U.S. patent application number 10/971116 was filed with the patent office on 2005-05-26 for microorganism for treatment or prevention of corpulence and diabetes mellitus, and pharmaceutical composition containing the same.
This patent application is currently assigned to BIONEER CORPORATION. Invention is credited to Bang, Young Bae, Joung, Hea Jung, Kim, Bong Cheol, Kim, Hang Rae, Park, Han Oh.
Application Number | 20050112112 10/971116 |
Document ID | / |
Family ID | 26637996 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112112 |
Kind Code |
A1 |
Park, Han Oh ; et
al. |
May 26, 2005 |
Microorganism for treatment or prevention of corpulence and
diabetes mellitus, and pharmaceutical composition containing the
same
Abstract
The present invention relates to microorganisms for the
treatment or the prevention of obesity or diabetes mellitus, which
reduce the amount of monosaccharide or disaccharide which may be
absorbed into human body by converting monosaccharides such as
glucose, fructose, galactose et al. and disaccharides into
polymeric materials which cannot be absorbed by the intestine, and
relates to a pharmaceutical composition containing the said
microorganisms.
Inventors: |
Park, Han Oh;
(Choongcheongbuk-Do, KR) ; Bang, Young Bae;
(Choongcheongbuk-Do, KR) ; Joung, Hea Jung;
(Choongcheongbuk-Do, KR) ; Kim, Bong Cheol;
(Choongcheongbuk-Do, KR) ; Kim, Hang Rae;
(Choongcheongbuk-Do, KR) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
BIONEER CORPORATION
|
Family ID: |
26637996 |
Appl. No.: |
10/971116 |
Filed: |
October 25, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10971116 |
Oct 25, 2004 |
|
|
|
09855836 |
May 16, 2001 |
|
|
|
6808703 |
|
|
|
|
Current U.S.
Class: |
424/93.45 ;
435/252.3; 435/252.9 |
Current CPC
Class: |
A61K 35/74 20130101;
A61K 35/747 20130101; C12R 2001/225 20210501; C12R 2001/02
20210501; Y10S 435/823 20130101; A61K 35/742 20130101; C12N 1/205
20210501; A61P 3/04 20180101; A61K 35/744 20130101; A61P 3/10
20180101; A61K 35/745 20130101; Y10S 435/853 20130101; A61K 35/74
20130101; A61K 2300/00 20130101; A61K 35/747 20130101; A61K 2300/00
20130101; A61K 35/745 20130101; A61K 2300/00 20130101; A61K 35/744
20130101; A61K 2300/00 20130101; A61K 35/742 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/093.45 ;
435/252.3; 435/252.9 |
International
Class: |
A61K 045/00; C12N
001/20 |
Foreign Application Data
Date |
Code |
Application Number |
May 17, 2000 |
KR |
10-2000-0026379 |
Aug 26, 2000 |
KR |
10-2000-0049805 |
Claims
1. (canceled)
2. (canceled)
3. A pharmaceutical composition comprising at least one
microorganism selected from the group consisting of Acetobacter
sp., Leuconostoc sp., Bacillus sp., Lactobacillus sp.,
Streptococcus sp., Bifidobactedum sp., Lactococcus sp. and
Pediococcus sp. bacteria in an amount effective to prevent or treat
obesity and a pharmaceutically acceptable carrier, wherein the
microorganism converts an oligosaccharide into a
polysaccharide:
4. The pharmaceutical composition according to claim 3, wherein
said microorganism is selected from the group consisting of
Acetobactor sp., Lactobacillus sp. and Lactococcus sp.
bacteria.
5. The pharmaceutical composition according to claim 3, wherein
said microorganism is selected from the group consisting of
Acetobacter xylinum, Acetobacter hansenii, Acetobacter
pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillus sp.,
Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus
bulgaricus, Lactobacillus casei, Lactobacillus kefir, Lactobacillus
keriranofaciens, Lactobacillus bifidus, Lactobacillus sake,
Lactobacillus reuteri, Lactobacillus lactis, Lactobacillus
delbrueckii, Lactobacillus helveticusglucos var. jugurti.,
Lactococcus cremoris, Bifidobacterium bifidium, Streptococcus
thermophilus and Pediococcus sp.
6. (canceled)
7. The pharmaceutical composition according to claim 3, which is a
formulation suitable for oral administration.
8. The pharmaceutical composition according to claim 3, which is a
formulation coated with enteric coating materials.
9. The pharmaceutical composition according to claim 7, which is a
formulation coated with enteric coating materials.
10. A pharmaceutical composition comprising at least one
microorganism selected from the group consisting of Acetobacter
sp., Leuconostoc sp., Bacillus sp., Lactobacillus sp.,
Streptococcus sp., Bifidobactedum sp., Lactococcus sp. and
Pediococcus sp. bacteria in an amount effective to prevent or treat
diabetes mellitus and a pharmaceutically acceptable carrier,
wherein the microorganism converts an oligosaccharide into a
polysaccharide.
11. The pharmaceutical composition according to claim 10, wherein
said microorganism is selected from the group consisting of
Acetobactor sp., Lactobacillus sp. and Lactococcus sp.
bacteria.
12. The pharmaceutical composition according to claim 10, wherein
said microorganism is selected from the group consisting of
Acetobacter xylinum, Acetobacter hansenii, Acetobacter
pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillus sp.,
Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus
bulgaricus, Lactobacillus casei, Lactobacillus kefir, Lactobacillus
keriranofaciens, Lactobacillus bifidus, Lactobacillus sake,
Lactobacillus reuteri, Lactobacillus lactis, Lactobacillus
delbrueckii, Lactobacillus helveticusglucos var. jugurti.,
Lactococcus cremoris, Bifidobacterium bifidium, Streptococcus
thermophilus and Pediococcus sp.
13. (canceled)
14. The pharmaceutical composition according to claim 10, which is
a formulation suitable for oral administration.
15. The pharmaceutical composition according to claim 10, which is
a formulation coated with enteric coating materials.
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to microorganisms for
preventing or treating obesity or diabetes mellitus, which are
capable of reducing an amount of monosaccharides or disaccharides
that can be absorbed into the intestine by converting those mono or
disaccharides into polymeric materials that cannot be absorbed in
the intestines. The present invention also relates to use of the
microorganisms for preventing or treating obesity or diabetes
mellitus and a pharmaceutical composition containing the
microorganisms.
BACKGROUND OF THE INVENTION
[0002] Obesity is well known as a chronic disease caused by various
factors whose origins have not yet been clearly discovered. It is
understood that obesity induces hypertension, diabetes mellitus,
coronary heart disease, gall bladder disease, osteoarthritis, sleep
apnea, respiratory disorder, endomerial, prostate, breast and colon
cancer and the like.
[0003] According to the NIH Report (THE EVIDENCE REPORT: Clinical
Guideline on the Identification, Evaluation, and Treatment of
Overweight and Obesity in Adults, 1999, NIH), about 97,000,000
Americans suffer from overweighting and obesesity, and the number
of patients of type II diabetes mellitus associated with obesity,
reaches about 15,700,000. Moreover, it is reported that about
200,000 people die of diseases associated with obesity each year
(Dan Ferber, Science, 283, pp 1424, 1999).
[0004] Diabetes mellitus is one of the most widespread chronic
diseases in the world, which impose a substantial expense on the
public as well as on patients of diabetes mellitus and their
families.
[0005] There are several types of diabetes mellitus that are caused
by various etiological factors and whose pathogenesis is different
from each other. For example, genuine diabetes mellitus is
characterized by high level of blood glucose and glycosuria, and is
a chronic disorder of carbohydrate metabolism due to a disturbance
of the normal insuline mechanism.
[0006] Non-Insulin-Dependent Genuine Diabetes Mellitus (NIDDM), or
the type II diabetes mellitus is found in adults who have
insulin-resistance in a peripheral target tissue, despite of normal
generation and function of insulin. Non-Insulin-Dependent Genuine
Diabetes Mellitus(NIDDM) can be caused by three important metabolic
disorders, i.e., insulin-resistance, fucntional disorder of insulin
secretion stimulated by nutrients, and overproduction of glucose in
liver. Failure to treat NIDDM, resulting in losing control of blood
glucose levels, leads to death of patients from diseases such as
atherosclerosis, and/or may cause late complications of diabetes,
such as retinopathy, nephropathy or neuropathy.
[0007] Accompanying diet-exercise therapy, NIDDM therapy uses
sulfonylurea and biguanidine compounds to control blood glucose
levels. Recently, therapeutic compounds such as metformin or
acarbose have been used for treating NIDDM. However, diet-exercise
therapy alone or even combined with chemotherapy using such
compounds fails to control hyperglycemia in some of the diabetes
mellitus patients. In such cases, these patients require exogenous
insulin.
[0008] Administration of insulin is very expensive and painful to
patients, and furthermore, may cause various detrimental results
and various complications in patients. For example, incidences,
such as, miscalculating insulin dosage, going without a meal or
irregular exercise, may cause insulin response (hypoglycemia) and
sometimes the insulin response occurs even without any particular
reasons. Insulin injection may also cause an allergy or
immunological resistance to insulin.
[0009] There are several methods for preventing or treating obesity
or diabetes mellitus, including diet-exercise therapy, surgical
operation and chemotherapy. Diet-exercise therapy involves a
low-calorie and low-fat diet accompanying aerobic exercise, but
this therapy requiring a regular performance is hard to continue
until achieving the goal.
[0010] Despite of instant effects, a surgery for physically
removing body fat has limitations due to the risk and cost involved
in a surgical operation and insufficient durability of the
effects.
[0011] As one of the most promising therapies currently developed,
pharmacotherapy can reduce blood glucose level, inhibit absorption
of glucose, strengthen the action of insulin or induce the decrease
of appetite. The medicines that have been developed so far use
various physiological mechanisms for the prevention and the
treatment of obesity and diabetes mellitus.
[0012] Some medicines, such as, sulfonylurea, mefformin,
pioglitazone or thiazolidindione derivatives and the like have been
developed to enhance the function of insulin. Although sulfonylurea
stimulates insulin-secretion from .beta.-cells in the pancreas, it
may accompany side effects, such as hypoglycemia resulting from
lowering blood glucose levels under normal levels.
[0013] Mefformin is mainly used for insulin-nondependent diabetes
mellitus patients who fail to recover after diet-exercise therapy.
This medicine inhibits hepatic gluconeogenesis and enhances glucose
disposal in muscle and adipose tissue. However, it suffers from
side effects, such as, nausea, vomiting and diarrhea.
[0014] Pioglitazone developed by Takeda in Japan, enhances the
function of insulin through increasing susceptibility of cells to
insulin (Kobayashi M. et al., Diabetes, 41(4), pp 476-483,
1992).
[0015] Beta 3-adreno receptor inhibitor (BRL-35135) known as a
medicine that stimulates the decomposition of body fats and that
convert body fats into heat with a specific action on adipose
cells, also suffers from lowerings blood glucose level.
[0016] The inhibitor of a pancreatic lipase (Orlistat produced by
Roche of Switzlend) inhibits and/or reduces absorption of body fats
by inhibiting pancreatic lipase. It, however, accompanies
undersirable inhibition of absorption of fat-soluble vitamin and
may also cause breast cancer.
[0017] Generally, medicines that decrease appetite affects
catecholamine in the brain. However, dexfenfluororamine and
fenfluoroamine have side effects of nerve toxicity and valvular
heart disease. Also, sibutramine has side effects of increasing
heart rate and blood pressure.
[0018] .alpha.-Glucosidase inhibitor (Acarbose produced by Bayer of
Germany), is known as a glucose absorbing inhibitor. Acarbose is
pseudo-monosaccharide which competitively inhibits the action of
various .alpha.-glucosidases existing in microvilli of the
gastrointestinal tract. However, taking a large amount of these may
induce diarrhea. (W. Puls et al., Front. Horm. Res. 2, 235,
1998).
[0019] Amylase inhibitor that inhibit converting carbohydrates into
oligosaccharides has been developed to prevent imbalance of
metabolism originated from excessive uptake of nutrient.
(Sanches-Monge R. et al. Eur. J. Biochem., 183, 003740, 1989).
[0020] Dietary fiber using diet with a large amount of vegetable
fiber is the easiest way to obtain inhibitory effect on obesity by
lowering glucose and/or fat amounts absorbed in the intestine.
However, such method also involves problems in requiring facility
and manpower for the production of dietary fiber with low
productivity.
[0021] Polymeric materials, such as, isomaltotriose, dextran and
pullulan, inhibit the increase of blood glucose level originated
from glucose. However, such materials also cause severe side
effects. For example, dextran may induce excessive bleeding by
delaying a blood coagulation time.
[0022] Among said various medicines, dietary fibers are the most
useful medicine for prevention or treatment of obesity because no
damage to the human metabolism-balance and use natural
substances.
[0023] Microorganism dietary fiber is produced using
microorganisms, such as, Gluconobacter sp., Agrobacterium sp.,
Acetobacterxylinum, A. hansenii, A. pasteurianus, A. aceti,
Rhizobium sp., Alcaligenes sp., Sarcina sp., Streptococcus
thermophilus, Lactococcus cremoris, Lactobacillus helveticus,
Lactobacillus bulgaricus, Lactobacillus sake, Lactobacillus
reuteri, Lactobacillus lactis, Lactobacillus delbrueckii subsp.,
Lactobacillus helveticusglucose var. jugurti, Leuconostoc
dextranicum, Bulgariscus sp., Campestris sp., Sphingomonas sp.
[0024] Dietary fiber produced by these microorganisms is used as
stabilizer, thickening agent, emulsifier, hygroscopic agent of
various foods and raw materials of cosmetics and pharmaceuticals.
Microorganism cellulose, xanthan, acetan, guar gum, locust bean
gum, carrageenan, alginate, and agar obtained from seaweed are
commercialized.
[0025] Lactobacillus sp. strain is the major component of normal
microbial flora in the human intestines. Its significant roles for
maintaining digestive organ and for healthy environment of the
vagina, have been well known. [Bible, D. J., ASM News,
54:661-665,1988; Reid G. and A. W. Bruce, In H Lappin-Scott (de.),
Bacterial biofilms, Cambridge University Press, Cambridge, England,
p. 274-281,1995; Reid G., A. W. Bruce, J. A. McGroarty, K. J.
Cheng, and J. W. Costerton, clin. Microbiol. Rev., 3:335-344,
1990]. Generally, Lactobacillus strain inhabits in digestive organs
(L. acidophilus, L. intestinalis, L. johnsonii, L. reuteri et
al.,), muscosa of the vagina (L. vanginals, L. gasseri), food
(wine-L. hilgardii), lactobacillus beverage (L. kefir, L.
kefiranofaciens), cheese (L. casei), vinegar (L. acetotolerance),
the oral cavity (L. oris), yeast (L. sake, L. homohiochi), fruit
juice (L. kunkeei, L. mali, L. suebicus), fermented sausages or
fish (L. farciminis, L. alimentarious) et al.
[0026] Many people take health complementary food containing a
Lactobacillus sp. strain in order to maintain healthy intestines
and to prevent urogenital tract infection. Recently, in addition to
the prevention of the diarrhea, constipation and urogenital tract
infection, various probiotic activities of Lactobacillus, such as,
control of immunity, control of cholesterol level in blood,
prevention of cancer, treatment of rheumatism, alleviation of
sensitivity on lactose or effect for atopic dermatitis, have been
reported and thus, have attracted more attention.
[0027] According to the U.S. Public Health Service Guideline, all
of the 262 Lactobacillus deposited in ATCC are classified as
"Bio-safety Level 1," which stands for no potential risk, which has
been known up to now, causing diseases in human or animals. There
is no harm to human body among approximately 60 strains of
Lactobacillus.
[0028] Recently, there has been a rapid progress in the research
for an extracellular dietary fiber produced by Lactobacillus. It
has been reported that a process of producing dietary fiber in
these strains are very complicated because a lot of genes are
mediated in the process, and the amount of dietary fiber thus
produced are very low (Int. J. Food Microbiol., Mar 3
40:1-2,.87-92, 1998; Current Opinion in Microbiology, 2:598-603,
1999; Appl. Environ. Microbiol., Feburary 64:2, 659-64, 1998; FEMS
Microbiol. Rev. April 23:2 153-77, 1999; FEMS Microbiol. Rev.
September 7:1-2, 113-30, 1990).
[0029] Also, various researches on the synthesis of cellulose by
Acetobacter sp. which is well known as a microorganism producing
dietary fiber, have been performed (Aloni Y., cohen R., Benziman
M., Delmer D, J Biological chemistry, 171:6649-6655, 1989; Ascher
M., J. Bacteriology, 33:249-252, 1937; Benziman M.,
Burger-Rachamimv H., J., Bacteriology, 84:625-630, 1962; Brown A M.
Journal of Polymer science, 59:155-169, 1962; Brown A M, Gascoigne
J A, Nature, 187:1010-1012, 1960; Calvin J R, Planta D P, Benziman
M., Padan E, PANS USA, 79:5282-5286,1982; Dehmer D P. Brown R M
Jr., Cooper J B, Lin F C, Science, 230:82-825, 1985).
[0030] Acetobacter is a strict aerobe but has characteristics of
surviving and living under the condition of infinitesimal oxygen,
and of being floated to seek for oxygen by means of synthesizing
cellulose dietary fiber itself under this condition of
infinitesimal oxygen. According to the research regarding the
amount and rate of converting glucose into cellulose dietary fiber
by Acetobacter (Brown et al.: Proc. Natl. Acad. Sci. USA, Vol 73
(12), 4565-4569), Acetobacter converts glucose into cellulose with
the speed rate of 400 mol/cell/hour. This is equivalent to the rate
that about 200 g glucose can be converted into cellulose dietary
fiber by 4.times.10.sup.15 cells per an hour.
[0031] Although Acetobacter that can metabolizes saccharose is
rare, Acetobacter converting sacchores in glucose, exists in nature
(PNAS, 9: pp 14-18). Presently, FDA of the United States has
approved Acetobacter xylinum for synthesizing acetic acid and
sorbose, and has classified it as generally safe microorganism
(GRAS: Generally Recognized As Safe).
[0032] As mentioned above, although there have been various
researches and efforts to develop drugs for treatment or prevention
of obesity and diabetes mellitus, their results were not
satisfactory. Various chemical substances mentioned above, have
been developed for treatment of obesity and diabetes mellitus, but
still suffer from various side effects. These drugs forcibly
discharge body fat together with valuable proteins. Consequently,
any one single drug for treatment or prevention of obesity and
diabetes mellitus at the origin thereof does not exist yet.
SUMMARY OF THE INVENTION
[0033] Therefore, the object of the present invention is to provide
microorganisms capable of living within the intestines and
converting oligosaccharides produced by the digestive enzymes into
non-digestable polysaccharides, and thereby remarkably reducing the
amount of oligosaccharide absorbed into the intestines.
[0034] Another object of the present invention is to provide a
pharmaceutical composition comprising at least one of said
microorganisms in an amount effective to prevent or treat obesity
and diabetes mellitus and a pharmaceutically acceptable carrier.
Another object of the present invention is to provide a method for
preventing or treating obesity, diabetes mellitus comprising
administering to a subject in need thereof capable of
pharmaceutical comprising a method for reducing weight gain,
controlling blood glucose level and control absorption of blood
lipod.
[0035] The microorganisms that can be used for the pharmaceutical
composition of the present invention preferably fall within
Acetobacter genus, Gluconobacter genus, Lactobacillus genus, and
Acrobacterium genus, which are capable of living in the intestine
and not harmful to human body, and are capable of converting
oligosaccharides into polysaccharides that cannot be absorbed into
human body. Specifically, the following microorganisms can be used
as microorganisms of the pharmaceutical composition of the present
invention, such as, Acetobacter xylinum, A. hansenii, A.
pasteurianus, A. aceti, Lactococcus cremoris, Lactobacillus
helveticus, L. bulgaricus, L. sake, L. reutari, L. lactis, the
subspecies of L. delbrueckii, L. delbrueckii subsp., and a variant
form of L. helveticusglucose. Preferably, the microorganisms can be
used as an active principle of the pharmaceutical composition of
the present invention is Lactobacillus sp. BC-Y009 (KCTC0774BP)
strain or Acetobacter sp. BC-Y058 (KCTC0773BP) strain.
[0036] The pharmaceutical composition of the present invention may
be administered in a form of tablet, capsule, suspension or
emulsion, which comprises excipients, pharmaceutically acceptable
vehicles and carriers which are selected depending on
administration routes. The pharmaceutical formulation of the
present invention may further comprises supplemental active
ingredients.
[0037] Lactose, dextrose, sucrose, sorbitol, mannitol, starch,
acacia gum, calcium phosphate, alginic acid salt, treguhkense
latex, gelatin, calcium silicate, finecrystalline cellulose,
polyvinylpyrolidon, cellulose, water, syrup, methylcellulose,
methylhydroxybenzoate and prophylhydroxybenzoate, talc, magnesium
stearate or mineral oil may be used as carriers, exipients or
diluents in the pharmaceutical composition of the present
invention.
[0038] In addition, the pharmaceutical composition of the present
invention may further comprises lubricants, moisturizer,
emulsifier, suspension stabilizer, preservative, sweetener and
flavor. The pharmaceutical composition of the present invention may
be in a form of an enteric coating formulation produced by various
methods which have been publicly known, in order to deliver the
active ingredients of the pharmaceutical composition, i.e.,
microorganisms, to the small intestines without degradation by
gastric juices in stomach.
[0039] Furthermore, microorganisms of the present invention may be
administered in a form of capsule prepared by conventional process.
For example, standard vehicles and lyophilized microorganisms of
the present invention are mixed together and prepared to pellets
and then, the pellets are filled into hard gelatin capsules. In
addition, the microorganisms of the present invention and
pharmaceutically allowable vehicles, for example, aqueous gum,
cellulose, silicate or oil are mixed to produce a suspension or
emulsion and then, this suspension or emulsion may be filled into
soft gelatin capsule.
[0040] The pharmaceutical composition of the present invention may
be prepared as an enterically coated tablets or capsules for oral
administration. The term "the enteric coating" of this application
includes all conventional pharmaceutically acceptable coating that
has resistance to gastric juice, however, in the small intestines,
can disintegrate sufficiently for a rapid release of the
microorganisms of the present invention.
[0041] The enteric coating of the present invention can be
maintained for more than 2 hours in synthetic gastric juice, such
as HCl solution of pH 1 at the temperature of 36.degree. C. to
38.degree. C. and desirably, decomposes within 0.5 hours in
synthetic intestinal juice, such as KH.sub.2PO.sub.4 buffer
solution of pH 6.8.
[0042] The enteric coating of the present invention applies to each
tablet with the amount of about 16 to 30 mg, desirably 16 to 25 mg,
more desirably 16 to 20 mg. The thickness of enteric coating of the
present invention is 5 to 100 .mu.m, desirably 20 to 80 .mu.m. The
components of the enteric coating are selected appropriately from
common polymeric materials which have been publicly well known. The
polymeric materials which may be employed for enteric coating of
the present invention are enumerated and described in the flowing
articles [The Theory and Practices of Industrial Pharmacy, 3rd
Edition, 1986, pp. 365-373 by L. Lachman, Pharmazeutische
Technologie, thieme, 1991, pp. 355-359 by H. Sucker, Hagers
Handbuch der Pharmazeutischen Praxis, 4th Edition, Vol. 7, pp. 739,
742, 766, and 778, (SpringerVerlag, 1971), and Remington's
Pharmaceutical Sciences, 13th Edition, pp.1689 and 1691 (Mack
Publ., Co., 1970)]. For example, cellulose ester derivative,
cellulose ether and copolymer of acryl and methyl acrylate or
maleic acid or phthalic acid derivative may be used in enteric
coating of the present invention.
[0043] The preferred enteric coating of the present invention are
prepared from polymers of cellulose acetate phthals or trimelitate
and methacrylic copolymer (for example, copolymer of more than 40%
of methacrylic acid and methacrylic acid which contains
hydroxyprophyl methylcellulose phthalate or derivatives from ester
thereof.
[0044] Endragit L 100-55 manufactured by Rohm GmbH of Germany may
be used as a raw material for the enteric coating of the present
invention.
[0045] Cellulose acetate phthalate employed in the enteric coating
of the present invention, has about 45 to 90 cP of viscosity, 17 to
26% of acetyl contents and 30 to 40% of phthalate contents. The
cellulose acetate trimelitate used in the enteric coating, has
about 15 to 21 cS of viscosity, 17 to 26% of acetyl contents, and
25 to 35% of trimelityl contents. The cellulose acetate trimelitate
which is manufactured by the Eastman Kodak Company may be used as a
material for the enteric coating of the present invention.
[0046] Hydroxyprophyl methylcellulose phthalate used in the enteric
coating of the present invention has molecular weight of generally
20,000 to 100,000 dalton, desirably 80,000 to 130,000 dalton and
has 5 to 10% of hydroxyprophyl contents, 18 to 24% of metoxy
contents, and 21 to 35% of phthalyl contents. Cellulose acetate
phthalate manufactured by the Eastman Kodak Company can be used as
a material for the enteric coating of the present invention.
[0047] Hydroxyprophyl methylcellulose phthalate used in the enteric
coating of the present invention is HP50 which is manufactured by
the Shin-Etsu Chemical Co. Ltd., Japan. The HP50 has 6 to 10% of
hydroxyprophyl contents, 20 to 24% of metoxy contents, 21 to 27% of
prophyl contents, and molecular weight is 84,000 dalton. Another
material for enteric coating manufactured by the Shin-Etsu Chemical
Co. Ltd., is HP55. HP55 can also be used as material for the
enteric coating of the present invention. The HP55 has 5 to 9% of
hydroxyprophyl contents, 18 to 22% of metoxy contents, 27 to 35% of
phthalate contents, and molecular weight is 78,000 dalton.
[0048] The enteric coating of the present invention is prepared by
using conventional methods of spraying the enteric coating solution
to the core. Solvents used in the process of the enteric coating
are alcohol such as ethanol, ketone such as acetone, halogenated
hydrocarbon such as dichloromethane, or the mixture thereof.
Softeners such as Di-n-butylphthalate and triacetin are added to
the enteric coating solution in the ratio of 1 part coating
material to about 0.05 or to about 0.3 part softner.
[0049] A spraying process is preferably performed continuously, and
the amount of materials sprayed may be controlled depending on the
condition of the coating process. Spraying pressure may be
regulated variously and, generally, desirable result can be
obtained under the pressure of average 1 to 1.5 bar.
[0050] "The effective amount" of this specification means the
minimum amount of the microorganisms of the present invention,
which can reduce the amount of oligosaccharide absorbed into the
body through the intestines of mammalian animals. The amount of
microorganisms administered into a body with the pharmaceutical
composition of the present invention may be adjusted depending on
the administration method and the administration subject.
[0051] The composition of the present invention may be administered
once or more per day on the subject. The unit of administration
amount means that it is separated physically and thus is suitable
for the unit administration for the human subjects and all other
mammalian animals. Each unit contains a pharmaceutically acceptable
carrier and the amount of the microorganisms of the present
invention which are effective in therapy.
[0052] An oral administration unit of an adult patient contains
microorganisms of the present invention in an amount, desirably,
0.1 g or more, and the composition of the present invention
contains 0.1 to 10 g per one time administration, desirably 0.5 to
5 g. The effective amount of microorganisms of the present
invention is 0.1 g per 1 day.
[0053] However, the administration amount can vary depending on the
weight and the severity of obesity of the patient, supplemental
active ingredients included and microorganisms used therein. In
addition, it is possible to divide up the. daily administration
amount and to administer continuously, if needed. Therefore, range
of the administration amount does not limit the scope of the
present invention in any way.
[0054] The "composition" of the present invention means not only as
medicinal products but also to serve as functional foods and health
complementary foods.
[0055] In case of taking the composition of the present invention
periodically, microorganisms form colony within the intestines and
interrupt absorption of oligosaccharide in the body competitively.
Also, non-digestable fibers produced by microorganisms make a
healthy condition for other useful microorganisms within the
intestines and stimulate the intestinal activity. Consequently, the
composition of the present invention functions to treat and prevent
obesity and diabetes mellitus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] The above objects and other advantages of the present
invention will become more apparent by describing in detail a
preferred embodiment thereof with reference to the attached
drawings, in which:
[0057] FIG. 1 is the graph illustrating the absorption rate of
glucose by the microorganisms of the present invention.
[0058] FIG. 2 is the graph illustrating the change of blood glucose
level after taking the microorganisms of the present invention.
[0059] FIG. 3 is the graph illustrating the change of energy
metabolism efficiency of obese mouse that has taken the
microorganism of the present invention.
[0060] FIG. 4 is the graph illustrating the change of energy
metabolism efficiency of control mouse that has taken the
microorganism of the present invention.
[0061] FIG. 5 is the graph illustrating the change of the body
weight of obese mouse induced by pharmacological prescription.
[0062] FIG. 6 is the graph illustrating the change of the metabolic
efficiency of obese mouse induced by pharmacological
prescription.
[0063] FIG. 7 is the phylogenetic analysis diagram of Lactobacillus
BC-Y009 based on 16 s rRNA nucleotide sequence of the present
invention.
[0064] FIG. 8 is the phylogenetic analysis diagram of Lactobacillus
BC-Y058 based on 16 s rRNA nucleotide sequence of the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0065] Hereinafter, the present invention will be described more in
detail.
[0066] The microorganisms which can be used in the pharmaceutical
composition of the present invention for preventing and treating
obesity and diabetes mellitus, or in a method therefore, should
satisfy the requirements of 1) being capable of proliferating
within the intestinal layers, 2) being capable of absorbing
oligosaccharide rapidly and of converting them into non-digestable
or hardly digestable high molecular weight materials, such as
fibrous materials, and 3) being harmless to human body and animals.
All microorganisms that can satisfy the above requirements can be
used as active principles of the pharmaceutical composition of the
present invention and for use of the pharmaceutical composition,
and may be obtained from the numerous microorganism depository
institutions in the world.
[0067] Therefore, the microorganisms of the pharmaceutical
composition of the present invention are Acetobacter xylinum,
Acetobacter BC-Y058, Acetobacter hansenii, Acetobacter
pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillus sp.,
Lactobacillus BC-Y009, Lactobacillus brevis, Lactobacillus
helveticus, Lactobacillus bulgaricus, Lactobacillus casei,
Lactobacillus kefir, Lactobacillus keriranofaciens, Lactobacillus
bifidus, Lactobacillus sake, Lactobacillus reuteri, Lactobacillus
lactis, Lactobacillus delbrueckii, Lactobacillus helveticusglucos
var. jugurti., Lactococcus cremoris, Bifidobacterium bifidium,
Streptococcus thermophilus or Pediococcus sp. Bacteria, which
produce polysaccharide. These microorganisms are described in the
following Articles:
[0068] Bart Degeest and Luc De Vuyst,
[0069] "Indication that the Nitrogen Source Influences Both Amount
and Size of Exopolysaccharides Produced by Streptococcus
thermophilus LY03 and Modelling of the Bacterial Growth and
Exopolysaccharide Production in a Complex Medium"
[0070] (Appl. Envir. Microbiol. 1999, 65: 2863-2870);
[0071] Stacy A. Kimmel, Robert F. Roberts and Gregory R.
Ziegler,
[0072] "Optimization of Exopolysaccharide Production by
Lactobacillus delbrueckii subsp. bulgaricus R R Grown in a
Semidefined Medium"
[0073] (Appl. Envir. Microbiol. 1998, 64: 659-664.);
[0074] P. L. Pham, I. Dupont, D. Roy, G. Lapointe and J.
Cerning,
[0075] "Production of Exopolysaccharide by Lactobacillus
rhamnosus
[0076] R and Analysis of Its Enzymatic Degradation during Prolonged
Fermentation"
[0077] (Appl. Envir. Microbiol. 2000, 66: 2302-2310.);
[0078] Petronella J. Looijesteijn, Ingeborg C. Boels, Michiel
Kleerebezem and Jeroen Hugenholtz,
[0079] "Regulation of Exopolysaccharide Production by Lactococcus
lactis subsp. cremoris by the Glucose Source"
[0080] (Appl. Envir. Microbiol. 1999, 65: 5003-5008);
[0081] G. H. Van Geel-Schutten, E. J. Faber, E. Smit, K. Bonting,
M. R. Smith, B. Ten Brink, J. P. Kamerling, J. F. G. Vliegenthart
and L. Dijkhuizen,
[0082] "Biochemical and Structural Characterization of the Glucan
and Fructan Exopolysaccharides Synthesized by the Lactobacillus
reuteri Wild-Type Strain and by Mutant Strains"
[0083] (Appl. Envir. Microbiol. 1999, 65: 3008-3014.);
[0084] G. J. Grobben, I. Chin-Joe, V. A. Kitzen, I. C. Boels, F.
Boer, J. Sikkema, M. R. Smith and J. A. M. de Bont,
[0085] "Enhancement of Exopolysaccharide Production by
Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 with a
Simplified Defined Medium"
[0086] (Appl. Envir. Microbiol. 1998, 64:1333-1337.);
[0087] Sandrine Petry, Sylviane Furlan, Marie-Jeanne Crepeau, Jutta
Cerning and Michel Desmazeaud,
[0088] "Factors Affecting Exocellular Polysaccharide Production by
Lactobacillus delbrueckii subsp. bulgaricus Grown in a Chemically
Defined Medium"
[0089] (Appl. Envir. Microbiol. 2000, 66: 3427-3431.);
[0090] Richard van Kranenburg, Iris I. van Swam, Joey D. Marugg,
Michiel Kleerebezem and Willem M. de Vos,
[0091] "Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO
B40: Functional Analysis of the Glycosyltransferase Genes Involved
in Synthesis of the Polysaccharide Backbone"
[0092] (J. Bacteriol. 1999, 181: 338-340.);
[0093] Deborah Low, Jeffrey A. Ahlgren, Diane Horne, Donald J.
McMahon, Craig J. Oberg and Jeffery R. Broadbent,
[0094] "Role of Streptococcus thermophilus MR-1C Capsular
Exopolysaccharide in Cheese Moisture Retention"
[0095] (Appl. Envir. Microbiol. 1998, 64: 2147-2151.);
[0096] Richard van Kranenburg and Willem M. de Vos,
[0097] "Characterization of Multiple Regions Involved in
Replication and Mobilization of Plasmid pNZ4000 Coding for
Exopolysaccharide Production in Lactococcus lactis"
[0098] (J. Bacteriol. 1998, 180: 5285-5290.);
[0099] F Stingele, J R Neeser, and B Mollet,
[0100] "Identification and characterization of the eps
(Exopolysaccharide) gene cluster from Streptococcus thermophilus
Sfi6"
[0101] (J. Bacteriol. 1996, 178: 1680-1690.);
[0102] M Kojic, M Vujcic, A Banina, P Cocconcelli, J Cerning and L
Topisirovic,
[0103] "Analysis of exopolysaccharide production by Lactobacillus
casei CG11, isolated from cheese"
[0104] (Appl. Envir. Microbiol. 1992, 58: 4086-4088.);
[0105] Christian Chervaux, S. Dusko Ehrlich and Emmanuelle
Maguin,
[0106] "Physiological Study of Lactobacillus delbrueckii subsp.
bulgaricus Strains in a Novel Chemically Defined Medium"
[0107] (Appl. Envir. Microbiol. 2000, 66: 5306-5311.);
[0108] J Lemoine, F Chirat, J M Wieruszeski, G Strecker, N Favre
and J R Neeser,
[0109] "Structural characterization of the exocellular
polysaccharides produced by Streptococcus thermophilus SFi39 and
SFi12"
[0110] (Appl. Envir. Microbiol. 1997, 63: 3512-3518.);
[0111] Bart Degeest and Luc De Vuyst,
[0112] "Correlation of Activities of the
Enzymes--Phosphoglucomutase, UDP-Galactose 4-Epimerase, and
UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis
by Streptococcus thermophilus LY03"
[0113] (Appl. Envir. Microbiol. 2000, 66: 3519-3527.);
[0114] Petronella J. Looijesteijn, Ingeborg C. Boels, Michiel
Kleerebezem and Jeroen Hugenholtz,
[0115] "Regulation of Exopolysaccharide Production by Lactococcus
lactis subsp. cremoris by the Glucose Source"
[0116] (Appl. Envir. Microbiol. 1999, 65: 5003-5008.);
[0117] G. J. Grobben, I. Chin-Joe, V. A. Kitzen, I. C. Boels, F.
Boer, J. Sikkema, M. R. Smith and J. A. M. de Bont,
[0118] "Enhancement of Exopolysaccharide Production by
Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 with a
Simplified Defined Medium"
[0119] (Appl. Envir. Microbiol. 1998, 64: 1333-1337.);
[0120] Richard van Kranenburg, Iris I. van Swam, Joey D. Marugg,
Michiel Kleerebezem and Willem M. de Vos,
[0121] "Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO
B40: Functional Analysis of the Glycosyltransferase Genes Involved
in Synthesis of the Polysaccharide Backbone"
[0122] (J. Bacteriol. 1999, 181: 338-340.);
[0123] Williams W S and Cannon R E,
[0124] "Alternative Environmental Roles for Cellulose Produced by
Acetobacter xylinum"
[0125] (Appl. Envir. Microbiol. 1989, 55:2448-2452.);
[0126] Brown A M and Gascoigne J A,
[0127] "Biosynthesis of cellulose by Acetobacter Acetigenum"
[0128] (Nature 1960, 187:1010-1012.);
[0129] Carr J G,
[0130] "A strain of acetobacter aceti giving a positive cellulose
reaction"
[0131] (Nature 1958, 182:265-266.);
[0132] Carr J G and Shimwell J L,
[0133] "Old and new cellulose-producing Acetobacter species"
[0134] (J. Inst. Brew. 1958, 64:477-484.);
[0135] Colvin J R and Leppard G G,
[0136] "The biosynthesis of cellulose by Acetobacter xylinum and
Acetobacter acetigenus"
[0137] (Can. J. Microbiol. 1977, 23:701-709.);
[0138] Colvin J R and Webb T E,
[0139] "The variable relation of oxygen consumption to cellulose
synthesis by Acetobacter xylinum"
[0140] (Can. J. Microbiol. 1964, 10:11-15.);
[0141] Cook K E and Colvin J R,
[0142] "Evidence for a Beneficial Influence of Cellulose Production
on Growth of Acetobacter xylinum in Liquid Medium"
[0143] (Curr. Microbiol. 1980, 3:203-205.);
[0144] Fiedler S, Fussel M and Sattler K,
[0145] "Production and application of bacterial cellulose"
[0146] (Zentralbl Mikrobiol. 1989, 144:473-484.);
[0147] Kauri T, Vladuttalor M and Kushner D J,
[0148] "Production of Glycocalyxes by Bacteria Grown in the
Presence of Cellulose"
[0149] (Abstract ASM Meeting 1986, 273);
[0150] Mounter L A,
[0151] "Observations on the formation and structure of bacterial
cellulose"
[0152] (Biochemical Journal 1951, 50:128-132.);
[0153] Valent B S and Albersheim P,
[0154] "The effect of pH on binding of xyloglucan to cellulose"
(Plant Physiol. 1973, 51 supp.:60.);
[0155] Valla S and Kjosbakken J,
[0156] "Isolation and characterization of a new extracellular
polysaccharide from a cellulose-negative strain of Acetobacter
xylinum"
[0157] (Can. J. Microbiol. 1981, 27:599-603.);
[0158] Valla S and Kjosbakken J,
[0159] "Isolation and characterization of a new extracellular
polysaccharide from a cellulose-negative strain of Acetobacter
xylinum"
[0160] (Can. J. Microbiol 1981, 27:599-603.);
[0161] Valla S, Kjosbakken J and Coucheron D H,
[0162] "Acetobacter xylinum contains several plasmids: evidence for
their involvement in cellulose formation"
[0163] (Archives of Microbiology 1983, 134:9-11.);
[0164] Walker T K and Kaushal R
[0165] "Formation of cellulose by Acetobacter acetigenum"
[0166] (Nature 1947, 160:572-573.);
[0167] Walker T K and Kaushal R,
[0168] "Formation of cellulose by certain species of Acetobacter"
(Biochemical J. 1951, 48:618-621.);
[0169] Webb T E and Colvin J R,
[0170] "The Variable Relation of Oxygen Consumption to. Cellulose
Synthesis by Acetobacter xylinum"
[0171] (Can. J. Microbiol. 1964, 10:11-15.);
[0172] Webb T E and Colvin J R,
[0173] "The extracellular proteins of Acetobacter xylinum and their
relationship to cellulose synthesis"
[0174] (Can. J. Biochemistry 1966, 45:465476.);
[0175] Williams W S and Cannon R E,
[0176] "Alternative environmental roles for cellulose produced by
Acetobacter xylinium"
[0177] (Appl. Environ. Microbiol. 1989, 55:2448-2452.);
[0178] Wong H C, et al.,
[0179] "Genetic organization of the cellulose in Acetobacter
xylinium"
[0180] (Proc. natl. acad. sci. USA 1990, 87:8130-8134.);
[0181] Higashimura M, Mulder-Bosman B W, Reich R, Iwasaki T and
Robijn G W,
[0182] "Solution properties of viilian, the exopolysaccharide from
Lactococcus lactis subsp. cremoris SBT 0495"
[0183] (Biopolymers 2000, August 54:2 143-158.);
[0184] Knoshaug E P, Ahlgren J A and Trempy J E,
[0185] "Growth associated exopolysaccharide expression in
Lactococcus lactis subspecies cremoris Ropy352"
[0186] (J. Dairy Sci. 2000, April 83:4 633-640.);
[0187] Micheli L, Uccelletti D, Palleschi C and Crescenzi V,
[0188] "Isolation and characterisation of a ropy Lactobacillus
strain producing the exopolysaccharide kefiran"
[0189] (Appl. Microbiol. Biotechnol. 1999, December 53:1
69-74.);
[0190] Looijesteijn P J, Boels I C, Kleerebezem M and Hugenholtz
J,
[0191] "Regulation of exopolysaccharide production by Lactococcus
lactis subsp. cremoris By the glucose source"
[0192] (Appl. Environ. Microbiol. 1999, November 65:11
5003-5008.);
[0193] Smitinont T, Tansakul C, Tanasupawat S, Keeratipibul S,
Navarini L, Bosco M and Cescutti P,
[0194] "Exopolysaccharide-producing lactic acid bacteria strains
from traditional Thai fermented foods: isolation, identification
and exopolysaccharide characterization"
[0195] (Int. J. Food Microbiol. 1999, October 15 51:2-3
105-111.);
[0196] Van Kranenburg R, van Swam I I, Marugg J D, Kleerebezem M
and de Vos W M,
[0197] "Exopolysaccharide biosynthesis in Lactococcus lactis NIZO
B40: functional analysis of the glycosyltransferase genes involved
in synthesis of the polysaccharide backbone"
[0198] (J. Bacteriol. 1999, January 181:1 338-340.);
[0199] Breedveld M, Bonting K and Dijkhuizen L,
[0200] "Mutational analysis of exopolysaccharide biosynthesis by
Lactobacillus sakei 0-1"
[0201] (FEMS Microbiol. Lett. 1998, December 15 169:2
241-249.);
[0202] De Vuyst L, Vanderveken F, Van de Ven S and Degeest B,
[0203] "Production by and isolation of exopolysaccharides from
Streptococcus thermophilus grown in a milk medium and evidence for
their growth-associated biosynthesis"
[0204] (J. Appl. Microbiol. 1998, June 84:6 1059-1068.);
[0205] Low D, Ahigren J A, Horne D, McMahon D J, Oberg C J and
Broadbent J R,
[0206] "Role of Streptococcus thermophilus MR-1C capsular
exopolysaccharide in cheese moisture retention"
[0207] (Appl. Environ. Microbiol. 1998, June 64:6 2147-2151.);
[0208] Kimmel S A and Roberts R F,
[0209] "Development of a growth medium suitable for
exopolysaccharide production by Lactobacillus delbrueckii ssp.
bulgaricus RR"
[0210] (Int. J. Food Microbiol. 1998, March 3 40:1-2 87-92.);
[0211] Duenas-Chasco M T, Rodriguez-Carvajal M A, Tejero-Mateo P,
Espartero J L, lrastorza-Iribas A and Gil-Serrano A M,
[0212] "Structural analysis of the exopolysaccharides produced by
Lactobacillus spp. G-77"
[0213] (Carbohydr. Res. 1998, Feburary 307:1-2 125-133.);
[0214] Espartero J L, Irastorza-lribas A, Gil-Serrano A M,
Duenas-Chasco M T, Rodriguez-Carvajal M A, Tejero Mateo P and
Franco-Rodriguez G,
[0215] "Structural analysis of the exopolysaccharide produced by
Pediococcus damnosus 2.6"
[0216] (Carbohydr. Res. 1997, October 7 303:4 453458.);
[0217] Stingele F, Lemoine J and Neeser J R,
[0218] "Lactobacillus helveticus Lh59 secretes an exopolysaccharide
that is identical to the one produced by Lactobacillus helveticus
TN-4, a presumed spontaneous mutant of Lactobacillus helveticus
TY1-2"
[0219] (Carbohydr. Res. 1997, August 7 302:3-4 197-202.);
[0220] Bubb W A, Urashima T, Fujiwara R, Shinnai T and Ariga H,
[0221] "Structural characterisation of the exocellular
polysaccharide produced by Streptococcus thermophilus OR 901"
[0222] (Carbohydr. Res. 1997, June 11 301:1-2 41-50.);
[0223] Staaf M, Widmalm G, Yang Z and Huttunen E,
[0224] "Structural elucidation of an extracellular polysaccharide
produced by Lactobacillus helveticus"
[0225] (Carbohydr. Res. 1996, Sepember 23 291: 155-164.);
[0226] Robijn G W, Gutierrez Gallego R, van den Berg D J, Haas H,
Kamerling J P and Vliegenthart J F,
[0227] "Structural characterization of the exopolysaccharide
produced by Lactobacillus acidophilus LMG9433"
[0228] (Carbohydr. Res. 1996, July 19 288: 203-218.);
[0229] Robijn G W, Wienk H L, van den Berg D J, Haas H, Kamerling J
P and Vliegenthart J F,
[0230] "Structural studies of the exopolysaccharide produced by
Lactobacillus paracasei 34-1"
[0231] (Carbohydr. Res. 1996, May 14 285: 129-139.);
[0232] Fontaine T, Wieruszeski J M, Talmont F, Saniez M H, Duflot
P, Leleu J B and Fournet B,
[0233] "Exopolysaccharide structure from Bacillus circulans"
[0234] (Eur. J. Biochem. 1991, Feburary 26 196:1 107-113.);
[0235] Osadchaia A I, Kudriavtsev V A and Safronova L A,
[0236] "The role of amino acids in intensification of Bacillus
subtilis exopolysaccharide biosynthesis in deep growth
conditions"
[0237] (Mikrobiologiia. 1995, Janurary -Feburary; 64(1):44-50.);
and
[0238] Mazza P,
[0239] "The use of Bacillus subtilis as an antidiarrhoeal
microorganism"
[0240] (Boll. Chim. Farm. 1994, Janurary; 133(1):3-18.),
[0241] which are hereby incorporated by reference in their
entirety, including any drawings, as if fully set forth herein.
[0242] In addition, the present inventors have isolated and
obtained novel microorganisms which can be used as an active
principle of the pharmaceutical composition of the present
invention.
[0243] In order to isolate and obtain novel microorganisms which
satisfy the requirements for an active principle of the
pharmaceutical composition of the present invention, the present
inventors have researched as follows:
[0244] Samples of microorganisms collected from the glucose factory
sewage and other locations were inoculated in MRS and BHS agar
mediums containing cycloheximide, and then cultured. Colonies
formed in agar medium were then inoculated into MRS and BHS liquid
medium and incubated without shaking. Microorganisms that formed a
matrix or a membrane shape on top layers of the medium were
selected. Formed membranes were separated and tested for whether or
not the separated membranes were decomposed by the intestinal
digestive enzyme. The results determined whether non-digestable (or
hardly digestable) high molecular-weight compounds were produced or
not. Among the microorganisms, BC-Y009 and BC-Y058 were selected
for their high productivity of extracellular polysaccharide
(dietary fiber).
[0245] Upon observing the morphology of BC-Y009 and BC-Y058 and
comparing with 16s rRNA's partial DNA sequences, it was confirmed
that each showed high percentage of homology sequence when compared
with Lactobacillus and Acetobacter. Based on the phenotype and 16s
rRNA DNA sequence analysis, it was ascertained that BC-Y009 is a
novel microorganism which falls within the Lactobaccilus genus and
BC-Y058 as a novel microorganism of Acetobacter genus.
[0246] Lactobacillus BC-Y009 and Acetobacter BC-Y058 of the present
invention were administered into a mouse which was induced to have
obesity and diabetes mellitus. The blood glucose level of a subject
mouse had been decreased approximately 70% after
administration.
[0247] According to these results, it was confirmed that
microorganisms of the present invention has an effect in decreasing
blood glucose level and thus it is effective for treating and
preventing against diabetes mellitus.
[0248] When microorganisms of the present invention, BC-Y009 and
BC-Y058 were administered into a mouse induced to have diabetes
mellitus and obesity, the feed consumption rate increased 17 to 24%
upon comparison with a control mouse. However, weight gain versus
feed consumption amount was decreased. The result thus indicates
that the microorganism composition of the present invention allows
for humans to consume without worrying about obesity or diabetes
mellitus.
[0249] From the observation that a blood lipid level is also lower
than that of control group in case of taking these microorganisms,
the microorganisms of the present invention is found to be capable
of controlling the occurrence of diabetes mellitus, obesity and
circulatory diseases, for example, arteriosclerosis or myocardial
infarction. Additionally, in case of a normal mouse, mouse
administered with the composition of the present invention consumed
more feed, thus energy efficiency had been decreased in comparison
with a control mouse. However, it was confirmed that there was no
side effects led from the administration upon observing that the
change of lipid content was negligible.
[0250] Hereinafter, the present invention will be further explained
with reference to the following examples. The examples are given
only for illustration of the invention and are not intended to
limit the scope of the present invention.
EXAMPLE 1
[0251] Selecting of Microorganism which Produces Extracellular
Polysaccharide from Samples
[0252] In order to isolate microorganisms which produce dietary
fibers, samples were collected from glucose factory sewage and
other locations. 10 g of the mixture thus collected were disrupted
and suspended in 90 ml of physiological saline solution(0.85%
NaCl). The said suspended samples were diluted to 10.sup.-2,
10.sup.-4, and 10.sup.-6 in physiological saline solution. These
diluted samples then smeared on MRS agar medium containing 1 mg of
cycloheximide per 100 ml medium(1% Peptone, 1% beef extract, 0.5%
yeast extract, 2% glucose, 0.1% Tween-80, 0.2% Citric Acid
Ammonium, 0.5% Sodium Acetate, 0.01% MgSO.sub.4, 0.005% MnSO.sub.4,
0.2% Sodium Phosphate pH6.5) and on BSH agar medium(2% glucose,
0.5% Peptone, 0.5% yeast extract, 0.27% Na.sub.2HPO.sub.4, 0.115%
Citric Acid pH 5.0)(Hestirin and Schramm, J. Gen. Microbiol.,
11:123,1954) and cultured in 30.degree. C. for 72 hours.
Approximately 2,000 colonies were selected and were initially
inoculated in 5 ml MRS liquid medium and BSH liquid medium at
30.degree. C. for 72 hours and cultured without shaking. The
microorganism which form a membrane shape on upper layer of the
liquid medium and the microorganism which form capsule-shaped
extracellular polysaccharide and of which medium was transparent,
were selected. These microorganisms were inoculated again in 5 ml
of MRS liquid medium and BSH liquid medium and stirred at
30.degree. C. and the absorbance thereof was measured at 600 nm by
spectrophotometer. Microorganisms were diluted with BSH liquid
medium until the absorbance thereof reached to 0.2. 10 ml of
microorganism thus diluted was inoculated into 100 ml of BSH liquid
medium at 30.degree. C. for 72 hours and cultured without
shaking.
[0253] In order to measure the amount of extracellular
polysaccharide (dietary fibers) thus produced, each medium were
centrifuged at 6,000 rpm in 4.degree. C. to obtain the
precipitation of microorganisms. Cell membrane were disrupted by
alkali lysis in 0.1 N NaOH solution and left alone in 800.degree.
C. for 30 minutes and centrifuged at 6,000 rpm in 4.degree. C. and
repeated multiple times, the above process in entirety.
Extracellular polysaccharide entangled like white strings were
isolated and lyophilized to be measured the amount thereof.
Microorganisms with high extracellular polysaccharide productivity
were selected and extracellular polysaccharide productivity was
compared with each other (Table 1).
1TABLE 1 Comparison of extracellular polysaccharide productivity
Amount of produced extracellular Selection polysaccharide Number
(dryweight g/l BSH) BC-Y 009 3.8 BC-Y 002 4.2 BC-Y 015 3.2 BC-Y 026
4.1 BC-Y 058 4.8 BC-Y 112 3.0 BC-Y 130 3.4 BC-Y 201 3.3
EXAMPLE 2
[0254] The Morphological Determination and Characteristics of the
Selected BC-Y009 and BC-Y058
[0255] Microorganisms which show high polysaccharide productivity
selected from the Example 1 were BC-YO09, BC-Y002, BC-Y015,
BC-Y026, BC-Y058, BC-Y112, BC-Y130, and BC-Y201. Upon observing
partial DNA sequences, BC-YO09, BC-Y002, BC-Y015 and BC-Y026 were
microorganisms of Lactobacillus genus, and BC-Y058, BC-Y112,
BC-Y130 and BC-Y201 were microorganisms of Acetobacter genus.
[0256] Among these bacteria, BC-Y009 and BC-Y058 which show high
polysaccharide productivity were inoculated in MRS and BSH liquid
mediums at 30.degree. C. for 72 hours and cultured in suspension.
Cultured mediums were centrifuged at 6,000 rpm in 4.degree. C. to
obtain microorganisms and the nucleic acids thereof were isolated
by means of using the CTAB/NaCl method. By using 16 s rRNA
consensus primer, 16 s rRNA was amplified by means of PCR method,
and the sequence thus obtained, was determined. BLAST analysis
(NCBI, USA) on the sequence thus determined, was performed and its
result showed high percentage of sequence homology with sequence of
Lactobacillus hilgardii, Acetobacter xylinum, Gluconobacter sp.,
numerous other Lactobacillus sp. and Acetobacter sp. (Tables 2 and
3).
2TABLE 2 Comparison of 16S rRNA nucleotide sequence of
Lactobacillus sp. BC-Y 009 L. delbrueckii Lactobacillus subsp. L.
helveticus L. acidophillus L. hilgardii sp. BC-Y009 ATCC9649
NCDO2712T ATCC4356 NCDO264 ATCC13133 BC-Y009 -- 145 136 146 3 4 L.
delbrueckii 88.93 -- 76 73 142 143 sp. ATCC9649 L. helveticus 89.16
93.94 -- 21 134 134 NCDO2712T L. acidophillus 88.85 94.43 98.33 --
144 144 ATCC4356 L. hilgardii 99.77 89.07 89.26 88.93 -- 1 NCDO264
Lactobacillus 99.69 88.97 89.21 88.90 99.92 -- sp. ATCC13133
[0257] Among 1,400 base pairs which are included in comparison, top
right of table indicates number of base pairs which show
difference, bottom left of table indicates % homology
3TABLE 3 Comparison of 16S rRNA nucleotide sequence of Acetobacter
sp. BC-Y 058 BC-Y 058 A. diazotrificus A. liqfaciens A. hansenii A.
xylinum A. europaeus BC-Y 058 -- 37 34 10 13 14 A. diazotrificus
97.20 -- 17 37 35 36 A. liqfaciens 97.42 98.71 -- 34 32 33 A.
hansenii 99.24 97.20 97.42 -- 15 16 A. xylinum 99.02 97.35 97.58
98.86 -- 3 A. europaeus 98.94 97.27 97.50 98.79 99.77 --
[0258] Among 1,320 base pairs which are included in comparison, top
right of table indicates number of base pairs which show
difference, bottom left of table indicates % homology
[0259] BC-Y009 is a gram-positive bacteria and 0.5 to 3.0 .mu.m in
size. It is a non-motile & short-rod shaped bacteria. It does
not form spores and is facultative anaerobic. The growth
temperature is between 20.degree. C. to 37.degree. C. and pH level
is 2.0 to 8.0 and optimal pH level is 4.0 to 7.0. The experimental
results showed that this microorganism was condensed in milk and
was negative (non-reactive) to catalase and formed white colored
colony in complex medium. It was precipitated in MRS liquid medium
and BSH liquid medium in form of white colored capsule. The
turbidity of the liquid medium was clear and the microorganism
produced extracellular polysacchardie in clear medium and in case
liquid medium was shaken, the extracellular polysacchride (dietary
fiber) were broken into small particles.
[0260] BC-Y058 is a gram-negative bacteria, rod shaped bacteria and
0.6 to 0.8 .mu.m in size and exists as single or a pair. It is also
a non-motile and does not form spores. Growth rate thereof is slow,
therefore 5 to 7 days of incubation time is needed and colonies
formed are small and hard. In liquid medium, clear cellulose
pellicle is formed. Ethanol, acetic acid, or lactic acid can be
used as substrates and showed positive response to catalase. This
microorganism produces acid by using glucose and in Hoier medium,
it can not grow.
[0261] Upon consideration of the result of analysis of phenotype
and 16s rRNA DNA sequence, BC-Y009 was named as Lactobacillus sp.
BC-Y009 and BC-Y058 as Acetobacter sp. BC-Y058. They were deposited
in KCTC(Korean Collection for Type Cultures) on May 30, 2000, and
the deposit number were granted as KCTC BC-Y009, KCTC BC-Y058,
respectively.
EXAMPLE 3
[0262] The Degree of Decomposition of Extracellular Polysacchride
(Dietary Fiber) bV Intestinal Digestive Enzymes
[0263] In order to determine whether or not dietary fiber produced
by said microorganisms is decomposed by intestinal digestive
enzyme, 1 g of porcine pancreatin that shows the activity of
3.times.U.S.Pharmacopia (manufactured by Sigma) and comprises
amylase, lipase, protease and nuclease, was suspended in buffer
solution (pH7.5) of 1 g of dried dietary fiber. This suspension was
incubated for 7 days at 40.degree. C. and the suspension was
collected once a day and the glucose therein was analyzed
quantitatively by using DNS(3,5-dinitrosalicylic acid). The result
thereof showed that dietary fibers has never been decomposed at
all.
[0264] Therefore, it was confirmed that the dietary fibers produced
by the microorganisms of the present invention do not decompose
within the intestine.
EXAMPLE 4
[0265] Glucose Absorption Rate of Bacteria
[0266] Glucose absorption rates of Lactobacillus acidophilus
(KCTC3140), L. hilgardii (KCTC3500) known as probiotics, and the
said Lactobacillus BC-Y009, Acetobacter BC-Y002, Acetobacter
BC-Y058 and E. coli., were measured in the condition of the
intestine. The results are represented in FIG. 1 and Table 4.
[0267] As illustrated in FIG. 1 and Table 4, the microorganisms of
the present invention are superior to the other lactic acid
bacteria in terms of glucose absorption rate.
4TABLE 4 Glucose concentration decreased by the bacteria of unit
O.D. per unit time. glucose concentration initial glucose glucose
decreased per initial concentration concentration unit time and
unit O.D.600 nm (mM) after 1 hour(mM) O.D.(mM/hr/O.D.) E. coli 3.0
.+-. 0.1 110 85 .+-. 0.5 8.3 .+-. 0.44 BC-Y009 3.0 .+-. 0.2 110 50
.+-. 0.3 20 .+-. 1.5 BC-Y002 3.0 .+-. 0.1 110 30 .+-. 0.7 26.6 .+-.
1.1 BC-Y058 3.0 .+-. 0.2 110 38.6 .+-. 0.3 23.8 .+-. 0.1 KCTC3500
3.0 .+-. 0.2 110 67.2 .+-. 0.3 14.2 .+-. 0.4 KCTC3140 3.0 .+-. 0.1
110 65.2 .+-. 0.4 14.4 .+-. 0.1
EXAMPLE 5
[0268] Concentration and Survival Rate of Microorganisms in the
Intestine After Adminstering Microorganisms
[0269] Mouse C57BL/6J Lep.sup.ob ob/ob genetically induced of
obesity and diabetes mellitus(hereinafter, "OB Mouse"), was starved
for 18. hours and fed the composition of the present invention (the
number of microorganism of the composition was 1.0.times.10.sup.13
CFU/g) containing 1% of Lactobacillus BC-Y009, Acetobacter BC-Y058
(w/w, drying weight) for 7 days, and then the bacterial
concentration in the duodenum, the jejunum, and the large intestine
of these mice were analyzed. In addition, the bacterial
concentration in the duodenum, the jejunum, and the large intestine
of the control OB mouse that had been fed the feed without
containing the microorganisms of the present invention, was
analyzed.
[0270] In order to measure the amount of Lactobacillus, the
duodenum, the jejunum, and the large intestine of the mouse that
had been fed Lactobacillus feed and the control mice were cut out.
Each surfaces of the organs were rinsed with physiological saline
solution and the contents were suspended in physiological saline
solution. Then, inoculated in MRS agar medium and incubated at
37.degree. C. Three (3) days later, the amount of bacteria was
measured by counting floc and by subtracting the amount of
Lactobacillus in the control group to determine the change of the
amount of bacteria (Table 5).
[0271] In order to confirm the existence of Acetobacter, the each
organs of mouse were cut out, then rinsed the surfaces of the
organs with physiological saline solution. The contents were
suspended in physiological saline solution, then inoculated in BSH
liquid medium and cultured at 37.degree. C. for 3 days. By checking
the pellicle appeared on top layer of the liquid medium, the
existence of fiber-producing Acetobacter was confirmed (Table
6).
[0272] According to the results represented in Table 5 and Table 6,
the said two kinds of microorganisms were both able to proliferate
in the intestine.
5TABLE 5 The amount of Lactobacillus sp. in the duodenum, the
jejunum, and the large intestine of mouse Existence of the region
of membrane intestine weight (g) bacterial number (CFU/g) formation
Duodenum 0.18 .+-. 0.03 83 .+-. 20 no Jejunum 0.29 .+-. 0.05 1.2
.times. 10.sup.3 .+-. 50 no large intestine 0.36 .+-. 0.07 5.1
.times. 10.sup.3 .+-. 30 yes
[0273]
6TABLE 6 The amount of Acetobacter sp. in the duodenum, the
jejunum, and the large intestine of mouse the region existence of
of intestine weight (g) membrane formation duodenum 0.20 .+-. 0.02
no jejunum 0.28 .+-. 0.04 yes large intestine 0.35 .+-. 0.03
yes
[0274] EXAMPLE 6
[0275] The Change in Blood Glucose Level Upon Feeding of BC-Y009
and BC-Y058
[0276] 100 g of mouse feed purchased from SAMYANG Co. and 400 g of
Korean rice were mixed to make a composition in which carbohydrate
content was 60%, then 5 g of dried Lactobaccillus BC-Y009 or
Acetobacter BC-Y058 were added thereto to prepare a lyophilized
tablet. Mice were fed this tablet with water.
[0277] All mice tested in this Example were female and OB mice.
Acetobacter feed group (OB-058), Lactobacillus feed group (OB-009),
and the control group (OB-con, which has no microorganism of the
present invention in the feed) were bred separately. The breeding
condition was that there was light every 12 hour intervals
(9:00-21:00 lighted, 21:00-9:00 no lighted) and maintained 20 to
24.degree. C. and 40 to 60% humidity.
[0278] Additionally, enteric coating solution was sprayed on dried
Lactobacillus BC-Y009 or Acetobacter BC-Y058 to produce the
compostion of the present invention which comprises enteric coated
microorganisms. The weight of the enteric coating of material on
the composition was approximately 16 to 30 mg or less per tablet.
The materials for the enteric coating were selected from common
high molecular weight materials, such as, cellulose acetate
phthalate, trimelitate, copolymer of methacrylic acid
(Methylacrylic acid 40% or more, especially methylacrylic acid
including hydroxypropyl methylcellulose phthalate and its ester
derivatives), or mixture thereof.
[0279] Methylacrylate used in the Example was Endragit L 100-55
manufactured by Rohm GmbH(Germany), cellulose acetate phthalate
with about 45 to 90 cP of viscosity, 17 to 26% of acetyl content
and 30 to 40% of phthalate content, or cellulose acetate
trimelitate manufactured by the Eastman Kodak Company
(approximately 15 to 20 cS of viscosity, 17 to 26% of acetyl
content and 25 to 35% of trimelityl content).
[0280] The enteric coating was produced by a conventional coating
process wherein the enteric coating solution was sprayed on a core.
Ethanol and acetone mixture was used as solvent and a softening
agent was added to the coating solution in a ratio of 1 to
approximately 0.005 or 0.3.
[0281] The enteric coating composition of the present invention
produced by means of the process was provided to the mice with
water for unrestricted taking. The blood glucose level of the mouse
which has taken the enteric coating composition, was measured.
[0282] Before measuring the blood glucose level of each mouse
group, each mouse was starved for 18 hours. Following 60 minutes
after starvation, sufficient amounts of feed were provided and
after a 60 minute period, serum was collected from the retroorbital
venous plexus by using anti-coagulating agent-free capillary
tubes.
[0283] The blood glucose level was measured by absorbance at 505
nm, using the Trinder kit (Cat. 315-500, Sigma, USA) which employs
enzyme coloring method. The statistical error of the results was
indicated by average .+-. standard deviation per experimental
group, and statistical significance of the average difference in
each group was tested through ANOVA (p<0.02).
[0284] Data for blood glucose level are illustrated in FIG. 2. As
illustrated in the FIG. 2, the blood glucose level for OB-con group
is approximately 500 mg/dl, whereas OB-058 blood glucose level is
low. Additionally, due to administration of Acetobacter BC-Y058 and
Lactobacillus BC-Y009, the blood glucose levels of each mouse had
been decreased to approximately 70% and 53% each (Table 7).
7TABLE 7 The change of blood glucose level after administration of
Acetobacter BC-Y058 and Lactobacillus BC-Y009 OB-009 OB-058 OB-con
Blood glucose 229 .+-. 16 141 .+-. 19 492 .+-. 60 level(mg/dl)
EXAMPLE 7
[0285] The Change of Weight and Amount of Diet Due to Taking
BC-Y058 and BC-Y009 and in Metabolic Efficiency
[0286] Mice were classified as OB-058 group, OB-009 group, OB-con
group, and Acetobacter BC-Y058 and Lactobacillus BC-Y009 were
administered on each group and the weight of each mouse was
measured in weekly intervals. Along with the measuring of changes
in weight, the weight of feed consumed by the mice was also
measured, therefore changes of metabolic efficiency of each group
were investigated.
[0287] The difference of weight change was apparent in each species
whose genetic characteristics were different, but the difference of
weight change, within the group having the same genetic
characteristics was negligible.
[0288] As indicated in Table 8, the weight change of OB mice within
the period of 7 weeks, regardless of the administration of
Acetobacter BC-Y058 or Lactobacillus BC-Y009, was approximately 47%
increase of weight. However, on the contrary, as indicated in
Tables 9 and 10, feed consumption percentage, depending on
microorganism administration, increased 17 to 24% in OB mice
group.
[0289] That is, the weight increase of the mice fed feed which
comprises the microorganisms of the prevent invention was the same
as that of the mice fed that does not contain the microorganism of
the prevent invention. The results indicate that because
Acetobacter BC-Y058 and Lactobacillus BC-Y009 suppress increase of
blood glucose levels after meal, increase of feed consumption
occurs as its compensation. In other words, with the same amount of
feed, increase of weight can be decreased by feeding the
microorganism of the present invention without causing no further
weight increase because of lower metabolic efficiency. Because of
the conversion of glucose into dietary fiber by BC-Y058 and BC-Y009
microorganism, metabolic efficiency has changed.
[0290] According to the formula represented below, the change of
energy efficiency depending on feed consumption, was calculated and
represented in Table 10.
[0291] energy metabolic efficiency=
[0292] (weight gain(g)/amount of feeding(g)).times.1,000
[0293] As represented in Table 10, when microorganisms were
administered to OB mouse, the energy metabolic efficiency was from
75 to 85% (FIG. 3) compared to that of the control group which was
not administered with the microorganisms of the present invention
(FIG. 4).
8TABLE 8 Change of the mouse weight(g) 1 2 3 4 5 6 7 week week week
week week week week OB-009 21.5 .+-. 3.21 26.53 .+-. 2.72 31.52
.+-. 3.01 34.91 .+-. 2.5 37.6 .+-. 2.53 40.1 .+-. 1.74 41.4 .+-.
1.47 OB-058 21.95 .+-. 5.3 26.75 .+-. 4.60 31.65 .+-. 2.33 35.8
.+-. 1.27 38.25 .+-. 0.78 40.35 .+-. 0.64 41.25 .+-. 0.21 OB-con
21.4 .+-. 2.83 26.3 .+-. 1.56 31.9 .+-. 0.99 35.8 .+-. 2.12 38.35
.+-. 2.33 40.1 .+-. 2.69 41.75 .+-. 3.61
[0294]
9TABLE 9 Change of amount of feed consumption according to the
administration of Acetobacter BC-Y058, Lactobacillus BC-Y009(g)
0-16 days 16-21 days 21-34 days 34-41 days Total OB-009 146.3 32.4
110.7 38.6 328 OB-058 157.4 34.3 115.3 41 348 OB-con 128.1 34.8
80.3 36.5 279.7
[0295]
10TABLE 10 Energy metabolic efficiency energy rate of Amount of
weight gain metabolic average weight feed (g) (g) efficiency weight
(g) increase OB-009 328 19.9 121 41.4 0.48 OB-058 348 19.3 111
41.25 0.47 OB-con 279.7 20.35 146 41.75 0.49
EXAMPLE 8
[0296] Change of Weight and Diet Amount of Obesity Mouse Induced by
GTG and Subsequent Change in M-tabolic Efficiency
[0297] Before feeding Acetobacter BC-Y058 and Lactobacillus
BC-Y009, each mouse was administered with 1 g/kg of goldthioglucose
(Cat. A-0632, Sigma, USA) in order to induce obesity. And every 3
or 4 weeks, weight change was measured and only obesity-induced
mice were selected. For accuracy of the experiment, a mouse of
which weight increase was too great or too little relatively, was
excluded from the experiment.
[0298] The target was female C57BL/6J mice and breeding environment
and conditions were the same as those in Example 6. The test
subjects were classified into BC-Y058 group, KCTC3140 group,
KCTC3500 group, and BC-Y009 group depending on microorganisms.
[0299] The weight changes of mice depending on microorganisms
administered with, are illustrated in FIG. 5 and it is confirmed
that when Acetobacter BC-YO58 and Lactobacillus BC-Y009 were
administered, the weight increase rate has decreased.
[0300] Additionally, as represented in Table 11 and FIG. 6, in case
that KCTC3140 and KCTC3500 which consume glucose but do not produce
dietary fibers, were administered, the energy efficiency of
obesity-induced mice was higher than that of the control group
which was not administered with the microorganisms of the present
invention. However, the mouse group which was administered with
BC-Y009 and BC-Y058 which produce dietary fibers, showed relatively
low energy efficiency, especially in case of BC-Y058. That is the
energy efficiency decreased to 55% compared with that of the
control group (Table 12).
11TABLE 11 Metabolism efficiency of obese mouse induced with drug
administration(g) energy metabolic Weight gain(g) amount of feed(g)
efficiency Carbohydrate 5.43 103.7 52 KCTC3140 6.65 92.4 72
KCTC3500 5.67 102.2 55 BC-Y009 4.38 104.4 42 BC-Y058 2.98 102.7
29
EXAMPLE 9
[0301] Lipid Level Changes when BC-Y058 and BC-Y009 were
Administered
[0302] After administration of the microorganisms of the present
invention, the change of blood lipid, especially cholesterol
change, was analyzed and confirmed whether or not the
microorganisms affected the circulatory disease, such as,
artheriosclerosis and myocardial infarction besides diabetes
mellitus and obesity.
[0303] Lipid analysis was performed by means of enzyme coloring
method as in Example 6, using TG-glycezyme-V (Young-Yeoun Chemical
Co., Japan), HDL-zyme-V (Young-Yeoun Chemical Co., Japan),
Cholestezyme-V (Young-Yeoun Co., Japan), LDL cholesterol (Cat.
61532, BioMeriux, France), to measure the absorbance at 505 to 570
nm with standard solution, and the amount of lipid in blood was
calculated.
[0304] As represented in Table 12, lipid concentration before feed
administration did not show any differences in obese mouse.
However, after Acetobacter BC-Y058 and Lactobacillus BC-Y009 were
administered, as indicated in Table 12, the change of lipid
concentration was apparent after 7 weeks.
[0305] In case of obese mice that have taken the microorganism, the
lipid level did not change in comparison with the data of early
steps in the present experiment and however, in case of control
mouse which had not been administered with the microorganisms,
overall lipid content in blood was increased.
12TABLE 12 Lipid amount in blood before administration of
feed(mg/dl) total cholesterol TG HDL-C LDL-C OB-009 130.22 .+-.
4.11 98.1 .+-. 11.4 98.73 .+-. 9.7 4.18 .+-. 2.36 OB-058 129.37
.+-. 4.24 101.6 .+-. 10.36 113.52 .+-. 15.47 3.35 .+-. 2.08 OB-con
127.57 .+-. 4.32 97.13 .+-. 14.64 96.86 .+-. 7.61 6.62 .+-. 2.78 n
= 4 TG: Triglyceride HDL-C: High Density Lipoprotein Cholesterol
LDL-C: Low Density Lipoprotein Cholesterol
[0306]
13TABLE 13 Lipid amount in blood after administration of
feed(mg/dl) Total cholesterol TG HDL-C LDL-C OB-009 167.04 .+-.
1.12 100.76 .+-. 3.2 157.71 .+-. 2.4 4.2 .+-. 2.08 OB-058 *135.25
.+-. 2.47 98.5 .+-. 2.83 135 .+-. 1.41 3.36 .+-. 1.31 OB-con *174
.+-. 1.41 110.5 .+-. 1.06 165.25 .+-. 1.06 3.19 .+-. 0.36 n = 4, *p
< 0.05 TG: Triglyceride HDL-C: High Density Lipoprotein
Cholesterol LDL-C: Low Density Lipoprotein Cholesterol
[0307] The Industrial Applicability of the Present Invention
[0308] The microorganisms of the present invention are capable of
living within the intestine and converting monosaccharides and
disaccharides into high molecular weight materials which cannot be
absorbed and hardly digestible in the intestine, thereby remarkably
reducing the amount of monosaccharide to be absorbed. Therefore,
the energy required for metabolic activity is provided from lipids
and protein accumulated in the body, thus effectively suppressesing
obesity and diabetes mellitus. In addition, the microorganisms of
the present invention produce dietary fibers within the intestine
and excreting harmful materials along with these dietary fibers, to
prevent appendicitis or large intestinal cancer, to suppress
cholesterol absorption and to clean the intestine.
[0309] While the present invention has been particularly shown and
described with reference to particular examples thereof, it will be
understood by those skilled in the art that various changes in form
and details may be conceived therefrom without departing from the
spirit and scope of the present invention as defined by the
appended claims.
[0310] This application claims priority from the Korean Patent
Application Nos. 10- 2000-0026379 (filed May 17, 2000) and
10-2000-0049805 (filed Aug. 26, 2000), the contents of which are
hereby incorporated by reference in their entirety, including the
specification, drawings and claims.
* * * * *