JP6562958B2 - Fermented milk and method for producing the same - Google Patents

Description

  The present invention relates to a novel fermented milk and a method for producing the same. By containing a specific milk component, the fermented milk of the present invention is useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism and arthritis.

  In recent years, various bone-related diseases such as osteoporosis, fractures, and back pain are increasing with the aging of the world on a global scale, which is a serious social problem. This is considered to be caused by insufficient intake of calcium, a decrease in calcium absorption capacity, and postmenopausal hormone imbalance. In order to prevent various bone diseases such as osteoporosis, fractures, and back pain, bone formation by osteoblasts is promoted from a young age to increase the amount of bone in the body as much as possible, and maximum bone mass and bone strength (bone density) + Bone quality) is considered effective. Bone quality refers to bone microstructure, turnover, micro fracture, and calcification. In addition, as a method for preventing various bone diseases such as osteoporosis, fractures, and back pain, it is conceivable to suppress bone resorption by osteoclasts. Bone repetitively undergoes balanced resorption and formation (remodeling), but due to changes in hormone balance after menopause, etc., bone resorption exceeds bone formation, which leads to various bone diseases such as osteoporosis, fractures, and back pain Cause. Therefore, it is possible to strengthen the bone as a result by suppressing bone resorption by osteoclasts and keeping the bone strength constant.

  Under these circumstances, for the purpose of strengthening bones, calcium salts such as calcium carbonate, calcium phosphate, and calcium lactate, and natural calcium preparations such as whey calcium, beef bone meal, eggshell, etc., alone for pharmaceuticals, foods and drinks, feeds, etc. What is added to is taken. Or these calcium agents are ingested together with substances having a calcium absorption promoting effect such as casein phosphopeptides and oligosaccharides in medicines, foods and drinks, feeds and the like. However, the absorption rate of calcium when ingesting these calcium salts and natural calcium preparations added to foods and drinks is 50% or less, and it is said that a lot of calcium is not absorbed and is discharged outside the body. Yes. In addition, even if calcium is absorbed by the body, the affinity to bone varies depending on its form and the type of other nutritional components that are taken at the same time, so it does not necessarily show an effect of improving bone metabolism or strengthening bone. There is also. In addition, female hormone preparations, active vitamin D3 preparations, vitamin K2 preparations, bisphosphonate preparations, calcitonin preparations, etc. are known as drugs for the treatment of osteoporosis and bone strengthening, and new drug development such as anti-RANKL antibodies has been developed. It is being advanced. However, these medicines may have side effects such as tinnitus, headache, and loss of appetite. Furthermore, these substances cannot be added to food and drink at present from the viewpoints of safety and cost. On the other hand, due to the nature of various bone diseases such as osteoporosis, fractures, and back pain, it can be taken orally for a long period of time, acting to promote bone formation and suppress bone resorption to increase bone strength and prevent or Development of food and drink that can be expected to have a therapeutic effect is desired.

JP-A-8-151331 Japanese Patent Laid-Open No. 10-7585 Japanese Patent Laid-Open No. 2000-28187

An object of the present invention is to provide fermented milk useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism and arthritis.

  The present inventors contain angiogenin and / or angiogenin degradation product, and contain cystatin and / or cystatin degradation product in a specific range of mass ratio with respect to angiogenin and / or angiogenin degradation product. It has been found that by ingesting fermented milk, an effect of effectively increasing bone density can be obtained, and the present invention has been completed.

That is, this invention consists of the following structures.
(1) 0.9 mg to 150 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 0.1. Fermented milk containing in the range of 006 to 1.7.
(2) A method for preventing a bone disease, wherein 100 g / day or more of the fermented milk according to (1) is ingested.
(3) The method for producing fermented milk according to the above (1), which comprises a step of adding angiogenin and / or angiogenin degradation product and cystatin and / or cystatin degradation product to a milk raw material, sterilizing, and fermenting.
(4) The method for producing fermented milk according to the above (1), comprising a step of adding angiogenin and / or angiogenin degradation product and cystatin and / or cystatin degradation product to the sterilized milk raw material and fermenting.

  The fermented milk of the present invention has an effect of strengthening bones, and is useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism and arthritis.

The features of the fermented milk of the present invention include angiogenin and / or angiogenin degradation products in a specific range of amounts, and angiogenin and / or angiogenin degradation products and cystatin in a specific range of mass ratio. And / or to contain a cystatin degradation product.
Generally, fermented milk contains about 0.2 to 0.8 mg / 100 g of angiogenin and / or angiogenin degradation products and about 0.4 to 1.1 mg / 100 g of cystatin and / or cystatin degradation products. Yes.
On the other hand, the fermented milk of the present invention is prepared by adding angiogenin and / or an angiogenin degradation product and cystatin and / or a cystatin degradation product to angiogenin and / or an angiogenin degradation product of 0.9 mg to 150 mg / 100 g is contained, and cystatin and / or cystatin degradation product is contained in a mass ratio of 0.006 to 1.7 with respect to angiogenin and / or angiogenin degradation product.

Angiogenin and / or angiogenin degradation products and cystatin and / or cystatin degradation products contained in the fermented milk of the present invention include angiogenin prepared from milk of mammals such as humans, cows, buffaloes, goats and sheep. And / or fractions containing angiogenin degradation products, fractions containing cystatin and / or cystatin degradation products, fractions produced by genetic engineering techniques, angiogenin purified from blood and organs and / or Angiogenin degradation products, cystatin and / or cystatin degradation products can be used. It is also possible to use purified and commercially available reagents for angiogenin and cystatin.
In addition, the above-mentioned angiogenin-containing fraction, angiogenin reagent or cystatin-containing fraction, cystatin reagent, etc., each containing an angiogenin degradation product or cystatin degradation product obtained by degradation with one or more proteolytic enzymes Is possible.

  On the other hand, containing protein materials prepared by extracting fractions containing angiogenin and / or angiogenin degradation products and cystatin and / or cystatin degradation products directly from milk or milk-derived raw materials such as skim milk and whey It is also possible to make it. Such a protein material is obtained by, for example, bringing milk or a milk raw material into contact with a cation exchange resin, and then eluting the milk-derived protein adsorbed on the resin at a salt concentration of 0.1 to 2.0 M to perform reverse osmosis. Proteins such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease after desalting and concentration with membranes, electrodialysis membranes, ultrafiltration membranes, microfiltration membranes, etc. It can be prepared by decomposing with a degrading enzyme so that the molecular weight becomes 8,000 or less. In addition, when decomposing | disassembling with a proteolytic enzyme, it is preferable that the minimum of molecular weight shall be 500 or more. In addition, the protein material obtained in this way can be dried by freeze drying, spray drying, or the like, and the dried material may be contained in fermented milk.

In the present invention, the above-mentioned angiogenin and / or angiogenin degradation product, cystatin and / or cystatin degradation product, protein material containing these, etc. are added to the milk raw material, and 0.9 mg of angiogenin and / or angiogenin degradation product is added. ˜150 mg / 100 g is contained, and cystatin and / or cystatin degradation product is contained in a mass ratio of 0.006 to 1.7 with respect to angiogenin and / or angiogenin degradation product.
As shown in the following test examples, as described above, by containing angiogenin and / or angiogenin degradation product, cystatin and / or cystatin degradation product, bone strengthening more effectively than ingesting each component alone The effect can be obtained.

The fermented milk of the present invention is not particularly limited except that it contains angiogenin and / or an angiogenin degradation product, cystatin and / or a cystatin degradation product in a specific amount, and may be produced according to a general method. The fermented milks produced by the present invention are all fermented milks such as fermented milk, dairy lactic acid bacteria beverages, and lactic acid bacteria beverages. For example, milk raw materials are mixed as necessary, angiogenin and / or angiogenin degradation products are blended in a specific amount range, and cystatin and / or cystatin degradation products are mixed with angiogenin and / or It is prepared by adding so as to have a mass ratio within a specific range with respect to the angiogenin degradation product. Here, in addition to milk, defatted concentrated milk, defatted powdered milk, whey, butter, cream, etc., milk beverages, processed milk, ingredient-adjusted milk, low-fat milk, non-fat mixed with these as necessary Milk is included. Then, Lactobacillus bulgaricus, Streptococcus thermophilus (Leptococcus thermophilus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus helveticus (Lactobacillus helveticus) After inoculating an appropriate amount of a starter culture prepared by inoculating and fermenting yeasts such as Kluyveromyces marxianus and Saccharomyces unisporus The fermented milk of the present invention can be prepared by fermenting according to a conventional method. It can also be sterilized after fermentation.

When added to a milk raw material, angiogenin and / or an angiogenin degradation product and cystatin and / or a cystatin degradation product may be added to an unsterilized milk raw material or a sterilized milk raw material. When adding to unsterilized milk raw materials, sterilization is performed after the addition. Sterilization is preferably performed by heat sterilization. However, when heat sterilization is performed after adding angiogenin and / or an angiogenin degradation product, cystatin and / or a cystatin degradation product to a milk raw material, it is preferable to make it at 130 degreeC for 2 second or less.
The fermented milk of the present invention includes angiogenin and / or an angiogenin degradation product and cystatin and / or a cystatin degradation product, in addition to the above-described milk raw materials, sugars, lipids, proteins, vitamins, minerals, Flavors and other raw materials usually used in foods and drinks can also be blended, and other components that exhibit bone strengthening action, such as calcium, vitamin D, vitamin K, isoflavone, etc., can be blended.

  The fermented milk of the present invention can strengthen bones by ingesting 100 g or more per kg of body weight in a test with experimental animals described later. The intake in this experimental animal corresponds to the intake per adult in the blood drug concentration (Mitsuyoshi Nakajima (1993) “Volume 8: Efficacy Evaluation”, Yodogawa Shoten, 2-18). Bone can be strengthened by ingesting 100 g or more of the fermented milk of the present invention per person per day, and the effects of prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism and arthritis can be particularly expected.

  Reference Examples, Examples and Test Examples are shown below, and the present invention will be described in more detail. However, these are merely illustrative, and the present invention is not limited by these.

[Reference Example 1]
(Preparation of angiogenin fraction 1)
A column packed with 30 kg of a cation exchange resin sulfonated chitopearl (Fuji Boseki Co., Ltd.) was thoroughly washed with deionized water, and then 1,000 L (pH 6.7) of non-sterilized skim milk was passed through the column. . The column was then thoroughly washed with deionized water and then eluted with a linear concentration gradient of 0.1-2.0 M sodium chloride. And the elution fraction containing angiogenin is fractionated by S-Sepharose cation exchange chromatography (manufactured by Amersham Biosciences), the obtained angiogenin-containing fraction is heated at 90 ° C. for 10 minutes, and centrifuged. The precipitate was removed by separation. Further, this angiogenin-containing fraction was treated with Superose 12 gel filtration chromatography. This eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 16.5 g of an angiogenin fraction having angiogenin purity of 90%. These series of treatments were repeated 30 times.

[Reference Example 2]
(Preparation of angiogenin fraction 2)
A column packed with 10 kg of heparin affinity sepharose (manufactured by GE Healthcare) was thoroughly washed with deionized water, and then 500 L (pH 6.7) of non-sterilized skim milk was passed through the column. Next, the column was thoroughly washed with 0.5 M sodium chloride solution and then eluted with 1.5 M sodium chloride solution. The eluate was desalted with a reverse osmosis membrane and then lyophilized to obtain 18 g of an angiogenin fraction having angiogenin purity of 5%. These series of treatments were repeated 50 times.

[Reference Example 3]
(Preparation of cystatin fraction)
100,000 L of a 5% whey protein solution was heated at 90 ° C. for 10 minutes and centrifuged to remove the precipitate. Further, a carrier in which carboxymethylated papain is bound to Tresyl-Toyopearl (Tosoh Corp.) is packed in a column, equilibrated with 0.5 M sodium chloride solution, and passed through the whey protein solution. Liquid. After the passage, the column was washed successively with a 0.5M sodium chloride solution and a 0.5M sodium chloride solution containing 0.1% Tween20. Subsequently, the cystatin-containing fraction was eluted with 20 mM acetic acid-0.5 M sodium chloride solution. The eluted fraction was immediately neutralized with 1M sodium hydroxide solution. The eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 9.6 g of a cystatin fraction having a cystatin purity of 90%. These series of treatments were repeated 20 times.

(Measurement of angiogenin and cystatin contained in fermented milk)
Measurement of angiogenin and angiogenin degradation products, cystatin and cystatin degradation products contained in fermented milk was carried out by modifying the method disclosed in Japanese Patent Application Laid-Open No. 2008-164511. That is, 86 μl of fermented milk was added to 5 ml of ultrapure water, and 1/1000 amount of formic acid was added thereto to prepare a sample solution. After 10 μl of this solution was dried up, it was dissolved in 20 μl of 0.1 M ammonium bicarbonate containing 8 M urea and 1 mM tris (carboxyethyl) phosphine (TCEP) and heated at 56 ° C. for 30 minutes. After returning to room temperature, 5 μl of a 100 mM iodoacetamide solution was added and allowed to react for 30 minutes in the dark. 54 μl of ultrapure water was added thereto, 10 μl of 0.1 μg / ml trypsin and 10 μl of 0.1 μg / ml lysyl endopeptidase were added, and the mixture was reacted at 37 ° C. for 16 hours. Thereafter, 3 μl of formic acid was added to stop the reaction to obtain a peptide solution for measurement sample. Each sample solution was diluted 6-fold with 10 fmol / μl of an internal standard peptide solution (containing 0.1% formic acid, 0.02% trifluoroacetic acid (TFA), 2% acetonitrile), and 2.5 μl of the diluted solution was diluted with LC. Analysis was performed with / MS / MS.

Peptide separation was gradient eluted using an HPLC system. Specifically, MAGIC C18 0.2 mm ID × 50 mm with a column 5 μl peptide trap was used, and the flow rate was 2 μl / min, using a MAGIC 2002 HPLC system. As the solvent for HPLC, A solution: 2% acetonitrile-0.05% formic acid and B solution: 90% acetonitrile-0.05% formic acid were used. As the elution conditions, the B solution was gradient eluted from 2% to 65% over 20 minutes.
The measurement target ion of cystatin is m / z 914.4 when the parent ion is NH2-QVSGMNYFLDVELGR-COOH, the target ion of MS / MS is NH ₂ -FLDVELGR-COOH, and m / z 948.7, and the measurement target ion of angiogenin is The parent ion was NH ₂ —YIHFLTQHYDAK-COOH, m / z 768.8, and the MS / MS target ion was NH 2 -FLTQHYDAK-COOH, m / z 1122.8. The internal standard peptide has a parent ion of NH 2 -ETTVFELPEK-COOH (wherein P carbon is labeled with C13 and nitrogen is labeled with N15) and m / z 656.9, and the MS / MS target ion is NH 2 -FENPEK- The reaction was carried out at m / z 882.4 with COOH (wherein P carbon was labeled with C13 and nitrogen was labeled with N15).
LCQ Advantage was used for MS. From the obtained chromatogram, the peak area of each protein was determined, and the concentration was determined from the ratio to the internal standard peptide.

[Example 1]
166 mg of angiogenin fraction of Reference Example 1 and 0.5 mg of cystatin fraction of Reference Example 3 are mixed with 100 g of 10% reduced skim milk powder sterilized by heating at 100 ° C. for 10 minutes, and the usual method And fermented milk (Example product 1) was obtained. The obtained fermented milk contains 150 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 0.00. 006.

[Example 2]
100 g of 10% reduced skim milk powder sterilized by heating at 100 ° C. for 10 minutes was mixed with 13.5 mg of the angiogenin fraction of Reference Example 2 and 1.2 mg of the cystatin fraction of Reference Example 3 Fermented milk (Example product 2) was obtained by fermentation according to a conventional method. The obtained fermented milk contains 0.9 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 1.7.

[Example 3]
13.5 mg of angiogenin fraction of Reference Example 1 and 1.2 mg of cystatin fraction of Reference Example 3 were mixed with 100 g of 10% reduced skim milk powder sterilized by heating at 100 ° C. for 10 minutes. Fermented milk (Example product 3) was obtained by fermentation according to a conventional method. The obtained fermented milk contains 12.4 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is It was 0.12.

[Comparative Example 1]
10% of reduced skim milk powder sterilized by heating at 100 ° C. for 10 minutes is mixed with 12 g of the angiogenin fraction of Reference Example 2 and 2.7 mg of the cystatin fraction of Reference Example 3 in 100 g of a normal method. And fermented milk (Comparative Example Product 1) was obtained. The obtained fermented milk contains 0.9 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 3.2.

[Comparative Example 2]
166 mg of the angiogenin fraction of Reference Example 1 and 0.25 mg of the cystatin fraction of Reference Example 3 are mixed with 100 g of 10% reduced skim milk powder that has been sterilized by heating at 100 ° C. for 10 minutes. And fermented milk (Comparative Example Product 2) was obtained. The obtained fermented milk contains 150 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 0.00. 0045.

[Test Example 1]
The bone strengthening effects of Examples 1 to 3 and Comparative Examples 1 and 2 were examined by animal experiments. For the experiment, C3H / HeJ male mice aged 5 weeks were used. After one week of pre-breeding, the mice were divided into 6 groups of 10 mice, and Examples 1 to 3 and Comparative Examples 1 and 2 were mixed with a sonde twice a day so that the mouse weight per kg was 100 g per day. Orally administered and reared for 2 weeks. Moreover, the thing which does not administer Example products 1-3 and the comparative example products 1 and 2 was made into the control group. After the end of administration (2 weeks), the bone density of the right tibia of the mouse was measured by micro CT (manufactured by Rigaku Corporation). The results are shown in Table 1. As shown in Table 1, the bone density was significantly increased in the group in which Examples 1 to 3 were orally administered for 2 weeks, as compared to the control group or the group to which Comparative Examples 1 and 2 were administered.

[Reference Example 4]
A column (diameter 4 cm × height 30 cm) packed with 400 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki Co., Ltd.) was thoroughly washed with deionized water, and 40 L of non-sterilized skim milk (pH 6.7) was added to this column. At a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water, and the protein adsorbed on the resin was eluted with a 0.02 M carbonate buffer solution (pH 7.0) containing 0.78 M sodium chloride. The eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 18 g of a powdered protein material (Reference Example Product 4).

[Reference Example 5]
4 g of the protein material of Reference Example 4 was dissolved in 800 ml of water, and trypsin (manufactured by Sigma) as a proteolytic enzyme was added so that the final concentration was 0.03% by weight, followed by enzyme treatment at 37 ° C. for 8 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized | freeze-dried and obtained 3.0g of powdery protein materials (reference example goods 5).

[Example 4]
40 mg of Reference Example Product 4 was mixed with 97 g of 10% reduced skim milk powder, and after heat sterilization at 93 ° C. for 6 minutes, 3 g of starter culture was added, fermented according to a conventional method, and fermented milk (Example Product 4 ) The obtained fermented milk contains 2.4 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 0.39.

[Example 5]
40 mg of Reference Example Product 5 was mixed with 97 g of 10% reduced skim milk powder, and after heat sterilization at 93 ° C. for 6 minutes, 3 g of starter culture was added and fermented according to a conventional method to produce fermented milk (Example Product 5). ) The obtained fermented milk contains 2.3 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 0.4.

[Comparative Example 3]
97 g of 10% reduced skim milk powder was mixed with 30 mg of Reference Example Product 4 and 10 mg of the cystatin fraction of Reference Example 3, and after heat sterilization at 93 ° C. for 6 minutes, 3 g of starter culture was added and fermented according to a conventional method. Thus, fermented milk (Comparative Example Product 3) was obtained. The obtained fermented milk contains 1.9 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 5.2.

[Test Example 2]
The bone strengthening effects of Examples 4 and 5 and Comparative Example 3 were examined by animal experiments. In the experiment, 40 51-week-old SD female rats were used. Rats were divided into 5 groups of 8 rats, 4 groups underwent oophorectomy, and the remaining 1 group underwent sham surgery. A 4-week recovery period was provided, and the rats 4 and 5 and Comparative Example 3 were orally administered with a sonde 6 times a day so that the rat weight per kg of the rat was 100 g per day. Raised for 16 weeks. A group that did not receive Examples 4 and 5 and Comparative Example 3 was used as a control group. In addition, after the recovery period of 4 weeks, rats subjected to sham operation were bred for 16 weeks as in the control group. After the end of administration (16 weeks), the bone density of the right femur of the rat was measured by micro CT (manufactured by Rigaku Corporation). The results are shown in Table 2. As shown in Table 2, the bone density was significantly increased in the group in which Example products 4 and 5 were orally administered for 16 weeks, compared with the control group or the group in which Comparative Example product 3 was administered, and the value was a sham operation. The level was close to the group.

[Example 6]
50 mg of Reference Example 4 was mixed with 98 g of 2.5% reduced skim milk powder sterilized by heating at 100 ° C. for 10 minutes, 2 g of starter culture was added, fermented according to a conventional method, and sterilized by heating at 130 ° C. for 2 seconds. Then, it cooled to 10 degreeC and fermented milk (Example product 6) was obtained. The obtained fermented milk contains 2.9 mg / 100 g of angiogenin and / or angiogenin degradation product, and the mass ratio of cystatin and / or cystatin degradation product to angiogenin and / or angiogenin degradation product is 0.32.

Claims (4)

Containing 0.9 to 150 mg / 100 g of angiogenin, and
Containing cystatin in a mass ratio of 0.006 to 1.7 with respect to angiogenin ,
Fermented milk for prevention or treatment of bone disease, wherein at least one of the angiogenin and the cystatin is purified . Containing 0.9 to 150 mg / 100 g of an angiogenin degradation product having a molecular weight of 500 to 8000, and
Containing a cystatin degradation product having a molecular weight of 500 or more and 8000 or less in a mass ratio of 0.006 to 1.7 with respect to the angiogenin degradation product ,
Fermented milk for prevention or treatment of bone disease, wherein at least one of the angiogenin degradation product and the cystatin degradation product is purified .   The fermented milk for prevention or treatment of bone disease according to claim 1, wherein the angiogenin and the cystatin are purified.   The fermented milk for prevention or treatment of bone disease according to claim 2, wherein the angiogenin degradation product and the cystatin degradation product are purified.