1 containing composition obtained in Examples 1 to 9 was added was 1.8 to 1.8 times less than the group to which no IGF-1 containing composition was added. 2. It showed a 7-fold osteoblast proliferation activity.
  • the experimental animals were 4-week-old SD female rats, and each test group consisted of 7 animals.
  • the osteoporosis model rat was preliminarily reared for one week, subjected to ovariectomy, and further reared on a calcium-deficient diet for five weeks before the experiment.
  • shamrats that had undergone only sham surgery and had no ovaries removed were also used in the experiment.
  • the osteoporosis model rat was divided into three groups: a control group (group A), a group administered with the IGF-1 containing composition (B), and a group containing the IGF-1 plus calcium (C). Each of the test feeds shown in 1 was bred for 3 weeks.
  • FIG. 2 shows the results. According to this, the femoral fracture stress showed a statistically significantly higher value in the group administered with the IGF-1 containing composition (B) than in the control group (A). Further, the group administered with the composition containing calcium IGF-1 and calcium (C) showed a statistically significantly higher value than the group administered with the composition containing calcium IGF-1 (B).
  • the IGF-1 composition which contains highly IGF-1 from milk or a raw material derived from milk. Since the composition containing bone IGF-1 has a bone strengthening effect, it is useful for preventing or treating various osteoarticular diseases, particularly osteoporosis. Also, By ingesting this IGF-1 composition during the human growth period, the maximum bone mass can be increased. Therefore, this composition containing IGF-1 is useful as a material for foods and drinks, medicines, feeds and the like.

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Abstract

A process for producing a composition containing bovine insulin-like growth factor 1 which comprises bringing milk or milk-derived material into contact with a cation exchanger and eluting the adsorbed bovine insulin-like growth factor 1. As the growth factor 1 can reinforce bones, the composition containing amply the same is useful as the material of food, drink, drug, feed and so forth effective in preventing or treating various osteoarticular diseases, particularly osteoporosis.

Description

明 細 書 ゥシイ ンスリ ン様増殖因子一 1含有組成物の製造法 技術分野  Description 法 Method for producing a composition containing linsulin-like growth factor-1
本発明は、 ィ ンスリ ン様増殖因子一 1含有組成物の製造法に関する。 このィ ンスリ ン様増殖因子一 1含有組成物は、 骨強化作用を有し、 骨粗 鬆症の予防や治療に有用である。 背景技術  The present invention relates to a method for producing a composition containing insulin-like growth factor-11. This insulin-like growth factor-11-containing composition has a bone strengthening effect and is useful for the prevention and treatment of osteoporosis. Background art
近年、 高齢化に伴い骨粗鬆症、 骨折、 腰痛などの各種骨疾患患者が增 加している。 これは、 カルシウムの摂取不足、 カルシウム吸収能力の低 下、 閉経後のホルモンアンバランスなどが原因であるとされている。 こ のような高齢化に伴う骨粗鬆症や骨折などの各種骨疾患を予防するため には、 骨量をできるだけ増加させて最大骨量 （p e a k b o n e m a s s ) を高めることが有効であるとされている。 そして、 最大骨量を高 めるということは、 まさしく骨を強化することに他ならない。  In recent years, with the aging of the population, patients with various bone diseases such as osteoporosis, fracture, and back pain have been increasing. This is thought to be due to insufficient calcium intake, decreased calcium absorption capacity, and hormonal imbalance after menopause. In order to prevent various bone diseases such as osteoporosis and fracture due to such aging, it is said that it is effective to increase the bone mass as much as possible to increase the maximum bone mass (peakbonemass). And increasing your maximum bone mass is just about strengthening your bones.
このような現状の中、 カルシウムの補絵を目的として、 炭酸カルシゥ ム、 乳酸カルシウム、 燐酸カルシウムなどのカルシウム塩や牛骨粉、 卵 殻、 魚骨粉などの天然のカルシウム剤が用いられている。 しかし、 これ らのカルシウムの中には、 消化管内で不溶性の塩を形成し、 体内に十分 吸収されないものもある。 また、 骨粗鬆症治療や骨強化のための医薬と して、 ビタ ミ ン D 3やカルシ 卜ニン製剤などが用いられているが、 これ らの医薬を用いた場合、 耳鳴り、 頭痛、 食欲不振などの副作用を伴うこ とがある。 したがって、 骨粗鬆症という疾病の性質上、 長期的に経口摂 取することができ、 その予防または治療効果を期待できるような食品の 開発が望まれている。 Under these circumstances, calcium salts such as calcium carbonate, calcium lactate, and calcium phosphate, and natural calcium agents such as bovine bone meal, eggshell, and fish bone powder are used for the purpose of supplementary painting of calcium. However, some of these calcium forms insoluble salts in the digestive tract and are not well absorbed by the body. Also, as a medicament for treating osteoporosis or bone augmentation, although such Vita Mi emissions D 3 and calcium Bok Nin formulation is used, the use of these medicines, tinnitus, headache, such as anorexia May have side effects. Therefore, due to the nature of the disease osteoporosis, long-term oral There is a demand for the development of foods that can be used and can be expected to have a preventive or therapeutic effect.
一方、 イ ンスリ ン様増殖因子一 1 (以下、 I G F— 1 と略記する） は、 ソマ トメ ジンのクラスに属する分子量約 7， 8 0 0のポリペプチドであ り、 骨芽細胞を活性化し、 骨量を増加させて骨を強化するなど骨代謝に おいて重要な役割を果たす因子であることが知られている。 また、 最近 になつて消化管に I G F— 1のレセプターが存在することが明らかにな り、 経口摂取された I G F— 1が消化管のレセブターを介して作用する ことが示唆されている 〔ホルモンと臨床、 第 3 9巻、 3 1〜 3 7頁、 1 9 9 1年〕 。 そして、 この I G F— 1については、 ヒ ト乳中に存在する ことが確認されており 〔 J . C l i n. E n d o c r i n o l . M e t a b . 、 第 5 8巻、 9 5 5〜 95 9頁、 1 9 8 4年〕 、 それ以外にも血 清や肝臓を含む全ての臓器に存在することが確認されている 〔P r 0 N a t l . A c a d. S c i . U S A、 第 8 1巻、 9 3 5〜 9 3 9頁、 1 9 8 4年〕 。  On the other hand, insulin-like growth factor-11 (hereinafter abbreviated as IGF-1) is a polypeptide having a molecular weight of about 7,800 belonging to the class of somatomedin, and activates osteoblasts. It is known to be a factor that plays an important role in bone metabolism, such as increasing bone mass and strengthening bone. In addition, it has recently been revealed that an IGF-1 receptor is present in the gastrointestinal tract, suggesting that orally ingested IGF-1 acts via a gastrointestinal receptor [hormones and Clinical, Vol. 39, pp. 31-37, 1991]. This IGF-1 has been confirmed to be present in human milk [J. Clin. Endocrinol. Metab., Vol. 58, pp. 955-959, 1 1984), but it was also confirmed to be present in all organs including serum and liver [Pr0N atl. Acad. Sci. USA, Vol. 81, 9] Pp. 35-93, 1984).
また、 この I G F— 1を調製する方法としては、 血清などから単離す る方法 〔 J . B i o l . C h e m. 、 第 2 6 1巻、 5 6 9〜5 75頁、 1 9 8 6年〕 や遺伝子組み換えによって生産する方法 〔特開昭 6 3 - 2 6 9 9 8 4号公報〕 などが知られている。 しかし、 骨粗鬆症の予防や治 療を目的として食品中に I G F— 1を添加することを考えると、 血清か ら単離したものや遺伝子組み換えで生産したものは、 コス トゃ安全性な どの点で問題がある。  As a method for preparing this IGF-1, a method for isolating it from serum or the like [J. Biol. Chem., Vol. 261, pp. 569-575, 1989] ] And a method of producing by genetic recombination [Japanese Patent Application Laid-Open No. 63-269984]. However, considering the addition of IGF-1 to foods for the purpose of preventing and treating osteoporosis, those isolated from serum and those produced by genetic modification are cost- and safety-related. There's a problem.
ところで、 牛乳中にも I G F— 1が存在することが確認されており、 ゥシ I G F— 1の化学構造はヒ ト I G F— 1 と全く同一であると報告さ れている 〔B i o c hm. J . 、 第 2 5 1巻、 9 5 ~ 1 0 3頁、 1 9 8 8年〕 。 これはゥシ I G F— 1がヒ 卜イ ンスリ ン様增殖因子一 1 と同じ 作用をもつことを示すものであり、 牛乳中に存在するゥシ I G F— 1で あれば、 食品中に添加しても安全上全く問題がないといえる。 By the way, it has been confirmed that IGF-1 is also present in milk, and it has been reported that the chemical structure of IGF-1 is exactly the same as that of human IGF-1 [Biochem. J , Vol. 251, pp. 95-103, 198 8 years]. This indicates that espresso IGF-1 has the same effect as human insulin-like growth factor-11, and if espresso IGF-1 is present in milk, it is added to food. It is safe to say that there is no problem at all.
なお、 牛乳中からゥシ I G F— 1を調製する方法としては、 ゥシ初乳 を酸抽出した後、 陽イオン交換ク口マ トグラフィ ー、 ゲル濾過、 逆相 H P L Cなどを組み合わせて処理する方法が知られている 〔 B i 0 c h e m. J . 、 第 2 3 3巻、 20 7〜 2 1 3頁、 1 9 86年〕 。 しかし、 実 際上、 この方法は工程が煩雑であり、 特に、 ゥシ初乳の酸抽出を行うこ とにより乳蛋白質の大部分が酸沈澱し、 この酸沈澱した乳蛋白質を有効 に利用することができないので、 工業的に好ましい方法とは言い難い。 また、 ゥシ I G F— 1の N末端 5残基が欠如したベプチドを牛乳中から 単離する方法が知られている 〔特表昭 6 3— 5 0 1 5 6 7号公報〕 。 し かし、 この方法も酸沈澱と陽ィォン交換ク口マトグラフィ 一などによる ものであり、 同様に工業的に好ましい方法とは言い難い。  As a method for preparing IGF-1 from milk, there is a method of extracting acid from colostrum and then combining it with cation exchange chromatography, gel filtration, reverse phase HPLC, etc. It is known [Bi0 chem. J., Vol. 233, 207-213, 1986]. However, in practice, this method is complicated in process, and in particular, most of the milk protein is precipitated by acid extraction of the colostrum, and the acid-precipitated milk protein is effectively used. It is difficult to say that it is an industrially preferable method. In addition, a method is known in which a peptide lacking the N-terminal 5 residues of PIG I-1 is isolated from milk (Japanese Patent Application Laid-Open No. 63-501567). However, this method is also based on acid precipitation and positive ion exchange chromatography, and is similarly not industrially preferable.
先に、 本発明者らは、 牛乳または牛乳由来の原料を加熱処理すること により、 I G F— 1を含有する組成物を調製する方法について提案した 〔特願平 5— 9 7 2 7 3号〕 。 しかしながら、 この I G F— 1含有組成 物の I G F— 1含量は 1 0〜25 // g g程度であり、 より I G F— 1 含量の高い組成物を調製する方法の開発が望まれている。 また、 この方 法においても、 加熱によって沈澱する乳蛋白質を有効に利用することが できないという問題もあった。  The present inventors have previously proposed a method for preparing a composition containing IGF-1 by heat-treating milk or a raw material derived from milk [Japanese Patent Application No. 5-97272]. . However, the IGF-1 content of the IGF-1 containing composition is about 10 to 25 // gg, 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 milk protein precipitated by heating cannot be used effectively.
本発明者らは、 上述の問題点を鑑み、 牛乳または牛乳由来の原料から ゥシ I G F— 1を分離する方法について、 鋭意研究を進めたところ、 陽 ィオン交換体を用いることにより、 ゥシ I G F— 1含量の高い組成物が 得られることを見出し、 本発明を完成するに至った。 したがって、 本発 明は、 ヒ ト I G F— 1と同一の化学構造を持ち、 骨強化作用や骨粗鬆症 の予防や治療に有用であるゥシ I G F— 1を高度に含有する組成物を牛 乳から効率良く分離する方法を提供することを課題とする。 発明の開示 In view of the above-mentioned problems, the present inventors have conducted intensive studies on a method of isolating IGF-1 from milk or a milk-derived material. The present inventors have found that a composition having a high content of 1 can be obtained, and have completed the present invention. Therefore, Akira has a chemical structure identical to that of human IGF-1 and is useful for bone strengthening and prevention and treatment of osteoporosis. The task is to provide Disclosure of the invention
本発明では、 ゥシ I G F— 1を高度に含有する組成物を製造するに際 して、 牛乳または牛乳由来の原料を陽イオン交換体と接触させてゥシ I G F— 1画分を吸着させた後、 その画分を適当な溶出液で溶出し、 回収 する。  In the present invention, in producing a composition containing a high amount of calcium IGF-1, milk or a raw material derived from milk was brought into contact with a cation exchanger to adsorb the calcium IGF-1 fraction. Thereafter, the fraction is eluted with an appropriate eluate and collected.
本発明で用いる牛乳または牛乳由来の原料とは、 例えば、 脱脂乳、 チ ーズホエー、 酸ホエー、 初乳などであり、 その他に、 ホエー蛋白質濃縮 物 （W P C ) 、 ホエー蛋白質単離物 （W P I ) 、 全粉乳、 脱脂粉乳、 ホ エー粉などを還元したものでも構わない。 また、 陽イオン交換体と接触 させる前に、 これらの原料を予め加熱しておく と良い。 すなわち、 原料 を加熱して乳中に存在するカゼイン、 α—ラク トアルブミ ン、 β —ラ ウ トグロプリ ンなどの主要な乳蛋白質を変性させることにより、 陽イオン 交換体に吸着する不純物の量を減少させることができる。 その結果、 組 成物中のゥシ I G F— 1含量を向上することになる。 この加熱処理を行 わなくてもゥシ I G F— 1は十分回収できるが、 カゼインゃホエー蛋白 質の混入が多く なり、 結果的に組成物中の I G F— 1含量を低下させる。 なお、 この加熱処理を行うに際しては、 次の式に従って加熱温度を設定 すれば良い。  The milk or raw material derived from milk used in the present invention includes, for example, skim milk, cheese whey, acid whey, colostrum, and the like, whey protein concentrate (WPC), whey protein isolate (WPI), Whole milk powder, skim milk powder, whey powder, etc. may be reduced. Further, it is preferable to heat these raw materials before they are brought into contact with the cation exchanger. In other words, the amount of impurities adsorbed on the cation exchanger is reduced by heating the raw materials and denaturing major milk proteins such as casein, α-lactalbumin, and β-lautoglopurin present in milk. Can be done. As a result, the content of IGF-1 in the composition is improved. Even if this heat treatment is not carried out, the IGF-1 can be sufficiently recovered, but the amount of casein / whey protein is increased, and as a result, the IGF-1 content in the composition is reduced. When performing this heat treatment, the heating temperature may be set according to the following equation.
Τ≥ - 5 ( ρ Η ) + 1 0 0  Τ≥-5 (ρ Η) + 1 0 0
(但し、 Τは摂氏温度を表し、 ρ Ηは 2≤ ρ Η≤ 7である。 ）  (However, Τ represents the temperature in degrees Celsius, and ρ 2 is 2≤ ρ Η≤7.)
陽ィオン交換体と接触させる原料の ρ Ηについては特に限定は無いが、 P Hが低すぎると夾雑する乳蛋白質の多くが陽ィォン交換体に吸着する ため、 結果的に組成物中のゥシ I G F— 1含量が低下する。 逆に p Hが 高すぎるとゥシ I G F— 1の陽ィォン交換体への吸着量が減少するので 好ましくない。 したがって、 通常の牛乳の p Hと同程度の中性域で陽ィ ォン交換処理を行うと良い。 There is no particular limitation on ρ の of the raw material to be brought into contact with the cation exchanger, If the pH is too low, many of the contaminating milk proteins will adsorb to the cation exchanger, resulting in a decrease in the IGF-1 content of the composition. Conversely, if the pH is too high, the amount of adsorbed IGF-1 to the ion exchanger decreases, which is not preferable. Therefore, it is advisable to perform the positive ion exchange treatment in a neutral region that is about the same as the pH of ordinary milk.
また、 陽イオン交換体と接触させる原料の塩濃度についても特に限定 は無いが、 通常行われるイオン交換処理と同様、 原料の塩濃度が高いと 陽イオン交換体への吸着が悪くなり、 原料からのゥシ I G F— 1回収率 が低下する。 したがって、 通常の牛乳と同程度の塩濃度か、 それ以下に 調整しておく ことが好ま しい。  In addition, there is no particular limitation on the salt concentration of the raw material to be brought into contact with the cation exchanger. However, as in the usual ion exchange treatment, if the salt concentration of the raw material is high, adsorption to the cation exchanger becomes poor, and The IGF-1 recovery rate decreases. Therefore, it is preferable to adjust the salt concentration to the same level as or less than that of ordinary milk.
さらに、 牛乳または牛乳由来の原料を陽イオン交換体と接触させる際 には、 予めクラ リファイヤーなどで処理を行い、 原料中に含まれる微細 な沈澱などを除去しておく ことが好ましい。  Further, when the milk or the milk-derived material is brought into contact with the cation exchanger, it is preferable to carry out a treatment with a clarifier or the like in advance to remove fine precipitates and the like contained in the material.
本発明では、 牛乳または牛乳由来の原料と陽ィオン交換体とを接触さ せる方法について特に制限はなく、 従来より行われている充填層型カラ ムを用いる方法、 回転型カラムを用いる方法、 あるいはバッチ法などの 方法に従って陽イオン交換を行うことができる。 また、 牛乳または牛乳 由来の原料と陽イオン交換体との接触時間についても特に制限は無く、 長時間接触させる方が良いが、 余り長時間接触させると原料が劣化する ので、 接触時間は 1 0分〜 24時間とすることが望ま しい。 さらに、 陽 イオン交換体と接触させる原料の温度についても特に制限はないが、 4 て〜 40°Cが望ましい。 温度が 40°C以上となると原料の劣化が著しく なる。  In the present invention, there is no particular limitation on the method of contacting milk or a raw material derived from milk with a cation exchanger, and a method using a conventional packed bed type column, a method using a rotary column, or Cation exchange can be performed according to a method such as a batch method. Also, there is no particular limitation on the contact time between the milk or the milk-derived material and the cation exchanger, and it is better to keep the contact for a long time, but if the contact is carried out for an excessively long time, the raw material deteriorates. It is desirable to set the time from minutes 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 to 40 ° C. When the temperature exceeds 40 ° C, the raw material deteriorates significantly.
本発明では、 陽イオン交換体と原料との割合を、 陽イオン交換体 Z原 料 = 1 1 0 (wZw) 〜1 3， 000 (w/w) 、 好ましく は、 陽 イオン交換体 Z原料 = 1 Z 1 6 (w/w) 〜； LZl, 000 (w/w) とする。 陽イオン交換体 Z原料の値が 1 Z1 0 (w/w) より大きくな ると陽イオン交換体のコス 卜が相対的に高く なる。 また、 陽イオン交換 体 原料の値が 1 3, 000より小さくなるとゥシ I G F— 1の回収 率が極端に悪く なる。 In the present invention, the ratio between the cation exchanger and the raw material is set as follows: the cation exchanger Z raw material = 110 (wZw) to 13,000 (w / w), preferably Ion exchanger Z raw material = 1 Z 16 (w / w) ~; LZl, 000 (w / w). When the value of the cation exchanger Z raw material is larger than 1 Z10 (w / w), the cost of the cation exchanger becomes relatively high. Further, when the value of the cation exchanger raw material is smaller than 13,000, the recovery rate of PG IGF-1 becomes extremely poor.
本発明で用いることのできる陽ィォン交換体と しては、 カルボキシメ チル基を交換基として持つ CM—セルロファイン、 CM—セルロース、 マイクロプレップ CMス トロングカチオンエクスチェンジサポー ト、 C M—セファ ロース、 CM—セフアデックス、 C一スフエロシルなどゃス ルホン酸基を交換基として持つスルホン化キトパール、 S P— トーョ一 パール、 S—セファロース、 S P—セフアデックス、 インディオン S 3、 S—スフエロシル、 マイクロブレップ Sス トロングカチオンェクスチヱ ンジサボ一 卜などを例示することができるが、 ゥシ I G F— 1含量のよ り高い組成物を得るためには、 スルホン酸基を交換基として持つ強酸性 陽イオン交換体を用いることが望ましい。  Examples of the cation exchanger that can be used in the present invention include CM—cellulofine, CM—cellulose, microprep CM strong cation exchange support, and CM—Sepharose having a carboxymethyl group as an exchange group. CM-Sephadex, C-Sulferosyl, etc. Sulfonated chitopearl having sulfonate group as an exchange group, SP-Toyo-Pearl, S-Sepharose, SP-Sephadex, Indion S3, S-Suefrosil, Microbrep Examples include S Strong Cation Extraction Sabot. However, in order to obtain a composition having a higher IGF-1 content, it is necessary to use a strongly acidic cation having a sulfonic acid group as an exchange group. It is desirable to use exchangers.
本発明では、 牛乳または牛乳由来の原料と陽イオン交換体とを接触さ せて、 陽ィオン交換体にゥシ I G F— 1を吸着させた後、 まず、 0. 1 M未満の塩濃度の溶液または脱イオン水などで陽ィォン交換体を洗浄す る。 この洗浄を行うと夾雑する乳蛋白質の一部を陽イオン交換体から除 去することができ、 結果的に組成物中のゥシ I G F— 1含量を向上させ ることができるので、 この処理を行うことが好ま しい。 その後、 陽ィォ ン交換体からゥシ I G F— 1を溶出する。 この溶出方法についても、 通 常行われている溶出方法に従って行えば良いが、 溶出に用いる溶出液の 塩濃度は 0. 1 M〜0. 3 Mの範囲のものを用いる必要がある。 溶出液 の塩濃度が 0. 1 M未満であると陽イオン交換体からのゥシ I G F— 1 の溶出が十分行えない。 また、 溶出液の塩濃度が 0 . 3 Mを超えると陽 イオン交換体に吸着したゥシ I G F— 1以外の蛋白質をも一緒に溶出さ せることになり、 結果的に組成物中のゥシ I G F— 1含量を低下させる。 なお、 この溶出液の p Hについては 5以上 8未満が良好であることを 実験により得ており、 したがって、 溶出液としては、 ト リス—塩酸緩衝 液、 リ ン酸緩衝液、 炭酸緩衝液など、 通常用いられている緩衝液に、 塩 化ナト リウム、 塩化力リウム、 酢酸アンモニゥムなどの中性の塩を溶解 したものを用いると良い。 また、 溶出液として、 塩濃度が 0 . 1 M以上 0 . 3 M以下で緩衝能を持たない中性の塩溶液を用いることもでき、 操 作性の向上ゃコス 卜の低減という意味では好ま しい。 In the present invention, after contacting milk or a raw material derived from milk with a cation exchanger to allow the cation exchanger to adsorb the calcium IGF-1, first, a solution having a salt concentration of less than 0.1 M Or wash the ion exchanger with deionized water. By performing this washing, a part of the contaminating milk protein can be removed from the cation exchanger, and as a result, the content of PG-IGF-1 in the composition can be improved. It is preferable to do it. Then, elute IGF-1 from the positive ion exchanger. This elution method may be performed according to a commonly used elution method, but the salt concentration of the eluate used for elution must be in the range of 0.1 M to 0.3 M. When the salt concentration of the eluate is less than 0.1 M, the concentration of IGF- 1 Cannot be eluted sufficiently. If the salt concentration of the eluate exceeds 0.3 M, proteins other than IGF-1 adsorbed on the cation exchanger will be eluted together, and as a result, Reduces IGF-1 content. Experiments have shown that the pH of this eluate is preferably 5 or more and less than 8, and therefore, eluents such as Tris-HCl buffer, phosphate buffer, carbonate buffer, etc. It is preferable to use a solution in which a neutral salt such as sodium chloride, potassium chloride, or ammonium acetate is dissolved in a commonly used buffer solution. In addition, a neutral salt solution having a salt concentration of 0.1 M or more and 0.3 M or less and having no buffer capacity can be used as the eluate, which is preferable in terms of improving operability and reducing costs. New
次に、 このようにして得られたゥシ I G F— 1画分を含む溶出液につ いては、 通常行われている方法、 例えば、 イオン交換樹脂、 逆浸透膜、 限外濾過膜、 透析膜、 電気透析膜、 ゲル濾過担体などを用いる方法によ り、 あるいは、 これらの方法を組み合わせた方法により、 脱塩および濃 縮を行うことができるが、 脱塩および濃縮の方法としては、 限外濾過お よびダイヤフィ ルトレーショ ンを組み合わせた方法が、 濃縮と脱塩を同 時に行えるので好ま しい。 なお、 この際に用いることのできる限外濾過 膜は、 分画分子量が 1 0 k D a以下のものであればどのような限外濾過 膜でも良い。 このゥシ I G F— 1含有組成物濃縮液をそのまま用いるこ ともできるが、 必要に応じて、 噴霧乾燥や凍結乾燥などの方法によりゥ シ I G F— 1含有組成物の乾燥粉末を得ることもできる。 さらに、 ゥシ I G F— 1は比較的熱に安定な性質を有するので、 通常行われているよ うな加熱殺菌の工程を加えることも可能である。  Next, the eluate containing the thus-obtained IGF-1 fraction is subjected to a conventional method, for example, ion exchange resin, reverse osmosis membrane, ultrafiltration membrane, dialysis membrane. Desalting and concentration can be performed by a method using an electrodialysis membrane, a gel filtration carrier, or the like, or by a method combining these methods. A combination of filtration and diamond filtration is preferred, since concentration and desalting can be performed simultaneously. The ultrafiltration membrane that can be used at this time may be any ultrafiltration membrane as long as the molecular weight cut off is 10 kDa or less. The concentrate of the composition containing the IGF-1 can be used as it is, but if necessary, a dry powder of the composition containing the IGF-1 can be obtained by a method such as spray drying or freeze drying. In addition, since PIG IGF-1 has a relatively heat-stable property, it is possible to add a heat sterilization step as is usually performed.
本発明の方法で得られたゥシ I G F— 1含有組成物中のゥシ I G F— 1含量を抗 I G F— 1抗体を用いた免疫学的測定法により測定したとこ ろ、 原料からの I G F— 1の回収率は平均 40 %程度であつた。 なお、 初乳から酸抽出と陽ィォン交換ク口マトグラフィ一を組み合わせて回収 したゥシ I G F— 1の回収率は、 文献 〔B i o c h e m. J . 、 第 23 3巻、 207~21 3頁、 1 986年〕 によると 25%であり、 本発明 の方法はそれを上回るものであった。 The IGF-1 content of the IGF-1 containing composition obtained by the method of the present invention was measured by an immunoassay using an anti-IGF-1 antibody. On the other hand, the recovery of IGF-1 from the raw material was about 40% on average. In addition, the recovery rate of PIG IGF-1 recovered from colostrum by a combination of acid extraction and positive ion exchange chromatography was described in literature [Biochem. J., Vol. 233, pp. 207-213, 1 986], 25%, and the method of the present invention exceeded it.
また、 本発明の方法によれば、 陽イオン交換体に吸着しなかった乳成 分を再利用することが可能であり、 工程も煩雑でないので、 酸抽出と陽 イオン交換ク口マ トグラフィ 一を組み合わせた方法より も実用的である。 なお、 このゥシ I G F— 1含有組成物中には、 カゼインゃホエー蛋白 質などの成分が含まれており、 特に、 ラク トフエリ ン、 ラク トパ一ォキ シダーゼ、 セク レタリーコンポーネン トなどの蛋白質が生理活性を有す るが、 これらの蛋白質は、 ゥシ I G F— 1の生理作用に何ら影響を与え るものではないので、 これらの蛋白質が含まれていても実質的な問題は ないが、 不都合がある場合は、 加熱などの処理によりこれらの蛋白質を 失活させることもできる。 また、 ラク トパ一ォキシダーゼに関しては、 再クロマ トグラフィ 一などの方法で分離するか、 酸性状態にして失活さ せることも可能である。  Further, according to the method of the present invention, it is possible to reuse milk components that have not been adsorbed on the cation exchanger, and the steps are not complicated, so that acid extraction and cation exchange chromatography are not necessary. It is more practical than the combined method. In addition, the composition containing casein and whey protein is contained in the composition containing the casein IGF-1. In particular, proteins such as lactoferrin, lactopaboxidase, and secretory components are included. Although these proteins have physiological activity, these proteins do not affect the physiological action of the IGF-1 at all, so there is no substantial problem with the inclusion of these proteins. If inconvenient, these proteins can be inactivated by treatment such as heating. In addition, lactopoxidase can be separated by a method such as rechromatography or inactivated by making it acidic.
本発明の方法によって得られたゥシ I G F— 1含有組成物は、 骨強化 作用を有するので、 飲食品、 医薬、 飼料などに添加し、 骨粗鬆症の予防 や治療などの効果を賦与することができる。 また、 このゥシ I G F— 1 含有組成物を含有する飲食品、 医薬、 飼料などに、 塩化カルシウム、 炭 酸カルシウム、 乳酸カルシウム、 卵殻、 あるいは乳由来のカルシウムな どの吸収性良好なカルシウムを添加することにより、 これらの効果をさ らに增すことが可能となる。 なお、 このゥシ I G F— 1含有組成物につ いては、 ラッ トによる動物試験の結果、 急性毒性は認められなかった。 図面の簡単な説明 The pharmaceutical composition containing IGF-1 obtained by the method of the present invention has a bone-strengthening effect, and thus can be added to foods and drinks, pharmaceuticals, feeds, and the like to impart effects such as prevention and treatment of osteoporosis. . In addition, calcium, calcium carbonate, calcium lactate, eggshell, or calcium with good absorbability, such as milk-derived calcium, is added to foods, drinks, medicines, feeds, etc., containing this composition containing IGF-1. As a result, these effects can be further enhanced. As a result of animal tests using rats, no acute toxicity was observed for the composition containing IGF-1. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 下記実施例 1から 9で得られたゥシ I GF— 1含有組成物の 骨芽細胞増殖促進効果について示したものである。  FIG. 1 shows the osteoblast proliferation-promoting effect of the composition containing PIG IGF-1 obtained in Examples 1 to 9 below.
図 2は、 下記実施例 5で得られたゥシ I G F— 1含有組成物の骨強化 作用について示したものである。 実施例  FIG. 2 shows the bone-strengthening action of the composition containing PIG IGF-1 obtained in Example 5 below. Example
次に、 実施例を挙げて本発明を具体的に説明する。  Next, the present invention will be specifically described with reference to examples.
実施例 1 Example 1
150 °C 5秒間加熱殺菌したチーズホエー （p H 6. 0) 50 1を、 脱イオン水で十分洗浄したスルホン化キトパール （富士紡績社製） 50 0 gを充填したカラムに、 流速 25 m 1 Z分で通液した。 通液後、 脱ィ オン水でスルホン化キトパールを十分洗浄した後、 0. 2 8M塩化ナト リウムを含む 0. 02M炭酸緩衝液 （p H 7. 0) で吸着したゥシ I G F— 1を溶出した。 そして、 この溶出液を分画分子量 10 k D aの限外 濾過膜で脱塩、 濃縮した後、 凍結乾燥して粉末状のゥシ I G F— 1含有 組成物 45 0 m gを得た。 このゥシ I G F— 1含有組成物中に含まれる ゥシ I G F— 1の含量をラジオィムノアッセィ （R I A) で測定したと ころ、 であることが判つた o  A column filled with 500 g of cheese whey (pH 6.0) 501 sterilized by heating at 150 ° C for 5 seconds and 500 g of sulfonated chitopearl (manufactured by Fuji Boseki Co., Ltd.) thoroughly washed with deionized water was applied at a flow rate of 25 m 1 The liquid was passed in Z minutes. After passage, wash the sulfonated chitopearl thoroughly with deionized water, and elute the IGF-1 adsorbed with 0.02M carbonate buffer (pH 7.0) containing 0.2M sodium chloride. did. The eluate was desalted and concentrated using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa, and then lyophilized to obtain 450 mg of a powdery IGF-1 containing composition. When the content of the PG IGF-1 contained in the PG I GF-1 containing composition was measured by a radioimmunoassay (RIA), it was found that
実施例 2 Example 2
未殺菌の脱脂乳 （p H 6. 5 ) 1, 000 1を、 脱イオン水で十分洗 浄した S P トーョーパール （東ソ一社製） 1 k gを充填したカラムに、 流速 30m l Z分で通液した。 通液後、 脱ィォン水で S P トーョ一パー ルを十分洗浄した後、 0. 1 5M塩化ナ ト リ ウムを含む 0. 05M炭酸 緩衝液 （ p H 7. 5 ) で吸着したゥシ I G F— 1を溶出した。 そして、 この溶出液を分画分子量 8 k D aの膜で限外濾過およびダイヤフィ ルト レ一シヨ ンを行って脱塩、 濃縮した後、 凍結乾燥して粉末状の I G F— 1含有組成物 50 gを得た。 このゥシ I G F— 1含有組成物中に含まれ るゥシ I G F— 1の含量をラジオィムノアッセィ （R I A) で測定した ところ、 65 /i gZ gであることが判った。 1,000 sterile non-sterilized skim milk (pH 6.5) is passed through a column packed with 1 kg of SP Toyo Pearl (manufactured by Tosoh I), thoroughly washed with deionized water, at a flow rate of 30 ml / min. Liquid. After passing the solution through, thoroughly wash the SP Toyo Par with deionized water, and then add 0.15M sodium chloride and 0.05M carbonate The adsorbed IGF-1 was eluted with a buffer (pH 7.5). The eluate is subjected to ultrafiltration and diafiltration using a membrane having a molecular weight cut-off of 8 kDa, desalted and concentrated, and then freeze-dried to obtain a powdery composition containing IGF-1 50. g was obtained. When the content of the PIG IGF-1 contained in the PIG IGF-1 containing composition was measured by a radioimmunoassay (RIA), it was found to be 65 / igZg.
実施例 3 Example 3
10重量％濃度となるようホエー蛋白質濃縮物 （WP C) を蒸留水で 十分溶解してホェ一蛋白質溶液 （ P H 6. 8) 40 1を調製した。 この ホェ一蛋白質溶液を、 脱イオン水で十分洗浄した CM—セルロフアイ ン (生化学工業社製） 400 gを充填したカラムに、 流速 2 Om 1 Z分で 通液した。 通液後、 0. 02 M塩化ナト リウムを含む 0. 03Mリ ン酸 緩衝液 （p H 7. 4) で CM—セル口ファイ ンを十分洗浄した後、 0. 20 M塩化ナ ト リウムを含む 0. 10 Mクェン酸緩衝液 （ p H 6. 2) で吸着したゥシ I G F— 1を溶出した。 そして、 この溶出液を電気透析 (E D) 法により脱塩、 濃縮した後、 凍結乾燥して粉末状のゥシ I G F 一 1含有組成物 1. 3 gを得た。 このゥシ I G F— 1含有組成物中に含 まれるゥシ I G F— 1の含量をラジオィムノアッセィ （R I A) で測定 したところ、 35 gZ gであることが判った。 Whey protein concentrate to be a 10% strength by weight (WP C) was prepared Hoe one protein solution (P H 6. 8) 40 1 fully dissolved in distilled water. This whey protein solution was passed through a column packed with 400 g of CM-cellulofine (manufactured by Seikagaku Corporation) sufficiently washed with deionized water at a flow rate of 2 Om 1 Z min. After passing the solution through, thoroughly wash the CM-cell fin with 0.03 M phosphate buffer (pH 7.4) containing 0.02 M sodium chloride, and then add 0.20 M sodium chloride. The adsorbed IGF-1 was eluted with a 0.10 M citrate buffer (pH 6.2). The eluate was desalted and concentrated by an electrodialysis (ED) method, and then freeze-dried to obtain 1.3 g of a powdery IGF-11-containing composition. The content of the IGF-1 contained in the composition containing the IGF-1 was measured by a radioimmunoassay (RIA) and found to be 35 gZg.
実施例 4 Example 4
1 0重量％濃度になるようホエー蛋白質単離物 （WP I ) を蒸留水で 十分溶解してホェ一蛋白質溶液 （p H6. 5) 80 1を調製した。 この ホエー蛋白質溶液を、 脱イオン水で十分洗浄した S P—セファロース (フアルマシア社製） 800 gを充填したカラムに、 流速 24m 1 Z分 で通液した。 通液後、 0. 01 M塩化ナ ト リ ウムを含む 0. 05M炭酸 緩衝液 （p H 7. 6) で S P—セファロースを十分洗浄した後、 0. 1 0 M塩化ナ ト リ ウムを含む 0. 20 Mクェン酸緩衝液 （ p H 5. 7) で 吸着したゥシ I G F— 1を溶出した。 そして、 この溶出液をイオン交換 クロマ トグラフィーにより脱塩した後、 噴霧乾燥して粉末状のゥシ I G F— 1含有組成物 29. 6 gを得た。 このゥシ I GF— 1含有組成物中 に含まれるゥシ I G F— 1の含量をラジオイムノアッセィ （R I A) で 測定したところ、 5 1. 2 gZgであることが判った。 The whey protein isolate (WPI) was sufficiently dissolved in distilled water to a concentration of 10% by weight to prepare a whey protein solution (pH 6.5) 801. This whey protein solution was passed through a column packed with 800 g of SP-Sepharose (manufactured by Pharmacia) thoroughly washed with deionized water at a flow rate of 24 m 1 Z minute. After passing through, 0.01 M containing sodium chloride 0.05 M carbonate After the SP-Sepharose was thoroughly washed with buffer (pH 7.6), it was adsorbed with 0.20 M citrate buffer (pH 5.7) containing 0.1 M sodium chloride. IGF-1 was eluted. Then, the eluate was desalted by ion exchange chromatography, and then spray-dried to obtain 29.6 g of a powdery IGF-1 containing composition. The content of the IGF-1 contained in the composition containing the IGF-1 was measured by radioimmunoassay (RIA) and found to be 51.2 gZg.
実施例 5 Example 5
1 2 1て、 30秒間加熱殺菌した酸カゼィ ンホエー 3 tを、 重炭酸ナ ト リ ウムで p H 6. 0に調整した後、 脱イオン水で十分洗浄した S P— セフアデックス （ファルマシァ社製） 30 k gを充填したカラムに、 流 速 1 0 1 分で通液した。 通液後、 脱ィォン水で S P—セフアデックス を十分洗浄した後、 0. 25 M塩化ナ ト リ ウムを含む 0. 05M炭酸緩 衝液 （p H 7. 0) で吸着したゥシ I G F— 1を溶出した。 そして、 こ の溶出液を逆浸透 （RO) 膜で脱塩、 濃縮した後、 噴霧乾燥して粉末状 のゥシ I G F— 1含有組成物 1 76 gを得た。 このゥシ I GF— 1含有 組成物中に含まれるゥシ I G F— 1の含量をラジオイムノアッセィ （R I A) で測定したところ、 1 06 z gZgであることが判つた。  1 2 3 Then, 3 t of acid casein whey heat-sterilized was adjusted to pH 6.0 with sodium bicarbonate, and then sufficiently washed with deionized water. SP-Sephadex (Pharmacia) The solution was passed through a column packed with 30 kg at a flow rate of 101 minutes. After passing the solution, the SP-Sephadex was thoroughly washed with deionized water, and then absorbed with a 0.05 M carbonate buffer (pH 7.0) containing 0.25 M sodium chloride. IGF-1 Was eluted. The eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and then spray-dried to obtain 176 g of a powdery IGF-1 containing composition in powder form. When the content of the calcium IGF-1 contained in the composition containing the calcium IGF-1 was measured by radioimmunoassay (RIA), it was found to be 106 z gZg.
実施例 6 Example 6
1 0重量％濃度になるようホエー蛋白質濃縮物 （WP C) を蒸留水で 十分溶解してホェ一蛋白質溶液 （PH 6. 8) を調製した後、 この溶液 を 90°Cで 1 0分間加熱し、 17, 000 X Gで遠心分離して得られた 上清 1 0 1を、 脱ィォン水で十分洗浄したィ ンデイオン S 3 (オルガノ 社製） 500 gを充填したカラムに、 流速 1 8m 1 /分で通液した。 通 液後、 0. 07 Mト リスー塩酸緩衝液でイ ンディオン S 3を十分洗浄し た後、 0. 3 M塩化ナト リゥム溶液 （ p H 7. 3 ) で吸着したゥシ I G F— 1を溶出した。 そして、 この溶出液をゲル濾過クロマ トグラフィー で脱塩した後、 凍結乾燥して粉末状のゥシ I G F— 1含有組成物 1. 4 gを得た。 このゥシ I G F— 1含有組成物中に含まれるゥシ I G F— 1 の含量をラジオイムノアツセィ （R I A) で測定したところ、 42 g Zgであることが判った。 A whey protein concentrate (WP C) is sufficiently dissolved in distilled water to a concentration of 10% by weight to prepare a whey protein solution (PH 6.8), and this solution is heated at 90 ° C for 10 minutes. The supernatant obtained by centrifugation at 17,000 XG was applied to a column packed with 500 g of Indeion S 3 (manufactured by Organo) thoroughly washed with deionized water at a flow rate of 18 m 1 / In minutes. After the infusion, wash Indion S 3 thoroughly with 0.07 M Tris-HCl buffer. After that, the adsorbed IGF-1 was eluted with a 0.3 M sodium chloride solution (pH 7.3). The eluate was desalted by gel filtration chromatography, and then lyophilized to obtain 1.4 g of a powdery IGF-1 containing composition. When the content of the IGF-1 contained in the composition containing the IGF-1 was measured by radioimmunoassay (RIA), it was found to be 42 g Zg.
実施例 7 Example 7
1 50°C、 5秒間加熱殺菌した初乳 （p H 6. 8) 2 1を、 脱イオン 水で十分洗浄した S—スフユロシル （ I B F社製） 1 00 gを充填した カラムに、 流速 20m l Z分で通液した。 通液後、 脱イオン水で S—ス フエ口シルを十分洗浄した後、 0. 3 M塩化ナト リゥム溶液 （ p H 7. 3 ) で吸着したゥシ I G F— 1を溶出した。 そして、 この溶出液を分画 分子量 1 0 k D aの膜で限外濾過およびダイャフィ ルトレーショ ンを行 つて脱塩、 濃縮した後、 凍結乾燥して粉末状のゥシ I G F— 1含有組成 物 1. 5 gを得た。 このゥシ I G F— 1含有組成物中に含まれるゥシ I GF— 1の含量をラジオィムノアッセィ （R I A) で測定したところ、 1 84 // g Z gであることが判った。  1 A column packed with 100 g of colostrum (pH 6.8) 21 sterilized by heating at 50 ° C for 5 seconds and 100 g of S-sufurosil (manufactured by IBF) thoroughly washed with deionized water was applied at a flow rate of 20 ml. The liquid was passed in Z minutes. After passing the solution through, the S-sphere mouth was thoroughly washed with deionized water, and the adsorbed IGF-1 was eluted with a 0.3 M sodium chloride solution (pH 7.3). The eluate is subjected to ultrafiltration and membrane filtration with a membrane having a molecular weight cut-off of 10 kDa, desalted and concentrated, and then lyophilized to form a powdered IGF-1 containing composition 1 .5 g were obtained. When the content of the calcium IGF-1 contained in the composition containing the calcium IGF-1 was measured by a radioimmunoassay (RIA), it was found to be 184 // g Z g.
実施例 8 Example 8
1 2 1°C、 30秒間加熱殺菌した脱脂乳 （p H 6. 5 ) 1 00 1を、 脱ィォン水で十分洗浄したマイクロレップ Sス トロングカチォンェクス チェンジサポー ト （バイオラッ ド社製） 0. 5 k gを充填したカラムに、 流速 35m l /分で通液した。 通液後、 0. 05 M炭酸緩衝液でマイク 口レップ Sス トロングカチオンエクスチェンジサポー トを十分洗浄した 後、 0. 20M塩化ナ ト リウムを含む 0. 1 0Mトリスー塩酸緩衝液 ( p H 7. 4 ) で吸着したゥシ I G F— 1を溶出した。 そして、 その溶 出液を逆浸透 （R0) 膜で脱塩、 濃縮した後、 噴霧乾燥して粉末状のゥ シ I G F— 1含有組成物 1. 3 gを得た。 このゥシ I G F— 1含有組成 物中に含まれるゥシ I G F— 1の含量をラジオイムノアッセィ （R I A) で測定したところ、 1 1 4 / g/gであることが判つた。 1 2 1 ° C, 30 seconds of heat sterilized skim milk (pH 6.5) 1001, Microrep S Strong Cationex Change Support (BioRad Co., Ltd.) thoroughly washed with deionized water The liquid was passed through a column packed with 0.5 kg at a flow rate of 35 ml / min. After passing the solution through, sufficiently wash the Microphone Rep S Strong Cation Exchange Support with 0.05 M carbonate buffer, then add 0.10 M Tris-HCl buffer containing 0.20 M sodium chloride (pH 7 The IGF-1 adsorbed in step 4) was eluted. And the melting The discharged liquid was desalted and concentrated by a reverse osmosis (R0) membrane, and then spray-dried to obtain 1.3 g of a powdery IGF-1 containing composition. The content of the IGF-1 contained in the composition containing the IGF-1 was measured by radioimmunoassay (RIA) and found to be 114 / g / g.
実施例 9 Example 9
未殺菌のチーズホエー （ p H 6. 5 ) 20 1を、 脱イオン水で十分洗 浄した C一スフヱロシル （ I B F社製） 300 gを充填したカラムに、 流速 25m lノ分で通液した。 通液後、 脱ィォン水で C一スフエロシル を十分洗浄し、 さらに、 0. 07 Mリ ン酸緩衝液 （ p H 7. 2 ) で十分 洗浄した後、 0. 25M塩化ナ ト リ ウムを含む 0. 05Mクェン酸緩衝 液 （ p H 6. 5 ) で吸着したゥシ I G F— 1を溶出した。 そして、 この 溶出液をナノフィ ルトレ一シヨ ン膜で脱塩、 濃縮した後、 凍結乾燥して 粉末状のゥシ I G F— 1含有組成物 890 m gを得た。 このゥシ I G F 一 1含有組成物中に含まれるゥシ I G F— 1の含量をラジオイムノアツ セィ （R I A) で測定したところ、 43 gZgであることが判った。 試験例 1  Unsterilized cheese whey (pH 6.5) 201 was passed through a column packed with 300 g of C-sfluorosyl (manufactured by IBF) thoroughly washed with deionized water at a flow rate of 25 ml. After passing through, wash C-sulferosyl thoroughly with deionized water, and further thoroughly with 0.07 M phosphate buffer (pH 7.2), then contain 0.25 M sodium chloride The adsorbed IGF-1 was eluted with a 0.05 M citrate buffer (pH 6.5). The eluate was desalted and concentrated using a nanofiltration membrane, and then lyophilized to obtain 890 mg of a powdery IGF-1 containing composition. When the content of the Pacific IGF-1 contained in the composition containing the Pacific IGF-11 was measured by radioimmunoassay (RIA), it was found to be 43 gZg. Test example 1
実施例 1から 9で得られたゥシ I GF— 1含有組成物について、 骨芽 細胞増殖効果を調べた。  The osteoblast-proliferating effects of the compositions containing PIG IGF-1 obtained in Examples 1 to 9 were examined.
培養骨芽細胞様株 （MC 3 T 3— Ε Γ) を 96穴の平底細胞培養ブレ 一卜に撒き込み、 0. 3重量％ゥシ血清を含む α— MEM培地 （F 1 0 w L a b o r a t o r i e s社製） で 1 8時間培養した。 なお、 この 培養に際しては、 培地 1 00 z l に対して、 ゥシ I G F— 1含有組成物 を 0. 5重量％濃度に溶解した溶液 2 1を添加した。 培養後、 ト リチ ゥムでラベルしたチミ ジンを添加し、 2時間後に細胞に取り込まれたチ ミ ジンの放射活性を測定することにより、 骨芽細胞の増殖活性を求めた。 その結果を図 1に示す。 なお、 図 1では、 培地にゥシ I G F— 1含有組 成物を添加しなかった群の放射活性を 1 00%とし、 放射活性からゥシ I G F— 1含有組成物を添加した群の細胞増殖活性を示した。 これによ ると、 実施例 1から 9で得られたゥシ I G F— 1含有組成物を添加した 群は、 ゥシ I G F— 1含有組成物を添加しなかった群と比べ、 1. 8〜 2. 7倍の骨芽細胞の ± 殖活性を示した。 A cultured osteoblast-like cell line (MC 3T3—Ε Γ) was inoculated into a 96-well flat bottom cell culture plate, and α-MEM medium containing 0.3% by weight of serum (F10 w Laboratories) was added. For 18 hours. At the time of this culturing, a solution 21 containing a composition containing the IGF-1 at a concentration of 0.5% by weight was added to 100 zl of the medium. After culture, thymidine labeled with tritium was added, and 2 hours later, the radioactivity of thymidine incorporated into the cells was measured to determine the osteoblast proliferation activity. Figure 1 shows the results. In FIG. 1, the radioactivity of the group to which the medium containing no IGF-1 was added was 100%, and the cell proliferation of the group to which the medium containing the IGF-1 was added was determined from the radioactivity. Showed activity. According to this, the group to which the IGF-1 containing composition obtained in Examples 1 to 9 was added was 1.8 to 1.8 times less than the group to which no IGF-1 containing composition was added. 2. It showed a 7-fold osteoblast proliferation activity.
試験例 2 Test example 2
実施例 5で得られたゥシ I G F— 1含有組成物について、 動物実験に より骨強化作用を調べた。  The bone strengthening effect of the composition containing PIG IGF-1 obtained in Example 5 was examined by animal experiments.
実験動物は 4週齢の S D系雌ラッ トを用い、 1試験群 7匹で行った。 骨粗鬆症モデルラッ トを 1週間予備飼育した後、 卵巣摘出手術を施し、 さらに、 カルシウム欠乏食で 5週間飼育して実験に供した。 また、 疑似 手術のみを施し、 卵巣を摘出しないシャムラッ トも実験に供した。 そし て、 骨粗鬆症モデルラッ トを対照群 （A群） 、 ゥシ I G F— 1含有組成 物投与群 （ B) およびゥシ I G F— 1含有組成物 +カルシウム投与群 (C) の 3群に分け、 表 1に示す被験飼料でそれぞれ 3週間飼育した。  The experimental animals were 4-week-old SD female rats, and each test group consisted of 7 animals. The osteoporosis model rat was preliminarily reared for one week, subjected to ovariectomy, and further reared on a calcium-deficient diet for five weeks before the experiment. In addition, shamrats that had undergone only sham surgery and had no ovaries removed were also used in the experiment. The osteoporosis model rat was divided into three groups: a control group (group A), a group administered with the IGF-1 containing composition (B), and a group containing the IGF-1 plus calcium (C). Each of the test feeds shown in 1 was bred for 3 weeks.
(A) 群 (B) 群 (C) 群 庶糖 51.05(%) 51.46(%) 50.62(%) カゼイ ン 20.0 18.0 18.0 コーンスターチ 15.0 15.0 15.0 セノレロース 5.0 5.0 5.0 コーン油 5.0 5.0 5.0 ビタ ミ ン混合 1.0 1.0 1.0 ミネラル混合 2.65 2.43 3.27(A) Group (B) Group (C) Group Sucrose 51.05 (%) 51.46 (%) 50.62 (%) Casein 20.0 18.0 18.0 Corn starch 15.0 15.0 15.0 Senorelose 5.0 5.0 5.0 Corn oil 5.0 5.0 5.0 Vitamin mixture 1.0 1.0 1.0 Mineral mixture 2.65 2.43 3.27
D L—メチォニン 0.3 0.3 0.3 ゥシ I G F— 1含有組成物 一 1.81 1.81 なお、 カゼィ ン 2重量％に相当する窒素量に置換して、 ゥシ I G F— 1含有組成物 1. 8 1重量％を添加した。 また、 飼料中のカルシウム量、 リ ン量およびマグネシゥム量については、 飼料 1 00 g当たり、 300 mg、 230m gおよび 50mgとした。 さらに、 （C) 群については、 カルシウム量およびリ ン量を飼料 100 g当たり、 520 mgおよび 4 00 m gとした。 DL-methionine 0.3 0.3 0.3 psi IGF-1 containing composition 1 1.81 1.81 Replace with the nitrogen equivalent to 2 wt% casein, and add 1.8 pt 1 wt% of the IGF-1 containing composition. did. The amounts of calcium, phosphorus and magnesium in the feed were 300 mg, 230 mg and 50 mg per 100 g of feed. Furthermore, for group (C), calcium and phosphorus were set to 520 mg and 400 mg per 100 g of feed, respectively.
そして、 3週間後に各群のラッ 卜の両側大腿骨を摘出し、 破断特性装 置で骨強度を測定した。 その結果を図 2に示す。 これによると、 大腿骨 破断応力は、 対照群 （A) に比べてゥシ I GF— 1含有組成物投与群 (B) で統計学的に有意に高い値を示した。 さらに、 ゥシ I G F— 1含 有組成物 +カルシウム投与群 （C) は、 ゥシ I G F— 1含有組成物投与 群 （B) に比べて統計学的に有意に高い値を示した。  Three weeks later, both femurs of the rats in each group were excised, and the bone strength was measured using a fracture characteristic device. Figure 2 shows the results. According to this, the femoral fracture stress showed a statistically significantly higher value in the group administered with the IGF-1 containing composition (B) than in the control group (A). Further, the group administered with the composition containing calcium IGF-1 and calcium (C) showed a statistically significantly higher value than the group administered with the composition containing calcium IGF-1 (B).
次に、 本発明の方法で製造したゥシ I G F— 1含有組成物を添加した 飲食品について、 参考例を示す。  Next, reference examples will be given of foods and drinks to which the composition containing the IGF-1 produced by the method of the present invention is added.
参考例 1 Reference example 1
常法に従い、 表 2に示す組成のゥシ I G F— 1含有組成物入り果汁飲 料を製造した。  In accordance with a conventional method, a fruit juice drink containing the composition containing the composition shown in Table 2 was produced.
表 2 混合異性化糖 5. 0 (重量％) 果汁 1 0 . 0 Table 2 Mixed isomerized sugar 5.0 (% by weight) Fruit juice 10.0
クェン酸 0 . 5  Cuic acid 0.5
ゥシ I G F 1含有組成物 0 . 5  Composition containing IGF1 0.5
香料 0 .  Spice 0.
カルシウム 0 .  Calcium 0.
水 7 3 . 5 参考例 2  Water 73.5 Reference example 2
常法に従い、 表 3に示す組成のゥシ I G F 含有組成物入りカルシ ゥム剤を製造した。  According to a conventional method, a calcium permeate containing the composition containing the composition shown in Table 3 was produced.
表 3 含水結晶ぶどう糖 7 3 . 5 (重量％) ゥシ I G F— 1含有組成物 2 0 . 0  Table 3 Hydrous dextrose 73.5 (% by weight) Composition containing IGF-1
カルシウム 5 . 0  Calcium 5.0
シュガーエステル 1 . 0  Sugar ester 1.0
香料 0 . 5  Spice 0.5
産業上の利用可能性 Industrial applicability
本発明の方法によると、 牛乳または牛乳由来の原料からゥシ I G F— 1を高度に含有するゥシ I G F— 1組成物を提供することが可能となる。 このゥシ I G F— 1含有組成物は、 骨強化作用を有することから、 各種 の骨関節疾患、 特に骨粗鬆症の予防あるいは治療に有用である。 また、 ヒ 卜の成長期にこのゥシ I G F— 1組成物を摂取させることにより、 最 大骨量を增加させることができる。 したがって、 このゥシ I G F— 1含 有組成物は、 飲食品、 医薬、 飼料などの素材として有用である。 ADVANTAGE OF THE INVENTION According to the method of this invention, it becomes possible to provide the IGF-1 composition which contains highly IGF-1 from milk or a raw material derived from milk. Since the composition containing bone IGF-1 has a bone strengthening effect, it is useful for preventing or treating various osteoarticular diseases, particularly osteoporosis. Also, By ingesting this IGF-1 composition during the human growth period, the maximum bone mass can be increased. Therefore, this composition containing IGF-1 is useful as a material for foods and drinks, medicines, feeds and the like.

Claims

請 求 の 範 囲 The scope of the claims
1. 牛乳または牛乳由来の原料を陽イオン交換体と接触させて、 ゥシ インスリ ン様増殖因子一 1を吸着させた後、 溶出して回収することを特 徴とするゥシィ ンスリ ン様増殖因子一 1含有組成物の製造法。 1. A cine-sulin-like growth factor characterized by contacting milk or a milk-derived material with a cation exchanger to adsorb ゥ -insulin-like growth factor-11 and then eluting and recovering it. A method for producing a 1-containing composition.
2. 溶出に用いる溶出液の塩濃度が 0. 1M以上、 0. 3M以下であ り、 p Hが 5. 6以上 8未満である請求の範囲第 1項記載の製造法。 2. The production method according to claim 1, wherein the salt concentration of the eluate used for elution is 0.1 M or more and 0.3 M or less, and the pH is 5.6 or more and less than 8.
3. 予め加熱した牛乳または牛乳由来の原料を用いる請求の範囲第 1 項または 2項記載の製造法。 3. The method according to claim 1 or 2, wherein milk or milk-derived raw material heated in advance is used.
4. 陽イオン交換体がスルホン酸基を交換基として持つ強酸性陽ィォ ン交換体である請求の範囲第 1〜 3項のいずれかに記載の製造法。 4. The process according to any one of claims 1 to 3, wherein the cation exchanger is a strongly acidic cation exchanger having a sulfonic acid group as an exchange group.
5. ゥシイ ンスリ ン様増殖因子一 1を含有する溶出液を、 さらに分画 分子量 1 0 k D a以下の限外瀘過膜で脱塩、 濃縮する請求の範囲第 1〜 4項のいずれかに記載の製造法。 5. The eluate containing ゥ -insulin-like growth factor-11 is further desalted and concentrated with an ultrafiltration membrane having a cut-off molecular weight of 10 kDa or less, wherein the salt is concentrated. Production method described in 1.
PCT/JP1995/000620 1994-03-31 1995-03-31 Process for producing composition containing bovine insulin-like growth factor 1 WO1995026984A1 (en)

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WO2000062785A1 (en) * 1999-04-15 2000-10-26 Takara Shuzo Co., Ltd. Remedies
CN106699869A (en) * 2008-05-14 2017-05-24 维多利亚农业服务控股公司 Angiogenin-enriched milk fractions

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CA2621739A1 (en) 2005-09-09 2007-03-15 Murray Goulburn Co-Operative Co. Limited Composition of whey growth factor extract for reducing muscle inflammation
CN101282763B (en) * 2005-09-09 2012-09-26 墨累古尔本合作有限公司 Milk derived composition and use to enhance muscle mass or muscle strength

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63501567A (en) * 1985-08-22 1988-06-16 グロペップ　プロプライエタリー　リミテッド growth factors

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63501567A (en) * 1985-08-22 1988-06-16 グロペップ　プロプライエタリー　リミテッド growth factors

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062785A1 (en) * 1999-04-15 2000-10-26 Takara Shuzo Co., Ltd. Remedies
CN106699869A (en) * 2008-05-14 2017-05-24 维多利亚农业服务控股公司 Angiogenin-enriched milk fractions

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