U.S. patent application number 10/067527 was filed with the patent office on 2002-12-05 for medicine, drink, food and feed having an action of strengthening bone.
Invention is credited to Aoe, Seiichiro, Kato, Ken, Takada, Yukihiro, Toba, Yasuhiro, Yamamura, Junichi.
Application Number | 20020183261 10/067527 |
Document ID | / |
Family ID | 14237144 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020183261 |
Kind Code |
A1 |
Takada, Yukihiro ; et
al. |
December 5, 2002 |
Medicine, drink, food and feed having an action of strengthening
bone
Abstract
The present invention is to provide a novel medicine, drink,
food or feed having an action of strengthening bone. More
specifically, the present invention is to provide a medicine,
drink, food or feed combined with collagen, fraction containing
collagen or degradation product thereof. Fraction containing
collagen was prepared by mincing skin corium layer into pieces,
defatting it and lyophylization or by pulverizing bone,
decalcifying it and lyophylization. Oral administration of these
can stimulate proliferation of osteoblast, inhibit bone resorption
and strengthen bone. They can be useful for improvement of
osteoporosis, bone fracture, bone pain, etc.
Inventors: |
Takada, Yukihiro;
(Kawagoe-shi, JP) ; Aoe, Seiichiro; (Sayama-shi,
JP) ; Kato, Ken; (Ohmiya-shi, JP) ; Toba,
Yasuhiro; (Kawagoe-shi, JP) ; Yamamura, Junichi;
(Kawagoe-shi, JP) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
14237144 |
Appl. No.: |
10/067527 |
Filed: |
February 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10067527 |
Feb 4, 2002 |
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08819110 |
Mar 17, 1997 |
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6344437 |
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Current U.S.
Class: |
514/5.5 ;
514/16.7; 514/17.2 |
Current CPC
Class: |
A61P 19/08 20180101;
A23L 29/284 20160801; A61K 38/39 20130101; A21D 2/261 20130101;
A61P 43/00 20180101; A23K 50/42 20160501; A23C 19/082 20130101;
A23L 33/17 20160801; A23J 1/10 20130101; A61P 3/00 20180101; A23L
2/66 20130101; A23L 29/256 20160801; A61P 19/10 20180101; A23K
20/147 20160501; A61P 3/14 20180101; A61P 19/00 20180101; Y10S
514/801 20130101; A23C 9/1315 20130101 |
Class at
Publication: |
514/21 |
International
Class: |
A61K 038/39 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 1996 |
JP |
JP 099059/1996 |
Claims
We claim,
1. A medicine, drink, food or feed having an action of
strengthening bone combined with collagen, fraction containing
collagen and/or degradation product thereof.
2. The medicine, drink, food or feed according to claim 1, wherein
said fraction containing collagen is skin protein fraction or bone
protein fraction.
3. The medicine, drink, food or feed according to claim 2, wherein
said skin protein fraction is a lyophylization product of
pulverized and fefatted skin corium layer.
4. The medicine, drink, food or feed according to claim 2, wherein
said bone protein fraction is a lyophylization product of
pulverized and decalcified bone.
5. The medicine, drink, food or feed according to claim 1, wherein
said fraction containing collagen is treated skin protein fraction
or bone protein fraction by soaking it in acid or alkaline
solution.
6. The medicine, drink, food or feed according to claim 1, wherein
said degradation product of fraction containing collagen is
degradation product with molecular weight of 2-150 kDa obtained by
hydrolysis of skin protein fraction or bone protein fraction with
proteolytic enzyme.
7. The medicine, drink, food or feed according to claim 1, wherein
said degradation product of fraction containing collagen is
degradation product with molecular weight of 2-150 kDa obtained by
hydrolysis of acid- or alkaline-treated skin protein fraction or
bone protein fraction with a proteolytic enzyme.
8. A medicine, drink, food or feed containing collagen, fraction
containing collagen and/or degradation product thereof combined
with calcium and/or vitamins.
9. The medicine, drink, food or feed according to claim 8, wherein
0.5-5.0 weight parts of collagen, fraction containing collagen or
degradation product thereof is combined with 1 weight part of
calcium.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a medicine, drink, food and
feed having an action of strengthening bone. The medicine, drink,
food or feed combined with collagen, fraction containing collagen,
and/or degradation product that has effects of promoting bone
formation, strengthening bone, preventing and treating bone
metabolic diseases such as osteoporosis, bone fracture, bone pain
and so on.
BACKGROUND OF THE INVENTION
[0002] Accompanying with the prolongation of human life span, the
incidence of metabolic bone diseases such as osteoporosis,bone
fracture,bone pain etc., recently increased. In bone tissue, bone
formation and bone resorption are always occurring. While the
balance of bone formation and bone resorption is kept in one's
youth, bone resorption exceeds bone formation due to various causes
as one's age increases (uncoupling). And when bone resorption
prolongs for a long duration, bone tissue becomes fragile, which
causes metabolic bone diseases such as osteoporosis, bone fracture,
bone pain, etc., are expected to be prevented.
[0003] As conventional methods of preventing or treating metabolic
one diseases by inhibiting uncoupling, (1) calcium supplemented
diets, (2) light exercise, (3) sunbathing, (4) medicinal therapy,
etc., are exemplified. As for calcium supplemented diets, calcium
salts such as calcium carbonate, calcium phosphate, etc., and
naturally occurring calcium-containing preparation, such as bovine
bone powder, egg shell, fish bone powder, etc. are used. They are,
however, not necessarily good enough for oral intake. As light
exercises, jogging or walking may be recommended. However, they are
troublesome to a person who becomes weak and quite difficult to an
immobilized aged person. Sunbathing is believed to be good for
supplement of active form of vitamin D.sub.3 but is not sufficient.
As medicinal therapy, 1 .alpha.-hydroxyvitamin D.sub.3 and/or
calcitonin may be used and they are known to be effective for
treating osteoporosis. However, they are medicines themselves and
can not be used as food sources.
[0004] On the other hand, collagen is known as a main protein
component composing animal connective tissue and occupies nearly
30% of total protein in mammalian, especially, human whole body.
This collagen is a protein of cellular matrix and can be defined to
be a substance having .alpha.-chain which is a helical portion
consisting of 3 polypeptide chains and forming a multi-molecular
complex. Further, collagen forms intercellular matrix with
glycoproteins, such as proteoglycan, fibronectin, laminin etc., and
is an essential component for exhibiting its function as assembled
tissue of cells in multi-cellular biological organism.
[0005] The present inventors have investigated to find out a
substance having action of strengthening bone which is useful for
food sources. Eventually, we found out that a fraction containing
collagen such as skin protein fraction or bone protein fraction has
an effect of promoting proliferation of osteoblast. Further, the
present inventors found that degradation products of the fraction
containing collagen also have an effect of promoting proliferation
of osteoblast and accomplished the present invention.
SUMMARY OF THE INVENTION
[0006] Accordingly, an object of the present invention is to
provide a medicine, drink, food and feed having an action of
strengthening bone. More specifically, the present invention is to
provide a medicine, drink, food and feed having an action of
strengthening bone combined with collagen, fraction containing
collagen and/or degradation product thereof.
[0007] Another object of the present invention is to provide a
medicine, drink, food and feed combined with collagen, fraction
containing collagen or degradation product thereof which is
combined with calcium and vitamins.
[0008] As a fraction containing collagen used in the present
invention, skin protein fraction, bone protein fraction,
lyophilization product of pulverized and skim corium layer,
lyophilization product of pulverized and decalcified bone, and
fraction which is produced by treating skin protein fraction or
bone protein fraction with acid or alkiline can be exemplified.
Further, degradation product which is produced by hydrolysis with a
proteolytic enzyme and has a molecular weight of 2-150 kDa can be
used.
BRIEF DESCRIPTION OF DRAWINGS
[0009] FIG. 1 represents an action of promoting collagen synthesis
in osteoblast by a fraction containing collagen or degradation
product thereof obtained in test example 2.
[0010] FIG. 2 represents breaking force of rat femur administered
orally with fraction containing collagen or degradation product
thereof obtained in test example 3.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0011] In the present invention, collagen, a fraction containing
fraction and/or degradation product thereof can be combined with a
medicine, drink, food or feed for having an action of strengthening
bone. This fraction containing collagen is a protein fraction
obtained from skin or bone. For example, when it is prepared from
skin, skin can be depilated and corium layer was cut off. The
tissue can be cut into pieces with high-speed cutter, defatted with
organic solvent and lyophilized to give a fraction containing
collagen. And when it is prepared from bone, bone can be
decalcified with acid etc., pulverized with a high-speed cutter,
decalcified, again, with acid etc., and lyophilized to give a
fraction containing collagen. Collagen can be more purified by
treating skin protein fraction or bone protein fraction containing
collagen with acid or alkaline. Treatment with acid or alkaline can
be carried out by soaking these fractions in a 5-30% of liquid
solution of acid, such as hydrochloric acid or nitric acid, or
alkaline, such as sodium hydroxide or potassium hydroxide, at
20-50.degree. C. for 0.5-3 hours. After soaking, these fractions
should be sufficiently washed with water. Hot water extraction of
these fractions can be carried out by treating them with
70-80.degree. C. hot water.
[0012] Further, these fractions containing collagen can be
hydrolyzed by a proteolytic enzyme, such as pepsin, trypsin,
chymotrypsin, etc., whose solubility can be enhanced and used.
Hydrolysis by a proteolytic enzyme can be carried out by
pulverizing fractions described before, suspending them in water,
adding about 1% of proteolytic enzyme thereto and keeping it at
37.degree. C. for 0.5-6 hours. After hydrolysis, the reaction
mixture was heated to deactivate proteolytic enzyme and
ultrafiltrated, followed by collection of filtrate. Hydrolysis can
be preferably carried out so that the molecular weight of
degradation product will be 2-200 kDa.
[0013] In addition, calcium or vitamins can be combined with these
fractions containing collagen. As a calcium source, calcium
chloride, calcium carbonate, calcium lactate, egg shell or
milk-derived calcium can be used. As combination rate of calcium
with fraction containing collagen, 0.5-5.0 weight parts of said
fraction will be preferable to 1 weight part of calcium. As
vitamins, any vitamin can be used and vitamin D.sub.3 or substance
containing vitamin D.sub.3 can be more preferably used. As raw
material containing substantial amount of collagen, fresh skin or
bone of cattle, pig, chicken, sheep and horse can be
exemplified.
[0014] In the present invention, collagen, fraction containing
collagen and/or degradation product thereof can be combined with a
medicine, such as tablet or powder, and with a drink or food, such
as milk, yogurt, ice cream, milk drink, coffee drink, juice, jelly,
noodle, cracker, bread or sausage, for endowing an action of
strengthening bone. Further, in the same manner, these fractions or
degradation product can be combined with a feed for endowing an
action of strengthening bone. In addition, calcium agent with good
absorptivity, such as calcium chloride, calcium carbonate, calcium
lactate, egg shell or milk-derived calcium,etc., or vitamin D can
be combined so that the action of strengthening bone thereof will
be enhanced.
[0015] In the present invention, since oral administration of
10-2,500 mg of collagen, fraction containing collagen and/or
degradation product thereof per day in an adult can exhibit an
action of strengthening bone, collagen, fraction containing
collagen, and/or degradation product thereof can be combined with a
medicine drink or food by considering the above effective amount.
Acute toxicity of fractions containing collagen, such as skin
protein fraction or bone protein fraction was not observed in
rat.
[0016] Since a medicine, drink, food or feed combined with
collagen, fraction containing collagen and/or degradation product
thereof has an action of strengthening bone, oral administration of
these will be useful for prevention and/or improvement of bone
metabolic diseases such as osteoporosis etc.
[0017] In addition, strengthening bone of livestock or poultry can
be also carried out by combining collagen, fraction containing
collagen and/or degradation product thereof with feed.
[0018] Preparation of fractions containing collagen and degradation
products thereof, and an action of strengthening bone will be
described by exemplifying reference examples and test examples as
below. Further, the present invention will be described by
exemplifying examples. However, these examples will not limit the
scope of the present invention.
REFERENCE EXAMPLE 1
[0019] Porcine skin (10 kg) was depilated and corium layer was
taken, minced into pieces with a high-speed cutter, defatted with a
solvent of hexane:ethanol (5:1) and lyophilized to yield 1,354 g of
skin protein fraction (fraction A) containing collagen. This
fraction contained 85% of protein and the molecular weight
distribution thereof was 50-200 kDa. The amount of protein was
determined according to Lowry method (Lowry O. H., et al. (1951) J.
Biol. Chem., 193, 265-275). Molecular weight distribution was
determined by SDS polyacrylamide electrophoresis. The amount of
protein and molecular weight distribution described below were also
determined by the same method as the above.
REFERENCE EXAMPLE 2
[0020] The skin protein fraction (1,000 g) containing collagen
obtained in reference example 1 was suspended in water so that its
concentration would be 5%, followed by soaking it in an liquid
solution of hydrochloric acid so that its concentration would be
10% and washed sufficiently with water. Then, it was again
suspended in water so that its concentration would be 10% and
heated at 90.degree. C. for 30 min., followed by lyophilization to
yield 620 g of skin protein fraction (fraction B) containing
collagen. This fraction contained 93% of protein and the molecular
weight distribution thereof was 50-200 kDa.
REFERENCE EXAMPLE 3
[0021] The skin protein fraction (300 g) containing collagen
obtained in reference example 2 was suspended so that its
concentration would be 5% and 1% pancreatin (Sigma) was added
thereto, which was kept at 37.degree. C. for 2 hours and, then,
heated at 80.degree. C. for 10 min. to deactivate pancreatin,
followed by lyophilization to yield 280 g of degradation product of
skin protein fraction (fraction C) containing collagen. This
fraction contained 95% of protein and the molecular weight
distribution was 2-50 kDa.
REFERENCE EXAMPLE 4
[0022] Bovine bone powder (10 kg) was suspended in water so that
its concentration would be 10% and decalcified with treatment of
hydrochloric acid so that its concentration would be 10%. Then, it
was minced into pieces with a high-speed chopper and decalcified by
soaking it in an liquid solution of hydrochloric acid so that its
concentration would be 10%. Further, it was sufficiently washed
with water, lyophilized and pulverized with a pulverizer to yield
2,210 g of bone protein fraction (fraction D) This fraction
contained 89% of protein and the molecular weight distribution
thereof was 50-150 kDa.
REFERENCE EXAMPLE 5
[0023] The bone protein fraction (1,000 g) containing collagen
obtained in reference example 4 was suspended in water so that its
concentration would be 5%, followed by soaking it in sodium
hydroxide solution so that its concentration would be 10% and
washed sufficiently with water to yield 540 g of bone protein
fraction (fraction E) containing collagen. This fraction contained
92% of protein and the molecular weight distribution was 50-150
kDa.
REFERENCE EXAMPLE 6
[0024] The bone protein fraction (300 g) containing collagen
obtained in reference example 5 was suspended so that its
concentration would be 5% and 1% pancreatin (Sigma) was added
thereto, which was kept at 37.degree. C. for 2 hours and, then,
heated at 80.degree. C. for 10 min. to deactivate pancreatin,
followed by lyophilization to yield 290 g of degradation product of
bone protein fraction (fraction F) containing collagen. This
fraction contained 95% of protein and the molecular weight
distribution was 3-70 kDa.
TEST EXAMPLE 1
[0025] Fractions A-F obtained in reference examples were
investigated with respect to an action of inhibiting bone
resorption. Long bone were extirpated from 10-20 days old ICR mice
and the whole bone marrow cells comprising osteoclast were obtained
by depriving soft tissue from the bones and mincing the bones in
.alpha.-modified minimum essential medium (.alpha.-MEM) containing
5% bovine fetal serum mechanically. About 2.times.10.sup.6 of these
cells in .alpha.-MEM containing 5% bovine fetal serum were placed
on a piece of dentinum. Two hours after then, a test sample in
.alpha.-MEM containing 5% bovine fetal serum was added so that the
final concentration would be 10 .mu.g/ml, which was cultured for 5
days and bone resorptive activity of osteoclast was investigated.
Analysis of bone resorption was carried out by depriving cells from
a piece of dentinum after cultivation thereof, staining them with
Hematoxylin dye and counting the number of bone resorptive pit by
morphometrical analysis with PIALA-555. As control, culture without
any addition was used and bone resorptive activity of each case was
calculated by defining 100% as that in the case of non-added group.
The results were represented in table 1.
[0026] Comparing with bone resorptive activity of non-added group,
any group with addition of fraction A-F which was skin protein
fraction or bone protein fraction and contained collagen obtained
in reference examples was found to have an action of inhibiting
bone resorption.
1 TABLE 1 Bone resorptive activity Control 100 (%) Fraction A 86
Fraction B 84 Fraction C 85 Fraction D 81 Fraction E 75 Fraction F
84
TEST EXAMPLE 2
[0027] Action of promoting collagen synthesis of fractions A-F
obtained in reference examples was studied. That is,
2.times.10.sup.4 cells/ml of osteoblastic cell line MC3T3-E1 in
.alpha.-MEM containing 10% bovine fetal serum (Flow Laboratories)
was inoculated in each well of 96-well plate and cultured at
37.degree. C. for 24 hours in the presence of 5% CO.sub.2. Then,
the medium was changed into .alpha.-MEM, which did not contain
bovine fetal serum, to which fractions A-F obtained in reference
examples was added so that the final concentration thereof would be
10 .mu.g/ml and cultured at 37.degree. C. for 3 days. After then,
the amount of synthesized collagen was measured by determining
hydroxyproline. Determination of hydroxyproline was carried out by
hydrolyzing suspension of punctured cultured cells with 6N
hydrochloric acid and using p-dimethylaminobenzaldehyde according
to Woessner's method (Woessner, J. F., Arch. Biochem. Biophys.,
vol. 93, pp 440-447, 1961) and the results were represented in FIG.
1. The amount of hydroxyproline in culture with addition of
fractions A-F obtained in reference examples which were skin
protein fraction or bone protein fraction and contained collagen
was higher than that in culture without addition of those
fractions, which suggested stimulation of collagen synthesis in
osteoblast by the fractions.
TEST EXAMPLE 3
[0028] Action of strengthening bone of fraction B, C, E and F were
studied in animal experiments. Osteoporotic model rats were made by
ovarectomy of 6-weeks-old female SD rats after raising for 1 week
and feeding with low calcium diet for 2 months and used in animal
experiments. These rats were divided into 5 groups consisting of 7
rats, that is, control group, 1.5% fraction B administered group (I
group), 1.5% fraction C administered group (II group), 1.5%
fraction E administered group (III group) and 1.5% fraction F
administered group (IV group) and fed with test diets consisting of
components represented in table 2 for 1 month. In addition, 7 sham
rats were also made by sham operation wherein ovary was not
extirpated and used in the same experiments. As calcium source of
mineral mixture in table 2, calcium carbonate was used.
2 TABLE 2 *I III Control g. *Sham g. group II group group IV group
Sucrose 49.3 49.3 49.3 49.3 49.3 49.3 (g/100 g) Casein 20.0 20.0
18.5 18.5 18.5 18.5 Corn starch 15.0 15.0 15.0 15.0 15.0 15.0
Cellulose 5.0 5.0 5.0 5.0 5.0 5.0 Corn oil 5.0 5.0 5.0 5.0 5.0 5.0
Vitamin mix. **1.2 1.2 1.2 1.2 1.2 1.2 (including choline) Mineral
mix. **4.5 4.5 4.5 4.5 4.5 4.5 Fraction B 1.5 Fraction C 1.5
Fraction E 1.5 Fraction F 1.5 *g.: group, **mix.: mixture
[0029] In addition, test diets consisting of components represented
in table 3 were also fed in another experimental groups, that is,
(1.5% fraction B+milk-derived calcium+vitamin D.sub.3 400 IU)
administered group(V group), (1.5% fraction E+milk-derived calcium
+vitamin D.sub.3 400 IU) administered group (VI group), 0.2%
fraction B administered group, 2.0% fraction B administered group.
As calcium source in mineral mixture, calcium carbonate was used in
VII group and VIII group and milk-derived calcium (Japanese
published unexamined patent application No. 6-125740) was used in V
group and VI group. In test diets in table 2 and table 3, the
amount of casein was adjusted so that nitrogen content in the all
test diets would be the same. And in 100 g of test diet, 400 mg of
calcium and 300 mg of phosphate were contained.
3 TABLE 3 V group VI group VII group VIII group Sucrose 49.3 49.3
49.3 49.3 (g/100 g) Casein 18.5 18.5 19.8 18.0 Corn starch 15.0
15.0 15.0 15.0 Cellulose 5.0 5.0 5.0 5.0 Corn oil 5.0 5.0 5.0 5.0
Vitamin mix.* 1.2 1.2 1.2 1.2 (including choline) Mineral mix.* 4.5
4.5 4.5 4.5 Fraction B 1.5 0.2 2.0 Fraction E 1.5 *mix.:
mixture
[0030] After 1 month administration, femurs of rats in each test
group were taken and breaking force thereof was determined by a
breaking force analyzer (Rheometer, Max RX-1600, I-thechno), whose
results were represented in FIG. 2.
[0031] As represented in this figure, fractions B, C, E and F which
were skin protein fractions or bone protein fractions and comprised
collagen were found to have significant action of strengthening
bone. Action of bone strengthening thereof were found to be
augmented by combination with milk-derived calcium having good
absorptivity and vitamin D.sub.3. Further, since there was
significant difference with respect to bone strength when the ratio
of calcium to collagen was 1.0:0.5-5.0, combination of 0.5-5.0
weight part of fraction containing collagen with 1 weight part of
calcium was found to be effective.
[0032] The present invention will be explained by exemplifying
examples.
EXAMPLE 1
[0033] A tablet having action of strengthening bone was prepared by
mixing raw materials represented in table 4 and formulating it
under pressure.
4 TABLE 4 Crystalline glucose hydrate 73.5 (weight %) Fraction F in
reference example 6 20.0 Calcium 5.0 Sugar ester 1.0 Flavor 0.5
EXAMPLE 2
[0034] A drink having action of strengthening bone was prepared by
mixing raw materials represented in table 5, packing it in a
container and sterilizing it by heating.
5 TABLE 5 Mixed isomerized saccharide 15.0 (weight %) Fruit juice
10.0 Citric acid 0.5 Fraction A in reference example 1 0.5 Flavor
0.1 Calcium 0.1 Water 73.8
EXAMPLE 3
[0035] A cracker having action of strengthening bone was prepared
by mixing raw materials represented in table 6, making dough,
formulating and baking it.
6 TABLE 6 Wheat powder 50.0 (weight %) Sugar 20.0 Sodium chloride
0.5 Margarine 12.5 Egg 12.1 Water 2.5 Sodium bicarbonate 0.1
Ammonium bicarbonate 0.2 Calcium carbonate 0.5 Fraction B in
reference example 1.2
EXAMPLE 4
[0036] A jelly having action of strengthening bone was prepared by
mixing raw materials represented in table 7, packing it in
container and sterilizing it by heating.
7 TABLE 7 Fructose 20.0 (weight %) Granulated sugar 15.0 Miller
jelly 5.0 Agar 1.0 Fraction A in reference example 1 0.5 Flavor 0.1
Calcium 0.1 Water 58.3
EXAMPLE 5
[0037] A processed cheese having action of strengthening bone was
prepared by mixing raw materials represented in table 8 and
emulsifying it at 85.degree. C.
8 TABLE 8 Gouda cheese 43.0 (weight %) Cheddar cheese 43.0 Sodium
citrate 2.0 Fraction B in reference example 2 0.5 Milk-derived
calcium 1.0 Water 10.5
EXAMPLE 6
[0038] After sterilizing 12 weight % reducing skim milk at
90.degree. C. for 20 min., Lactobacillus acidophilus and
Streptococcus thermophilus were inoculated to give 2 kinds of
starter culture, which were mixed in the same amount. A yogurt
having action of strengthening bone was prepared by mixing raw
materials represented in table 9 and fermenting it.
9 TABLE 9 Yogurt mix 96.5 (weight %) Starter culture 3.0 Fraction C
in reference example 3 0.5
EXAMPLE 7
[0039] A powder milk for infant having action of strengthening bone
was prepared by mixing raw materials represented in table 10.
10 TABLE 10 Skim milk 75.5 (weight %) Whey protein concentrate 2.4
Lactose 13.5 Mineral mix 0.3 Water soluble vitamin mix 0.3 Fat
containing fat-soluble vitamin 7.5 Fraction F in reference example
6 0.5
Example 8
[0040] A feed for dog having action of strengthening bone was
prepared by mixing raw materials represented in table 11.
11 TABLE 11 Soy bean cake 12.0 Skim milk powder 14.0 Soy bean oil
4.0 Corn oil 2.0 Palm oil 28.0 Corn starch 15.0 Wheat powder 8.0
Wheat bran 2.0 Vitamin mix 9.0 Mineral mix 2.0 Cellulose 3.0
Fraction A in reference example 1 1.0
* * * * *