U.S. patent application number 10/599446 was filed with the patent office on 2007-09-06 for functional food for ameliorating engogenous melatonin secretion rhythm and functional food for ameliorating circadian rhythm.
This patent application is currently assigned to Calpis Co., Ltd. Invention is credited to Toshiyuki Kai, Akihiro Masuyama, Yoshinori Moriyama, Toshiaki Takano, Seiji Tsuboi, Keita Ueno.
Application Number | 20070207134 10/599446 |
Document ID | / |
Family ID | 35063515 |
Filed Date | 2007-09-06 |
United States Patent
Application |
20070207134 |
Kind Code |
A1 |
Moriyama; Yoshinori ; et
al. |
September 6, 2007 |
FUNCTIONAL FOOD FOR AMELIORATING ENGOGENOUS MELATONIN SECRETION
RHYTHM AND FUNCTIONAL FOOD FOR AMELIORATING CIRCADIAN RHYTHM
Abstract
The invention provides agents for phase-adjusting or enhancing
the amplitude of an endogenous melatonin secretion rhythm and for
improving a circadian rhythm, which may be taken daily and
continuously, have excellent safety, and may effectively prevent or
ameliorate disorders of an endogenous melatonin secretion rhythm or
of a circadian rhythm without administration of exogenous
melatonin, as well as functional food containing such an agent,
which may prevent or ameliorate various symptoms, such as sleep
disorder or prolonged sleep latency. The agent for phase-adjusting
or enhancing the amplitude of an endogenous melatonin secretion
rhythm according to the invention contains whey as the active
component. The functional food for improving an endogenous
melatonin secretion rhythm according to the invention contains the
agent for phase-adjusting or enhancing the amplitude of an
endogenous melatonin secretion rhythm. The functional food for
improving a circadian rhythm contains the agent for improving a
circadian rhythm.
Inventors: |
Moriyama; Yoshinori;
(Okayama-shi, JP) ; Tsuboi; Seiji; (Okayama-shi,
JP) ; Masuyama; Akihiro; (Sagamihara-shi, JP)
; Takano; Toshiaki; (Kawasaki-shi, JP) ; Ueno;
Keita; (Tokyo, JP) ; Kai; Toshiyuki;
(Machida-shi, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
Calpis Co., Ltd
|
Family ID: |
35063515 |
Appl. No.: |
10/599446 |
Filed: |
March 31, 2005 |
PCT Filed: |
March 31, 2005 |
PCT NO: |
PCT/JP05/06244 |
371 Date: |
February 8, 2007 |
Current U.S.
Class: |
424/93.45 ;
514/17.7; 514/5.6 |
Current CPC
Class: |
A61K 35/20 20130101;
A61P 43/00 20180101; A61P 25/20 20180101; A61P 25/00 20180101; A23L
33/10 20160801; A61K 35/747 20130101; A61P 5/04 20180101 |
Class at
Publication: |
424/093.45 ;
514/012 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61K 38/17 20060101 A61K038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2004 |
JP |
2004-106153 |
Jun 4, 2004 |
JP |
2004-166498 |
Claims
1. An agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm comprising whey as an active
component.
2. The agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm according to claim 1, wherein
said whey is at least one of acid whey and cheese whey
3. The agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm according to claim 2, wherein
said acid whey comprises fermented milk whey obtained by
fermentation of milk with bacteria including lactic acid
bacteria.
4. The agent for phase-adjusting or enhancing amplitude of an
endogenous melatonin secretion rhythm according to claim 3, wherein
said lactic acid bacteria are of the genus Lactobacillus sp.
5. The agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm according to claim 4, wherein
said lactic acid bacteria of the genus Lactobacillus are of the
species Lactobacillus helveticus.
6. The agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm according to claim 5, wherein
said Lactobacillus helveticus is of a strain Lactobacillus
helveticus CM4 (deposited at National Institute of Advanced
Industrial Science and Technology, International Patent Organism
Depositary under Accession Number FERM BP-6060).
7. The agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm according to claim 2, wherein
said acid whey is casein whey containing an aqueous fraction
obtained by adding acid to milk.
8. An agent for improving a circadian rhythm comprising whey as an
active component.
9. The agent for improving a circadian rhythm according to claim 8,
wherein said whey is at least one of acid whey and cheese whey.
10. The agent for improving a circadian rhythm according to claim
9, wherein said acid whey comprises fermented milk whey obtained by
fermentation of milk with bacteria including lactic acid
bacteria.
11. The agent for improving a circadian rhythm according to claim
10, wherein said lactic acid bacteria are of the genus
Lactobacillus sp.
12. The agent for improving a circadian rhythm according to claim
11, wherein said lactic acid bacteria of the genus Lactobacillus
are of the species Lactobacillus helveticus.
13. The agent for improving a circadian rhythm according to claim
12, wherein said Lactobacillus helveticus is of a strain
Lactobacillus helveticus CM4 (deposited at National Institute of
Advanced Industrial Science and Technology, International Patent
Organism Depositary under Accession Number FERM BP-6060).
14. The agent for improving a circadian rhythm according to claim
9, wherein said acid whey is casein whey containing an aqueous
fraction obtained by adding acid to milk.
15. Functional food for improving an endogenous melatonin secretion
rhythm comprising an agent for phase-adjusting or enhancing an
amplitude of an endogenous melatonin secretion rhythm according to
any one of claims 1 to 7.
16. The functional food according to claim 15, wherein said
improvement of an endogenous melatonin secretion rhythm comprises
prevention or amelioration of sleep disorder or prolonged sleep
latency.
17. Functional food for improving a circadian rhythm comprising an
agent for improving a circadian rhythm according to any one of
claims 8 to 14.
18. The functional food according to claim 17, wherein said
improvement of a circadian rhythm comprises prevention or
amelioration of sleep disorder or prolonged sleep latency.
Description
FIELD OF ART
[0001] The present invention relates to functional food for
improving an endogenous melatonin secretion rhythm and functional
food for improving a circadian rhythm, which are expected to
prevent or ameliorate sleep disorder or prolonged sleep latency, a
material for the active component of such food, and an agent for
phase-adjusting or enhancing the amplitude of an endogenous
melatonin secretion rhythm and an agent for improving a circadian
rhythm, which may prevent or ameliorate various disorders
associated with disorders of the endogenous melatonin secretion
rhythm or the circadian rhythm.
BACKGROUND ART
[0002] People in the modern world tend to lead irregular lifestyle
under the influence of recent advances and complication of
technologies as well as fast-moving social situation. The advent of
the 24-hour society has forced people into an irregular mode of
life, which leads to circadian rhythm disorder, such as sleep
disorder, or disorder of an endogenous melatonin secretion rhythm,
which is a possible factor for the circadian rhythm disorder
[0003] It has been confirmed that entrainment of a circadian rhythm
is controlled in the suprachiasmatic nucleus (SCN) in the
hypothalamus. A typical substance that is known to regulate a
circadian rhythm controlled by the suprachiasmatic nucleus is
melatonin (N-acetyl-5-methoxytryptamine) secreted mainly from the
corpus pineale. Melatonin is a hormone synthesized from tryptophan
through serotonin under the action of NAT (N-acetyltransferase) as
a rate-limiting enzyme. This hormone is believed to be involved in
introduction of photoperiodic information in photoperiodic mammals
and exert an influence on reproduction, body weight, metabolic
regulation, circadian rhythm control, as well as nervous and
endocrine functions. Further, since the melatonin secretion level
of the corpus pineale is low in the day time and high in the night
time, melatonin is believed to be one of the sleep-modulatory
substances.
[0004] It is known that the melatonin secretion rhythm in patients
with sleep disorder or circadian rhythm disorder is different from
that of healthy people in that the amplitude is decreased or the
phase is either advanced or delayed.
[0005] For overcoming such disorders of a circadian rhythm or a
melatonin secretion rhythm to treat or prevent sleep disorder,
administration of exogenous melatonin has been proposed. For
example, Patent Publication 1 reports that oral administration of
melatonin from an external source to artificially regulate a
melatonin rhythm has an effect on non-24-hour sleep-wake syndrome,
jet lag syndrome, shift-work sleep syndrome, delayed sleep phase
syndrome, or the like, accompanied by prolonged sleep latency,
insomnia, waking in bad mood, jet lag, reverse of day and night, or
the like.
[0006] However, the safety of melatonin products, including the
side effect of exogenous melatonin products per se, has not been
fully assured.
[0007] Patent Publication 2 proposes a composition for improving
the quality of sleep, containing as the active component muramyl
peptide prepared by hydrolysis from the cell wall of non-pathogenic
lactic acid bacteria. However, the improvement in the quality of
sleep taught in this publication is achieved by increasing the
length of the non-REM sleep phase through induction of sleep by the
immune system, so that this way of improving the quality of sleep
is completely different from the improvement of the endogenous
melatonin secretion level. [0008] Patent Publication 1:
JP-06-502259-T [0009] Patent Publication 2: JP-2003-517828-T
SUMMARY OF THE INVENTION
[0010] It is an object of the present invention to provide an agent
for phase-adjusting or enhancing the amplitude of an endogenous
melatonin secretion rhythm and an agent for improving a circadian
rhythm, which may be taken daily and continuously, have excellent
safety, and may effectively prevent or ameliorate disorders of an
endogenous melatonin secretion rhythm or of a circadian rhythm
without administration of exogenous melatonin.
[0011] It is another object of the present invention to provide
functional food, such as foods for specified-health uses, which may
be taken daily and continuously, has excellent safety, and may
effectively prevent or ameliorate various disorders including sleep
disorder or prolonged sleep latency possibly caused by disorders of
an endogenous melatonin secretion rhythm or of a circadian rhythm,
without administration of exogenous melatonin.
[0012] According to the present invention, there is provided an
agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm comprising whey as an active
component.
[0013] According to the present invention, there is also provided
an agent for improving a circadian rhythm comprising whey as an
active component.
[0014] According to the present invention, there is further
provided functional food for improving an endogenous melatonin
secretion rhythm, such as preventing or ameliorating sleep disorder
or prolonged sleep latency, comprising the above-mentioned agent
for phase-adjusting or enhancing an amplitude of an endogenous
melatonin secretion rhythm.
[0015] According to the present invention, there is also provided
functional food for improving a circadian rhythm, such as
preventing or ameliorating sleep disorder or prolonged sleep
latency, comprising the above-mentioned agent for improving a
circadian rhythm.
[0016] According to the present invention, there is provided a
method for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm comprising the step of orally
administering to an animal in need thereof an effective amount of
an agent for phase-adjusting or enhancing an amplitude of an
endogenous melatonin secretion rhythm comprising whey as an active
component.
[0017] According to the present invention, there is also provided a
method for improving a circadian rhythm comprising the step of
orally administering to an animal in need thereof an effective
amount of an agent for improving a circadian rhythm comprising whey
as an active component.
[0018] According to the present invention, there is further
provided use of whey for the manufacture of an agent for
phase-adjusting or enhancing an amplitude of an endogenous
melatonin secretion rhythm, or for the manufacture of functional
food for phase-adjusting or enhancing an amplitude of an endogenous
melatonin secretion rhythm.
[0019] According to the present invention, there is also provided
use of whey for the manufacture of an agent for improving a
circadian rhythm, or for the manufacture of functional food for
improving a circadian rhythm.
[0020] Since the agent for phase-adjusting or enhancing the
amplitude of an endogenous melatonin secretion rhythm, and the
agent for improving a circadian rhythm according to the present
invention contain whey, which has been taken as food, as the active
component, the present agents may be taken daily and continuously,
are excellently safe, and are expected to prevent or ameliorate
sleep disorder or prolonged sleep latency, such as non-24-hour
sleep-wake syndrome, jet lag syndrome, shift-work sleep syndrome,
sleep apnea syndrome, and middle-age sleep disorder, which are
believed to be associated with the disorders of such rhythms.
[0021] Since the functional food according to the present invention
contains the agent for phase-adjusting or enhancing the amplitude
of an endogenous melatonin secretion rhythm or the agent for
improving a circadian rhythm of the present invention, the present
functional food may prevent or ameliorate various symptoms such as
sleep disorder or prolonged sleep latency, without administration
of exogenous melatonin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a graph illustrating the comparison among the
groups of the melatonin concentration in the corpus pineale at
12:00 performed in Examples 1 and 2.
[0023] FIG. 2 is a graph illustrating the comparison among the
groups of the melatonin concentration in the corpus pineale at 0:00
performed in Examples 1 and 2.
[0024] FIG. 3 is a graph illustrating the comparison among the
groups of the NAT activity in the corpus pineale at 12:00 performed
in Examples 3 and 4.
[0025] FIG. 4 is a graph illustrating the comparison among the
groups of the NAT activity in the corpus pineale at 0:00 performed
in Examples 3 and 4.
PREFERRED EMBODIMENTS OF THE INVENTION
[0026] The present invention will now be explained in detail.
[0027] The agent for phase-adjusting or enhancing the amplitude of
an endogenous melatonin secretion rhythm according to the present
invention is capable of preventing or ameliorating various
disorders caused by disorder of an endogenous melatonin secretion
rhythm, exhibits at least one of a phase-adjusting effect and an
amplitude enhancing effect on the rhythm, and contains whey as the
active component.
[0028] The agent for improving a circadian rhythm according to the
present invention exhibits an effect of preventing or ameliorating
various disorders caused by circadian rhythm disorder, and contains
whey as the active component.
[0029] The active component, whey, includes an aqueous fraction of
milk obtained by removing all or most of the casein protein and the
like from milk according to a common procedure, and may be, for
example, acid whey and/or cheese whey. Examples of the acid whey
may include fermented milk whey obtained by fermentation of milk
with lactic acid bacteria, and casein whey containing an aqueous
fraction of milk obtained by adding acid to milk to remove all or
most of the casein protein and the like according to a common
procedure. Fermented milk whey is particularly preferred for its
excellent ability to phase-adjust or enhance the amplitude of an
endogenous melatonin secretion rhythm or to improve a circadian
rhythm.
[0030] The fermented milk whey may usually be a fermented milk whey
prepared by fermentation of milk with lactic acid bacteria, or by
symbiotic fermentation of milk with lactic acid bacteria and a
yeast. The starting material milk may be animal milk, such as cow's
milk, goat's milk, or sheep's milk; vegetable milk, such as soy
bean milk; or processed milk thereof, such as skim milk,
reconstituted milk, powdered milk, or condensed milk. The milk may
be in the form of a mixture.
[0031] The solid content of the milk is not particularly limited.
For example, for skim milk, the solid non-fat content is typically
about 9 mass %. On the other hand, considering the per-plant
productivity, the solid non-fat content may be increased to some
extent. The fermented milk whey obtained in the production of
fermented milk may be separated from other milk components before
use, but when the fermented milk whey is to be made into the
functional food or the like to be discussed later, such other milk
components are not necessarily separated.
[0032] The lactic acid bacteria may be those of the genus
Streptococcus, Lactococcus, Lactobacillus, Bifidobacterium, or the
like, with Lactobacillus being preferred. Specific examples of
Lactobacillus may include Lactobacillus bulgaricus, Lactobacillus
helveticus, Lactobacillus casei, Lactobacillus acidophilus, and
Lactobacillus fermentum, with Lactobacillus helveticus being
particularly preferred. More specifically, Lactobacillus helveticus
ATCC 15009, Lactobacillus helveticus ATCC 521, and Lactobacillus
helveticus CM4 strain (deposited at National Institute of Advanced
Industrial Science and Technology, International Patent Organism
Depositary under Accession Number BERM BP-6060 on Aug. 15, 1997)
(referred to as CM4 hereinbelow) may be used, with CM4 being
particularly preferred. CM4 has been deposited under the
above-mentioned accession number under the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. All restrictions on the availability
to the public of this strain will be irrevocably removed upon the
granting of a patent.
[0033] The lactic acid bacteria are preferably in the form of a
pre-cultured starter having sufficiently high activity. The initial
cell count may preferably be about 10.sup.5-10.sup.7 cells/ml.
[0034] When the fermented milk whey is to be used in functional
food, such as foods for specified health uses, yeast may be used
for symbiotic fermentation for improved flavor and palatability.
The strain of the yeast is not particularly limited, and may
preferably be, for example, yeast of the genus Saccharomyces, such
as Saccharomyces cerevisiae. The content of the yeast may suitably
be selected depending on the purpose.
[0035] The fermentation may be carried out by culturing one or more
kinds of the lactic acid bacteria in a medium, or culturing a
mixture of one or more kinds of the lactic acid bacteria and one or
more kinds of the yeast in a medium. The medium may be those
composed only of one or more kinds of the milk components mentioned
above, or those optionally contain additional components, such as
yeast extract; vitamins, e.g. ascorbic acid; amino acids, e,g.
cysteine; salts, e.g. sodium chloride; sugars, e.g. glucose,
sucrose, raffinose, or stachyose; stabilizers, e.g. gelatine; and
flavoring agents.
[0036] The fermentation may be performed usually by static or
stirred culture, for example at 20 to 50.degree. C., preferably 30
to 45.degree. C., at the initial pH of 6.0 to 7.0, and may be
terminated when the cell count becomes 10.sup.7 cells/ml or higher
at pH 5.0 or lower. The milk may be subjected to high-temperature
pasteurization before fermentation.
[0037] The fermented milk whey may be separated from curd by means
of a common separating operation. On the other hand, when the
fermented milk whey as the active component is to be used in the
functional food to be discussed later, the fermented milk
containing the whey may be used as it is without separation, if so
desired, or the extent of separation may suitably be decided
[0038] The casein whey may be prepared by, when solid milk, such as
whole milk or skim milk is used, dissolving the milk in distilled
water, adding, for example, lactic acid, citric acid, acetic acid,
tartaric acid, fumaric acid, malic acid, gluconic acid, or adipic
acid to adjust the acidity to a level suitable for removing
protein, typically casein, and separating the whey component
(aqueous fraction) by a common procedure, such as membrane
filtration. Here, the milk maybe subjected to high temperature
pasteurization before the acid is added. The acid may usually be
added in an amount for achieving 1.0 to 4.0% acidity, depending on
the kind of the acid or the like.
[0039] The cheese whey may be prepared in the ordinary cheese
production, by coagulating milk with rennet to form curd, and
separating the whey component from the curd by centrifugation or
the like. Here, the milk may be subjected to high temperature
pasteurization before the rennet is added.
[0040] The dose of the sour milk whey as the active component in
the agent for phase-adjusting or enhancing the amplitude of an
endogenous melatonin secretion rhythm or in the agent for improving
a circadian rhythm, is not particularly limited, taking the
continuity of administration into account, and may usually be not
less than 0.001 g per kg body weight per day, preferably not less
than 0.01 g per kg body weight per day, in terms of freeze-dried
powder. Further, the agent for phase-adjusting or enhancing the
amplitude of an endogenous melatonin secretion rhythm or the agent
for improving a circadian rhythm of the present invention may
optionally contain components others than the whey as desired,
having the function of phase-adjusting or enhancing the amplitude
of an endogenous melatonin secretion rhythm or improving a
circadian-rhythm.
[0041] The agent for phase-adjusting or enhancing the amplitude of
an endogenous melatonin secretion rhythm or the agent for improving
a circadian rhythm according to the present invention may be in the
form of whey with or without processing, for example, a whey
concentrate obtained by concentrating whey through vacuum
concentration or the like process, or a dried whey powder obtained
by drying whey through freeze-drying or spray drying.
[0042] The agent for phase-adjusting or enhancing the amplitude of
an endogenous melatonin secretion rhythm or the agent for improving
a circadian rhythm according to the present invention may be
administered usually through an oral route. For example, the agent
may be administered even after the symptoms of sleep disorder
caused by disorder of an endogenous melatonin secretion rhythm or
of a circadian rhythm are developed, or in the seasons to prevent
such symptoms, either continuously daily or intermittently.
[0043] The functional food for improving an endogenous melatonin
secretion rhythm according to the present invention contains the
agent for phase-adjusting or enhancing the amplitude of an
endogenous melatonin secretion rhythm, and the functional food for
improving a circadian rhythm according to the present invention
contains the agent for improving a circadian rhythm.
[0044] The functional food may be functional food, such as foods
for specified health uses, claiming prevention or amelioration of
symptoms caused by disorder of an endogenous melatonin secretion
rhythm or a circadian rhythm, such as prevention or amelioration of
sleep disorder or prolonged sleep latency.
[0045] The functional food may optionally contain additives, such
as sugars, proteins, lipids, vitamins, minerals, flavoring agents,
or mixtures thereof. Further, the milk components from which the
whey is separated, may also be contained.
[0046] In the functional food of the present invention, the content
of the whey as the active component may suitably be selected
depending on the form or kind of the food. The content may suitably
be selected also depending on the continuity of intake of the
functional food or the like factors, and is not particularly
limited. A suitable content may be usually 1 to 100 mass %.
[0047] The various functional food mentioned above may be in the
form of, for example, fermented milk products, such as yogurt or
lactic acid bacteria beverage, processed food and beverage
containing whey, dry powders, tablets, capsules, granules, or the
like,
[0048] The dose and the timing of administration of the various
functional food of the present invention are not particularly
limited, and it is preferred to take the functional food in such an
amount that the above-mentioned dose of the active component is
generally achieved For example, the present functional food may be
taken continuously or intermittently before or after the symptoms
of sleep disorder or the like caused by disorder of an endogenous
melatonin secretion rhythm or a circadian rhythm are developed.
EXAMPLES
[0049] The present invention will now be explained in more detail
with reference to Examples, which do not intend to limit the
present invention.
Examples 1 and 2
[0050] Commercially available skim milk was dissolved in distilled
water at a solid content of 9 mass %, subjected to high temperature
pasteurization in an autoclave at 105.degree. C. for 10 minutes,
allowed to cool to the room temperature, inoculated with 3 mass %
of a pre-cultured CM4 starter, and cultured at 37.degree. C. for 24
hours, to thereby obtain fermented milk. This fermented milk was
centrifuged at 12000 G for 20 minutes for removing the solids, to
prepare fermented milk whey.
[0051] On the other hand, commercially available skim milk was
dissolved in distilled water at a solid content of 9 mass %,
subjected to high temperature pasteurization in an autoclave at
105.degree. C. for 10 minutes, and allowed to cool to the room
temperature. Lactic acid was added to increase the acidity to 2.2%.
Then the product was centrifuged at 12000 G for 20 minutes for
removing the solids, to prepare casein whey.
[0052] Each of the obtained fermented milk whey (Example 1) and
casein whey (Example 2) was diluted with distilled water to 10 mass
%, and used in the following animal test as a drinking water. As a
control, distilled water without whey was also used in the
test.
[0053] Fifty one male Wistar rats at 3 weeks of age were pre-bred
for 1 week. During the pre-breeding, the rats were allowed free
access to solid feed (trade name MF, manufactured by ORIENTAL YEAST
CO., LTD.) and distilled water. The daily light-dark cycle during
the pre-breeding was set such that the light cycle was from 8:00 to
20:00 and the dark cycle was for 12 hours after that. After the
pre-breeding, the rats were divided into three groups of 17 animals
each, i.e., Group (1) taking distilled water (control), Group (2)
taking 10 mass % fermented milk whey (Example 1), and Group (3)
taking 10 mass % casein whey (Example 2), and bred with free access
to the respective drinks and solid feed for 1 month. After 1 month
of breeding, 9 animals in each group was slaughtered at 12:00, and
the remaining 9 animals in each group at 0:00, and the corpus
pineale of each animal was taken out of the brain. 200 .mu.l of
0.01 M perchloric acid was added, and the mixture was homogenized
and centrifuged. The melatonin content in the resulting supernatant
was measured, whereas the precipitate was collected for
quantification of proteins.
[0054] The melatonin content was measured using Melatonin EIA Kit
(trade name, manufactured by IBL Hamburg). The proteins were
quantified by the Bradford method using Bio-Rad Protein Assay
(trade name, manufactured by Bio-Rad). The results of the
measurement of melatonin content at 12:00 are shown in FIG. 1, and
those at 0:00 in FIG. 2. The statistical significance was
determined by the student-newman-keuls test.
[0055] As seen from FIGS. 1 and 2, in Group (1) taking distilled
water (control), the melatonin concentration was low at 12:00 and
high at 0:00, showing the behavior of melatonin that is low in day
time and high in night time. As seen from FIGS. 1 and 2, in Group
(2) taking the fermented milk whey and Group (3) taking the casein
whey, the melatonin concentration was lower at 12:00 and higher at
0:00, compared to those in Group (1) taking distilled water.
Particularly, in Group (2) taking the fermented milk whey, the
melatonin concentration at 12:00 was significantly lower than that
in Group (1) taking distilled water (significance level p<0.05),
and the melatonin concentration at 0:00 was significantly higher
than that in Group (1) taking distilled water (significance level
p<0.05) and that in Group (3) taking the casein whey
(significance level p<0.01).
[0056] The above results suggest that intake of fermented milk whey
or casein whey enhances the behavior of melatonin concentration
that is low in day time and high in night time. In particular, the
amplitude of enhancement was large when the fermented milk whey was
taken, suggesting that the fermented milk whey has still stronger
effect. That is, by taking fermented milk whey or casein whey, the
endogenous melatonin secretion rhythm is phase-adjusted or the
amplitude thereof is enhanced. The amplitude of enhancement is
larger for fermented milk whey than for casein effect.
Examples 3 and 4
[0057] Thirty three male Wistar rats at 3 weeks of age were
pre-bred for 1 week. During the pre-breeding, the rats were allowed
free access to solid feed (trade name MF, manufactured by ORIENTAL
YEAST CO., LTD.) and distilled water. The daily light-dark cycle
during the pre-breeding was set such that the light cycle was from
8:00 to 20:00 and the dark cycle was for 12 hours after that. After
the pre-breeding, the rats were divided into three groups (1) to
(3) of 11 animals each in the same way as in Examples 1 and 2, and
bred with free access to the respective drinks and solid feed for 1
month. After 1 month of breeding, 5 animals in each group was
slaughtered at 12:00, and the remaining 6 animals in each group at
0:00, and the corpus pineale of each animal was taken out of the
brain. The NAT activity in the corpus pineale was measured by the
method of Thomas et al.
[0058] Specifically, 100 ml of 0.25 M calcium phosphate buffer (pH
1.5) containing 1.5 mM of acetyl CoA was added to the corpus
pineale, and homogenized to prepare an enzyme solution. To 30 ml of
the enzyme solution, 70 ml of 0.25 M calcium phosphate buffer
containing 1.5 mM of acetyl CoA and 20 mM of tryptamine was added
and reacted at 37.degree. C. for 30 minutes, to which 1 ml of a
toluene/isoamyl alcohol/1M HCl (99:0.660:0.33) solution was added.
After stirring, the mixture was centrifuged at 500 G for 10
minutes, and 750 ml of the supernatant was evaporated in a
centrifugal evaporator to obtain a dried product. 100 ml of a
mobile phase (0.1 M citric acid, 0.1 M sodium acetate, 35% methanol
(pH 4.1)) was added and stirred, and the resulting solution was
measured for N-acetyltryptamine by a high performance liquid
chromatography with fluorescence detector. The measurement was
performed at the excitation wavelength of 285 nm, detection
wavelength of 360 nm, and flow rate of 1 ml/min, using a colum,
Wakosil-II 5C18 RS (4.6 mm.times.150 mm). The proteins were
quantified by the Bradford method against BSA standards.
[0059] The results of measurement of the NAT activity at 12:00 are
shown in FIG. 3, and those at 0:00 in FIG. 4. The statistical
significance was determined by the student-newman-keuls test.
[0060] As seen from FIGS. 3 and 4, no difference in the NAT
activity at 12:00 was observed among the groups, but at 0:00 the
NAT activity in Group (2) taking the fermented milk whey (Example
3) was significantly higher than that in Group (1) taking distilled
water (control) and that in Group (3) taking the casein whey
(Example 4) (significance level p<0.01). Further, the NAT
activity in Group (3) taking the casein whey was significantly
higher than that in Group (1) taking distilled water (significance
level p<0.05). Considering the fact that NAT is a rate-limiting
enzyme in melatonin synthesis, it is confirmed from these results
that NAT supports the change in melatonin concentration in the
corpus pineale.
Prescription Example 1
[0061] 90 mass % of fermented milk containing the fermented milk
whey prepared in Example 1, 0.05 mass % of Aspartame (trade name,
manufactured by AJINOMOTO K.K.) for drinkability, 0.05 mass % of
Yogurt Flavor cw-3186 (manufactured by T. HASEGAWA CO., LTD.) and
0.1 mass % each of Yogurt Flavor DY 4449 and Sugar Flavor HASE
SF-5531 (manufactured by T. HASEGAWA CO., LTD.) as flavoring
agents, 0.5 mass % of a stabilizer, and 9.2 mass % of distilled
water were mixed as starting materials. The mixture was homogenized
and pasteurized at 90.degree. C. The resulting product was
hot-filled into brown bottles by 100 g, and pasteurized by heating
at 80.degree. C. for 10 minutes, to thereby obtain a fermented sour
milk drink.
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