U.S. patent application number 14/418261 was filed with the patent office on 2015-12-03 for beverage, and method of producing the same.
This patent application is currently assigned to MEGMILK SNOW BRAND CO., LTD.. The applicant listed for this patent is Yuko Ishida, Ken Kato, Hiroaki Matsuyama, Yoshikazu Morita, Takayuki Nara, Aiko Ohmachi, Atsushi Serizawa, Hiroshi Ueno, Hiroshi Urazono. Invention is credited to Yuko Ishida, Ken Kato, Hiroaki Matsuyama, Yoshikazu Morita, Takayuki Nara, Aiko Ohmachi, Atsushi Serizawa, Hiroshi Ueno, Hiroshi Urazono.
Application Number | 20150343030 14/418261 |
Document ID | / |
Family ID | 50027410 |
Filed Date | 2015-12-03 |
United States Patent
Application |
20150343030 |
Kind Code |
A1 |
Ohmachi; Aiko ; et
al. |
December 3, 2015 |
BEVERAGE, AND METHOD OF PRODUCING THE SAME
Abstract
The invention relates to a drink includes angiogenin and/or
angiogenin hydrolysate in an amount of more than 0.8 mg/100 ml and
not more than 150 mg/100 ml, and lactoperoxidase and/or
lactoperoxidase hydrolysate in the mass ratio to the angiogenin
and/or angiogenin hydrolysate of 0.3 to 23.
Inventors: |
Ohmachi; Aiko; (Saitama,
JP) ; Matsuyama; Hiroaki; (Saitama, JP) ;
Morita; Yoshikazu; (Saitama, JP) ; Ishida; Yuko;
(Saitama, JP) ; Nara; Takayuki; (Saitama, JP)
; Kato; Ken; (Saitama, JP) ; Serizawa;
Atsushi; (Sapporo, JP) ; Ueno; Hiroshi;
(Saitama, JP) ; Urazono; Hiroshi; (Saitama,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ohmachi; Aiko
Matsuyama; Hiroaki
Morita; Yoshikazu
Ishida; Yuko
Nara; Takayuki
Kato; Ken
Serizawa; Atsushi
Ueno; Hiroshi
Urazono; Hiroshi |
Saitama
Saitama
Saitama
Saitama
Saitama
Saitama
Sapporo
Saitama
Saitama |
|
JP
JP
JP
JP
JP
JP
JP
JP
JP |
|
|
Assignee: |
MEGMILK SNOW BRAND CO.,
LTD.
Hokkaido
JP
|
Family ID: |
50027410 |
Appl. No.: |
14/418261 |
Filed: |
July 31, 2012 |
PCT Filed: |
July 31, 2012 |
PCT NO: |
PCT/JP2012/069394 |
371 Date: |
April 30, 2015 |
Current U.S.
Class: |
424/94.2 |
Current CPC
Class: |
A61P 19/00 20180101;
A23L 2/52 20130101; A23V 2002/00 20130101; C12Y 301/27 20130101;
A23C 9/1526 20130101; A61K 38/465 20130101; C12Y 111/01007
20130101; A23V 2002/00 20130101; A23C 9/1512 20130101; A61K 38/44
20130101; A61P 43/00 20180101; A23V 2200/306 20130101; A23C 9/1465
20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; A23C 9/152 20060101 A23C009/152; A23L 2/52 20060101
A23L002/52; A61K 38/44 20060101 A61K038/44 |
Claims
1. A drink comprising angiogenin and/or angiogenin hydrolysate in
an amount of more than 0.8 mg/100 ml and not more than 150 mg/100
ml and lactoperoxidase and/or lactoperoxidase hydrolysate in the
mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.3
to 23.
2. A method of preventing bone diseases comprising ingesting the
drink according to claim 1 in an amount of 200 ml/day or more.
3. A method of producing the drink according to claim 1, comprising
mixing angiogenin and/or angiogenin hydrolysate and lactoperoxidase
and/or lactoperoxidase hydrolysate with a drink raw material, and
sterilizing the obtained mixture.
4. A method of producing the drink according to claim 1, comprising
adding angiogenin and/or angiogenin hydrolysate and lactoperoxidase
and/or lactoperoxidase hydrolysate to a sterilized drink raw
material.
Description
TECHNICAL FIELD
[0001] This invention relates to a beverage and a method of
producing the same. The drink includes a specific milk component,
and may be useful for prevention and treatment of various bone
diseases such as osteoporosis, fracture, rheumatism, and
arthritis.
BACKGROUND ART
[0002] In recent years, various bone diseases, such as
osteoporosis, fracture, and backache have increased on a global
basis along with aging of society and the like, and have become a
serious social problem. These diseases are caused by insufficient
calcium intake, depression of calcium absorption ability, hormone
imbalance after menopause, and the like. It is considered that
increase the body bone mass as much as possible by activating the
osteoblast and bone formation from the early stage of life, and
increase the maximum bone mass and the bone strength (bone
density+bone quality) is effective in preventing various bone
diseases, such as osteoporosis, fracture, and backache. Note that
the term "bone quality" refers to the bone microstructure,
metabolic turnover, microfracture, and calcification. It is thought
that various bone diseases, such as osteoporosis, fracture, and
backache may be prevented by suppressing osteoclastic bone
resorption. Bones are always repeatedly resorbed and formed in a
balanced manner (remodeling). However, various bone diseases, such
as osteoporosis, fracture, and backache may occur when bone
resorption exceeds bone formation due to a change in hormone
balance after menopause, and the like. Therefore, bones can be
strengthened by suppressing osteoclastic bone resorption and
maintaining the bone strength at a constant level.
[0003] In view of the above situation, a drug, food, drink, feed,
or the like, in which a calcium salt, such as calcium carbonate,
calcium phosphate, or calcium lactate or a natural calcium product,
such as whey calcium, bovine bone powder, or eggshell is added
individually, has been taken in order to strengthen bones. A drug,
food, drink, feed, or the like that contains such a calcium product
together with a substance having a calcium absorption-promoting
effect, such as casein phosphopeptide or oligosaccharide has also
been used to strengthen bones. However, the calcium absorption rate
is 50% or less when a food or drink that contains a calcium salt or
a natural calcium product is taken, and the large part of the
calcium taken may be discharged from the body without being
absorbed. Moreover, even if calcium is absorbed into the body, it
does not necessarily exhibit the bone metabolism-improving effect
or a bone-strengthening effect, since the affinity to bones may
differ according to its form or the type of nutritional ingredient
taken together. An estrogen product, an active vitamin D.sub.3
product, a vitamin K.sub.2 product, a bisphosphonate product, a
calcitonin product, and the like have been known as a drug for
treating osteoporosis or strengthening bones, and new drugs such as
an anti-RANKL antibody have been also developed. However, these
drugs may have side effects such as buzzing in the ear, a headache,
or loss of appetite. Moreover, the above substances are in a
situation that they cannot be added to a food or drink at present
from the viewpoint of safety, cost, and the like. Therefore, in
light of the nature of various bone diseases, such as osteoporosis,
fracture, and backache, development of such a food or drink that
can be taken orally for a long time, increases the bone strength by
promoting bone formation and suppressing bone resorption, and may
be expected to have the effect of preventing or treating the
various bone diseases has been desired.
PRIOR-ART DOCUMENT
Patent Document
[Patent Document 1] JP-A-H08-151331
[Patent Document 2] JP-A-H10-7585
[Patent Document 3] JP-A-2004-238320
[Patent Document 4] JP-A-2005-60321
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0004] The invention relates to provide a drink that may be useful
for prevention and treatment of various bone diseases such as
osteoporosis, fracture, rheumatism, and arthritis.
Means for Solving the Problems
[0005] The present inventors have found that the bone density can
be effectively increased by ingesting a drink that includes
angiogenin and/or angiogenin hydrolysate, and includes
lactoperoxidase and/or lactoperoxidase hydrolysate in a specific
mass ratio with respect to angiogenin and/or angiogenin
hydrolysate. This finding has led to the completion of the
invention.
[0006] Specifically, the invention includes following aspects:
[0007] (1) A drink including angiogenin and/or angiogenin
hydrolysate in an amount of more than 0.8 mg/100 ml and not more
than 150 mg/100 ml and lactoperoxidase and/or lactoperoxidase
hydrolysate in the mass ratio to the angiogenin and/or angiogenin
hydrolysate of 0.3 to 23.
[0008] (2) A method of preventing bone diseases including ingesting
the drink according to (1) in an amount of 200 nil/day or more.
[0009] (3) A method of producing the drink according to (1),
including mixing angiogenin and/or angiogenin hydrolysate and
lactoperoxidase and/or lactoperoxidase hydrolysate with a drink raw
material, and sterilizing the obtained mixture.
[0010] (4) A method of producing the drink according to (1),
including adding angiogenin and/or angiogenin hydrolysate and
lactoperoxidase and/or lactoperoxidase hydrolysate to a sterilized
drink raw material.
Effects of the Invention
[0011] The drink of the invention exhibits a bone-strengthening
effect, and may be useful for prevention and treatment of various
bone diseases such as osteoporosis, fracture, rheumatism, and
arthritis.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0012] A drink of the invention is characterized in that the drink
includes angiogenin and/or angiogenin hydrolysate in a specific
amount, and further includes lactoperoxidase and/or lactoperoxidase
hydrolysate in a specific mass ratio with respect to angiogenin
and/or angiogenin hydrolysate.
[0013] Cow milk generally contains angiogenin and/or angiogenin
hydrolysate in an amount of about 0.2 to 0.8 mg/100 ml, and
lactoperoxidase and/or lactoperoxidase hydrolysate in an amount of
about 1.5 to 6.0 mg/100 ml.
[0014] In contrast, the drink of the invention is added with
angiogenin and/or angiogenin hydrolysate and lactoperoxidase and/or
lactoperoxidase hydrolysate, and the drink contains angiogenin
and/or angiogenin hydrolysate in an amount of more than 0.8 mg/100
ml and not more than 150 mg/100 ml, and lactoperoxidase and/or
lactoperoxidase hydrolysate in a mass ratio with respect to
angiogenin and/or angiogenin hydrolysate of 0.3 to 23.
[0015] A fraction containing angiogenin and/or angiogenin
hydrolysate that is prepared from milk of a mammal, such as human,
cow, buffalo, goat, or sheep, a fraction containing lactoperoxidase
and/or lactoperoxidase hydrolysate that is prepared from milk of a
mammal, such as human, cow, buffalo, goat, or sheep, a fraction
containing angiogenin and/or angiogenin hydrolysate that is
produced by a genetic engineering, a fraction containing
lactoperoxidase and/or lactoperoxidase hydrolysate that is produced
by a genetic engineering, angiogenin and/or angiogenin hydrolysate
purified from blood or an organ, lactoperoxidase and/or
lactoperoxidase hydrolysate purified from blood or an organ, or the
like may be used as the angiogenin and/or angiogenin hydrolysate
and the lactoperoxidase and/or lactoperoxidase hydrolysate included
in the drink of the invention. A commercially available purified
angiogenin or lactoperoxidase reagent may also be used.
[0016] The drink of the invention may include angiogenin
hydrolysate or lactoperoxidase hydrolysate obtained by digesting a
fraction containing angiogenin, an angiogenin reagent, a fraction
containing lactoperoxidase, a lactoperoxidase reagent, or the like
using one or more proteases.
[0017] The drink of the invention may include a protein material
prepared by extracting a fraction containing angiogenin and/or
angiogenin hydrolysate and lactoperoxidase and/or lactoperoxidase
hydrolysate directly from milk or a material derived from milk,
such as skim milk or whey. Such a protein material may be prepared
as follows, for example. Specifically, milk or a material derived
from milk is brought into contact with a cation-exchange resin, and
milk-derived proteins adsorbed on the resin is eluted at a salt
concentration of 0.1 to 2.0 M, desalted and concentrated using a
reverse osmosis membrane, an electrodialysis membrane, an
ultrafiltration membrane, a microfiltration membrane, or the like,
and optionally subjected to proteolysis to a molecular weight of
8000 or less using a protease, such as trypsin, pancreatin,
chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin,
or V8 protease. When subjecting to proteolysis using a protease,
the lower limit of the molecular weight is preferably 500 or more.
The protein material thus obtained may be dried by freeze-drying,
spray drying, or the like, and the dried product may be added in
the drink.
[0018] The drink of the invention is produced by adding angiogenin
and/or angiogenin hydrolysate, and lactoperoxidase and/or
lactoperoxidase hydrolysate and a protein material that contains
angiogenin and/or angiogenin hydrolysate and lactoperoxidase and/or
lactoperoxidase hydrolysate, or the like to a drink raw material so
that the drink includes angiogenin and/or angiogenin hydrolysate in
an amount of more than 0.8 mg/100 ml and not more than 150 mg/100
ml, and includes lactoperoxidase and/or lactoperoxidase hydrolysate
in a mass ratio with respect to angiogenin and/or angiogenin
hydrolysate of 0.3 to 23.
[0019] As shown in the test examples described below, when the
drink includes angiogenin and/or angiogenin hydrolysate and
lactoperoxidase and/or lactoperoxidase hydrolysate as described
above, the bone-strengthening effect can be obtained more
effectively than the case of ingesting angiogenin and/or angiogenin
hydrolysate or lactoperoxidase and/or lactoperoxidase hydrolysate
separately.
[0020] The drink of the invention may be produced in the usual
manner as long as the drink includes the angiogenin and/or
angiogenin hydrolysate and lactoperoxidase and/or lactoperoxidase
hydrolysate in specific amounts, respectively. For example, the
drink of the invention is produced by adding angiogenin and/or
angiogenin hydrolysate to a drink raw material, such as a material
derived from milk so that the drink includes angiogenin and/or
angiogenin hydrolysate in a specific amount, and adding
lactoperoxidase and/or lactoperoxidase hydrolysate to the mixture
so that the mass ratio of lactoperoxidase and/or lactoperoxidase
hydrolysate to angiogenin and/or angiogenin hydrolysate is within
the specific range. Note that as the drink raw material, such as a
material derived from milk, cow milk, concentrated skim milk, skim
milk powder, whey, butter, cream, fermented milk, a dairy lactic
acid bacteria beverage, a lactic acid bacteria beverage, or the
like can be given, further a milk drink mixed thereof as
appropriate, processed milk, composition-modified milk, low-fat
milk, fat-free milk, or the like can be given, for example.
[0021] When adding angiogenin and/or angiogenin hydrolysate and
lactoperoxidase and/or lactoperoxidase hydrolysate to a drink raw
material, such as a material derived from milk, angiogenin and/or
angiogenin hydrolysate and lactoperoxidase and/or lactoperoxidase
hydrolysate may be added to either unsterilized drink raw material,
or a sterilized drink raw material. When adding to an unsterilized
drink raw material, sterilization may be conducted after the
addition. In this instance, heat sterilization is preferable. For
example, the drink raw material itself, or the drink raw material
to which angiogenin and/or angiogenin hydrolysate and
lactoperoxidase and/or lactoperoxidase hydrolysate are added, may
be heated to 70.degree. C., homogenized using a homogenizer at a
pressure of 15 MPa, sterilized at 130.degree. C. for 2 seconds, and
cooled to 5.degree. C. When sterilizing a mixture prepared by
adding angiogenin and/or angiogenin hydrolysate and lactoperoxidase
and/or lactoperoxidase hydrolysate to a drink raw material, it is
preferable to sterilize at 130.degree. C. for 2 seconds or
less.
[0022] It may be possible that the drink of the invention may be
added with a raw material or the like that is commonly used for a
food or drink, such as a saccharide, a lipid, a protein, a vitamin,
a mineral, or a flavor, in addition to angiogenin and/or angiogenin
hydrolysate, lactoperoxidase and/or lactoperoxidase hydrolysate,
other than the above drink raw material, and may also be added with
another bone-strengthening component such as calcium, vitamin D,
vitamin K, or isoflavone.
[0023] The drink of the invention can strengthen bones when taken
orally in an amount of 200 ml or more per kg of body weight, as
shown in the animal experiments described below. Since the intake
for the experiment animal corresponds to the intake for adults in
terms of blood drug concentration (see Mitsuyoshi Nakajima (1993),
"Yakkou Hyoka Vol. 8", Hirokawa-Shoten Ltd., pp. 2-18), it is
expected that bones can be strengthened, and especially various
bone diseases, such as osteoporosis, fracture, rheumatism, and
arthritis can be prevented or treated by ingesting the drink of the
invention in an amount of 200 ml/day or more per adult.
[0024] The invention is further described below in more detail by
way of reference examples, examples, and test examples. Note that
the following examples are intended for illustration purposes only,
and should not be construed as limiting the invention.
Reference Example 1
Preparation (1) of Angiogenin Fraction
[0025] A column filled with 30 kg of cation-exchange resin
(Sulfonated Chitopearl; manufactured by Fuji Spinning Co., Ltd.)
was thoroughly washed with deionized water, and 1000 liters of
unpasteurized skim milk (pH 6.7) was then applied to the column.
After thoroughly washed the column with deionized water, the
absorbed protein was eluted with a linear gradient of 0.1 to 2.0 M
sodium chloride. The eluted fraction containing angiogenin was
fractionated using an S-Sepharose cation-exchange chromatography
(manufactured by Amersham Bioscientific), and the resulted
angiogenin-containing fraction was heat-treated at 90.degree. C.
for 10 minutes, and centrifuged to remove a precipitate. The
angiogenin-containing fraction was further subjected to gel
filtration chromatography (column: Superose 12). The eluate
obtained was desalted using a reverse osmosis membrane, and the
desalted eluate was freeze-dried to obtain 16.5 g of an angiogenin
fraction having an angiogenin purity of 90%. These successive
operations were repeated 30 times.
Reference Example 2
Preparation (2) of Angiogenin Fraction
[0026] A column filled with 10 kg of Heparin Sepharose
(manufactured by GE Healthcare) was thoroughly washed with
deionized water, and 500 liters of unpasteurized skim milk (pH 6.7)
was then applied to the column. After thoroughly washing the column
with a 0.5 M sodium chloride solution, the column was eluted with a
1.5 M sodium chloride solution. The eluate was desalted using a
reverse osmosis membrane, and the desalted eluate was freeze-dried
to obtain 18 g of an angiogenin fraction having an angiogenin
purity of 5%. The above successive operations were repeated 50
times.
Reference Example 3
Preparation of Lactoperoxidase Fraction
[0027] A column (diameter: 5 cm, height: 30 cm) filled with 600 g
of cation-exchange resin (sulfonated Chitopearl; manufactured by
Fuji Spinning Co., Ltd.) was thoroughly washed with deionized
water, and 360 liters of unpasteurized skim milk (pH 6.7) was
applied to the column at a flow rate of 25 ml/min. After thoroughly
washing the column with deionized water, the absorbed protein was
eluted with a 0.02 M carbonate buffer (pH 7.0) containing 2.0 M
sodium chloride. The eluted fraction containing lactoperoxidase was
adsorbed on an S-Sepharose FF column (manufactured by Amersham
Bioscientific), and the column was thoroughly washed with deionized
water. After equilibration with a 10 mM phosphate buffer (pH 7.0),
the adsorbed fraction was eluted with a linear gradient of 0 to 2.0
M sodium chloride to collect a fraction containing lactoperoxidase.
The fraction was subjected to gel filtration chromatography using a
HiLoad 16/60 Superdex 75pg (manufactured by Amersham
Bioscientific). The eluate obtained was desalted using a reverse
osmosis membrane, and freeze-dried to obtain 27 g of a
lactoperoxidase fraction having a lactoperoxidase purity of 90%.
These successive operations were repeated 25 times.
Measurement of Angiogenin and Lactoperoxidase Contained in
Drink
[0028] The content of angiogenin, angiogenin hydrolysate,
lactoperoxidase and lactoperoxidase hydrolysate in the drink was
measured according to the method described in JP-A-2008-164511 with
modification. Specifically, 106 .mu.l of the drink was added to 5
ml of ultrapure water, and a 1/1000-equivalent amount of formic
acid was added to the mixture to prepare a sample solution. Ten
microliter (10 .mu.l) of the sample solution was dried up, and
dissolved in 20 .mu.l of 0.1 M ammonium bicarbonate containing 8 M
urea and 1 mM tris(carboxyethyl)phosphine (TCEP). The solution was
heated at 56.degree. C. for 30 minutes. After returning the
solution to room temperature, 5 .mu.l of 100 mM iodoacetamide
solution was added to the solution, and the mixture was reacted for
30 minutes in the dark. After the addition of 54 .mu.l of ultrapure
water, 10 .mu.l of 0.1 .mu.g/ml trypsin and 10 .mu.l of 0.1
.mu.g/ml Lysyl Endopeptidase were added to the mixture. The mixture
was reacted at 37.degree. C. for 16 hours. The reaction was then
terminated by adding 3 .mu.l of formic acid and used as the sample
peptide solution for measurement. The sample solution was diluted
6-fold with 10 fmol/.mu.l internal standard peptide solution
containing 0.1% formic acid, 0.02% trifluoroacetic acid (TFA), and
2% acetonitril, and 2.5 .mu.l of the diluted solution was subjected
to LC/MS/MS analysis.
[0029] The peptides were separated by gradient elution using an
HPLC system. More specifically, the peptides were separated using a
column (MAGIC C18, 0.2 mm (ID).times.50 mm) equipped with a 5
.mu.l-peptide trap on a MAGIC 2002 HPLC system at a flow rate of 2
.mu.l/min. A solution A (2% acetonitrile-0.05% formic acid) and a
solution B (90% acetonitrile-0.05% formic acid) were used as eluant
for HPLC. Gradient elution was conducted under the elution
condition from 2 to 65% using the solution B over 20 minutes.
[0030] As object ions for measuring lactoperoxidase, parent ion was
NH.sub.2-IHGFDLAAINLQR-COOH (m/z 734.4), and the MS/MS target ion
was NH.sub.2-IHGFDLA-COOH (m/z 754.4). As object ions for measuring
angiogenin, parent ion was NH.sub.2-YIHFLTQHYDAK-COOH (m/z 768.8),
and the MS/MS target ion was NH.sub.2-FLTQHYDAK-COOH (m/z 1122.8).
Regarding the internal standard peptide parent ion was
NH.sub.2-ETTVFENLPEK-COOH (wherein, P was labeled with .sup.13C and
.sup.15N) (m/z 656.9.), and the MS/MS target ion was
NH.sub.2-FENLPEK-COOH (wherein, P was labeled with .sup.13C and
.sup.15N) (m/z 882.4).
[0031] A system "LCQ Advantage" was used for MS. The peak area of
each protein was calculated from the resulting chromatogram, and
the concentration was calculated from the ratio with respect to the
internal standard peptide.
Example 1
[0032] Three hundred and thirty milligrams (330 mg) of the
angiogenin fraction obtained in Reference Example 1 and 90 mg of
the lactoperoxidase fraction obtained in Reference Example 3 were
mixed with 200 ml of cow milk. The mixture was sterilized at
130.degree. C. for 2 seconds, and cooled to 10.degree. C. to obtain
a drink (example product 1). The resulting drink contained
angiogenin and/or angiogenin hydrolysate in an amount of 150 mg/100
ml, and the mass ratio of lactoperoxidase and/or lactoperoxidase
hydrolysate to angiogenin and/or angiogenin hydrolysate in the
drink was 0.3.
Example 2
[0033] Twenty four milligrams (24 mg) of the angiogenin fraction
obtained in Reference Example 2 and 30 mg of the lactoperoxidase
fraction obtained in Reference Example 3 were mixed with 200 ml of
cow milk. The mixture was sterilized at 130.degree. C. for 2
seconds, and cooled to 10.degree. C. to obtain a drink (example
product 2). The resulting drink contained angiogenin and/or
angiogenin hydrolysate in an amount of 0.81 mg/100 ml, and the mass
ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to
angiogenin and/or angiogenin hydrolysate in the drink was 23.
Example 3
[0034] Twenty four milligrams (24 mg) of the angiogenin fraction
obtained in Reference Example 1 and 30 mg of the lactoperoxidase
fraction obtained in Reference Example 3 were mixed with 200 ml of
cow milk. The mixture was sterilized at 130.degree. C. for 2
seconds, and cooled to 10.degree. C. to obtain a drink (example
product 3). The resulting drink contained angiogenin and/or
angiogenin hydrolysate in an amount of 11 mg/100 ml, and the mass
ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to
angiogenin and/or angiogenin hydrolysate in the drink was 1.7.
Comparative Example 1
[0035] Twenty milligrams (20 mg) of the angiogenin fraction
obtained in Reference Example 2 and 34 mg of the lactoperoxidase
fraction obtained in Reference Example 3 were mixed with 200 ml of
cow milk. The mixture was sterilized at 130.degree. C. for 2
seconds, and cooled to 10.degree. C. to obtain a drink (comparative
example product 1). The resulting drink contained angiogenin and/or
angiogenin hydrolysate in an amount of 0.7 mg/100 ml, and the mass
ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to
angiogenin and/or angiogenin hydrolysate in the drink was 29.
Comparative Example 2
[0036] Three hundred and sixty milligrams (360 mg) of the
angiogenin fraction obtained in Reference Example 1 and 60 mg of
the lactoperoxidase fraction obtained in Reference Example 3 were
mixed with 200 ml of cow milk. The mixture was sterilized at
130.degree. C. for 2 seconds, and cooled to 10.degree. C. to obtain
a drink (comparative example product 2). The resulting drink
contained angiogenin and/or angiogenin hydrolysate in an amount of
162 mg/100 ml, and the mass ratio of lactoperoxidase and/or
lactoperoxidase hydrolysate to angiogenin and/or angiogenin
hydrolysate in the drink was 0.19.
Test Example 1
[0037] The bone-strengthening effects of the example products 1 to
3 and the comparative example products 1 and 2 were determined by
animal experiments. C3H/HeJ mice (5 weeks old, male) were used for
the animal experiments. After 1 week acclimation, the mice were
divided into six groups (10 mice/group). The mice were orally
administered each product of the example products 1 to 3 and the
comparative example products 1 and 2 in an amount of 200 ml per 1
kg of mouse weight once a day for 2 weeks using a tube. The control
group was not administrated any example products 1 to 3 and the
comparative example products 1 and 2 were not administered. After
completion of administration (second week), the bone density of the
right tibia of each mouse was measured using a micro-CT
(manufactured by Rigaku Corporation). The results are shown in
Table 1. As shown in Table 1, the groups that were orally
administered the example products 1 to 3 showed a significant
increase in bone density compared with the control group and the
comparative example groups that were orally administered the
comparative example product 1 or 2.
TABLE-US-00001 TABLE 1 Bone density (mg/cm.sup.3) Control group
1239 .+-. 8 Example product 1 1264 .+-. 12 Example product 2 1271
.+-. 12 Example product 3 1267 .+-. 13 Comparative example product
1 1243 .+-. 6 Comparative example product 2 1244 .+-. 6
Reference Example 4
[0038] A column (diameter: 4 cm, height: 30 cm) filled with 400 g
of cation-exchange resin (Sulfonated Chitopearl; manufactured by
Fuji Spinning Co., Ltd.) was thoroughly washed with deionized
water, and 40 liters of unpasteurized skim milk (pH 6.7) was
applied to the column at a flow rate of 25 ml/min. After thoroughly
washing the column with deionized water, proteins adsorbed on the
resin were eluted using a 0.02 M carbonate buffer (pH 7.0)
containing 0.78 M sodium chloride. The eluate was desalted using a
reverse osmosis membrane, and the desalted eluate was freeze-dried
to obtain 18 g of a powdery protein material (reference example
product 4).
Reference Example 5
[0039] Four grams (4 g) of protein material of the reference
example product 4 was dissolved in 800 ml of water. After the
addition of trypsin (manufactured by Sigma), which is a protease,
so as to obtain the final concentration of 0.03 wt %, the mixture
was subjected to enzymatic treatment at 37.degree. C. for 8 hours.
After inactivating the protease through heat-treatment at
90.degree. C. for 5 minutes, the mixture was freeze-dried to obtain
3.0 g of a powdery protein material (reference example product
5).
Example 4
[0040] Forty milligrams (40 mg) of the reference example product 4
was mixed with 200 ml of sterilized cow milk to obtain a drink
(example product 4). The resulting drink contained angiogenin
and/or angiogenin hydrolysate in an amount of 1.2 mg/100 ml, and
the mass ratio of lactoperoxidase and/or lactoperoxidase
hydrolysate to angiogenin and/or angiogenin hydrolysate in the
drink was 7.5.
Example 5
[0041] Forty milligrams (40 mg) of the reference example product 5
was mixed with 200 nil of sterilized cow milk to obtain a drink
(example product 5). The resulting drink contained angiogenin
and/or angiogenin hydrolysate in an amount of 1.15 mg/100 ml, and
the mass ratio of lactoperoxidase and/or lactoperoxidase
hydrolysate to angiogenin and/or angiogenin hydrolysate in the
drink was 7.4.
Comparative Example 3
[0042] Fifteen milligrams (15 mg) of the reference example product
4 and 25 mg of the lactoperoxidase fraction obtained in Reference
Example 3 were mixed with 200 ml of sterilized cow milk to obtain a
drink (comparative example product 3). The resulting drink
contained angiogenin and/or angiogenin hydrolysate in an amount of
0.7 mg/100 ml, and the mass ratio of lactoperoxidase and/or
lactoperoxidase hydrolysate to angiogenin and/or angiogenin
hydrolysate in the drink was 25.4
Test Example 2
[0043] The bone-strengthening effects of the example products 4 and
5 and the comparative example product 3 was determined by animal
experiments. Forty SD female rats (51 weeks old) were used for the
animal experiments. The rats were divided into five groups (8
rats/group). Four groups underwent ovariectomy, and the remaining
one group sham surgery. After a 4-weekrecovery period, the
ovariectomized rats were orally administered the example products 4
or 5 or the comparative example product 3 in an amount of 200 ml
per 1 kg of rat weight daily in six divided dose for 16 weeks using
a tube. The control group was not administrated any example
products 4 and 5 and the comparative example product 3. After
4-week recovery period, the rats underwent sham surgery were fed
for 16 weeks in the same manner as the control group. After
completion of administration (sixteenth week), the bone density of
the right tibia of each rat was measured using a micro-CT
(manufactured by Rigaku Corporation).
[0044] The results are shown in Table 2. As shown in Table 2, the
groups that were orally administered the example products 4 and 5
showed a significant increase in bone density as compared with the
control group and the group that was orally administered the
comparative example product 3. Moreover, the bone density
approached that of the sham surgery group.
TABLE-US-00002 TABLE 2 Bone density (mg/cm.sup.3) Control group 551
.+-. 11 Sham surgery group 602 .+-. 9 Example product 4 595 .+-. 10
Example product 5 596 .+-. 14 Comparative example product 3 554
.+-. 13
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