U.S. patent number RE48,652 [Application Number 15/978,986] was granted by the patent office on 2021-07-20 for lactic acid bacteria isolated from mother's milk with probiotic activity and inhibitory activity against body weight augmentation.
This patent grant is currently assigned to BIONEER CORPORATION. The grantee listed for this patent is BIONEER CORPORATION. Invention is credited to Ji Hee Kang, Han Oh Park, Byeung Il You, Sung Il Yun.
United States Patent |
RE48,652 |
Kang , et al. |
July 20, 2021 |
Lactic acid bacteria isolated from mother's milk with probiotic
activity and inhibitory activity against body weight
augmentation
Abstract
The present invention relates to a lactic acid bacterium
isolated from human mother's milk, more precisely a Lactobacillus
gasseri BNR17 strain that is isolated from Korean mother's milk and
has excellent probiotic activity including acid resistance, bile
acid resistance and antimicrobial activity and weight gaining
inhibitory effect as well. Again, the Lactobacillus gasseri BNR17
of the present invention has excellent acid resistance, bile acid
resistance, enteric absorption activity and antimicrobial activity
against pathogenic microorganisms, in addition to the weight
gaining inhibitory effect by synthesizing indigestible
polysaccharides from monosaccharides included in food taken and
releasing the synthesized polysaccharides out of the body.
Therefore, the strain of the invention, owing to such beneficiary
effects, can be effectively used not only for the production of
fermented milk, other fermented food products and animal feeds but
also for the production of live cell products and food additives
for preventing weight gaining.
Inventors: |
Kang; Ji Hee (Daejeon,
KR), You; Byeung Il (Daejeon, KR), Yun;
Sung Il (Daejeon, KR), Park; Han Oh (Daejeon,
KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
BIONEER CORPORATION |
Daejeon |
N/A |
KR |
|
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Assignee: |
BIONEER CORPORATION (Daejeon,
KR)
|
Family
ID: |
1000005415204 |
Appl.
No.: |
15/978,986 |
Filed: |
May 14, 2018 |
PCT
Filed: |
May 14, 2007 |
PCT No.: |
PCT/KR2007/002363 |
371(c)(1),(2),(4) Date: |
March 17, 2010 |
PCT
Pub. No.: |
WO2008/016214 |
PCT
Pub. Date: |
February 07, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14537565 |
Jun 26, 2018 |
RE46912 |
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Reissue of: |
12376368 |
May 14, 2007 |
8309076 |
Nov 13, 2012 |
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Reissue of: |
12376368 |
May 14, 2007 |
8309076 |
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Foreign Application Priority Data
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Aug 4, 2006 [KR] |
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10-2006-0073722 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/335 (20130101); A23L 33/18 (20160801); A23C
9/1234 (20130101); A23L 33/135 (20160801); C12N
1/20 (20130101); C12N 1/205 (20210501); A61K
38/00 (20130101); C12R 2001/225 (20210501); A23Y
2220/37 (20130101) |
Current International
Class: |
A23C
9/123 (20060101); A23L 33/18 (20160101); A23L
33/135 (20160101); C07K 14/335 (20060101); C12N
1/20 (20060101); A61K 38/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2005-080636 |
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Mar 2005 |
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JP |
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2004/003235 |
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Jan 2004 |
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WO |
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Other References
Kitazawa. H., International Journal of Food Microbiology 40 (1998)
169-175 (Year: 1998). cited by examiner .
Martin et al., J Hum Lact 21(1), 2005 (Year: 2005). cited by
examiner .
Hammami, Cell. Mol. Life Sci. vol. 70: pp. 2947-2967 (Year 2013).
cited by examiner .
Kirjavainen et al., FEMS Immunol. And Med. Micro., 26:131-35, 1999.
cited by applicant .
Usman et al., J. of Dairy Res., 2001:617-624. cited by applicant
.
StrainInfobeta F. Gasser AM 63. cited by applicant .
Accessions AF243173, AF243144, AF243156, 519171. cited by applicant
.
Jackson et al., 2002, J. of Microbiol. Methods 51:313-321. cited by
applicant .
Mosca et al., Human milk: composition and health benefits, La
Pedriatria Medica e Chirurgica, v.39:155, pp. 47-52, 2017. cited by
applicant .
Pavlova et al 2002, J. Appl. Microbiol. 92 (3), 451-459. cited by
examiner .
Supplementary European Search Report issued in EP Appln. No. 07 74
6512, dated Aug. 17, 2010, 2 pages. cited by applicant .
A.C. Majhenic, et al.: "DNA analysis of the genes encoding acidocin
LF221 A and acidocin LF221 B, two bacteriocins produced by
Lactobacillus gasseri LF221," Appl. Microbiol. Biotechnol., vol.
63, Sep. 18, 2003, pp. 705-714. cited by applicant .
NCBI GenBank Accession No. AY297947: "Lactobacillus gasseri
putative complement factor, acidocin LF221B, and putative immunity
protein genes, complete cds; and unknown genes," Nov. 4, 2005, 2
pages. cited by applicant .
Y. Kawai et al.: "Primary Amino Acid and DNA Sequences of
Gassericin T, a Lactacin F-Family Bacteriocin Produced by
Lactobacillus gasseri SBT2055," in Biosci. biotechnol. Biochem,
vol. 64(10), Jun. 6, 2000, pp. 2201-2208. cited by applicant .
NCBI GenBank Accession No. AB029612: "Lactobacillus gasseri
gassericin T gene region (ORF1, ORF2, ORF3, gatA, gatX, ORF6),
complete cds," Oct. 27, 2000, 3 pages. cited by applicant .
Majhenic, A.G. et al., DNA analysis of the genes encoding acidocin
LF221 A and acidocin LF221 B, two bacteriocins produced by
Lactobacillus gasseri LF221, Appl. Microbiol. Biotechnol., vol. 63,
pp. 705-714. (2004). cited by applicant.
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Primary Examiner: Ponnaluri; Padmashri
Attorney, Agent or Firm: Sughrue Mion, PLLC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a .Iadd.Reissue application of U.S. Pat. No.
8,309,076 and Continuation Reissue application of U.S. application
Ser. No. 14/537,565, filed Nov. 10, 2014 (issued as U.S. Pat. No.
RE16,912 on Jun. 26, 2018), which is a Reissue application of U.S.
Pat. No. 8,309,076 issued on Nov. 13, 2012, which is issued from
the .Iaddend.35 U.S.C. .sctn.371 National Phase Entry Application
from PCT/KR2007/002363, filed May 14, 2007, and designating the
United States, which claims priority under 35 U.S.C. .sctn. 119 to
Korean Patent Application No. 10-2006-0073722 filed Aug. 4, 2006,
which is incorporated herein in its entirety.
Claims
What is claimed is:
.[.1. A Lactobacillus gasseri BNR17 strain of a biologically pure
culture deposited at Korean Collection for Type Culture of Korea
Research Institute of Biotechnology and Bioscience under the
Accession number of KCTC 10902BP..].
.[.2. The Lactobacillus gasseri BNR17 strain according to claim 1,
wherein the strain contains 16S rRNA sequence represented by SEQ ID
NO: 1..].
.[.3. A composition containing an effective dose of the
Lactobacillus gasseri BNR17 of claim 1..].
.[.4. The composition according to claim 3, wherein the composition
is selected from the group consisting of food, food additive,
animal feed and animal feed additive..].
.[.5. The composition according to claim 4, wherein the animal feed
additive contains at least one selected from the group consisting
of other non-pathogenic microorganisms, enzymes and a mixture
thereof..].
.[.6. A pharmaceutical composition comprising an effective dose of
the Lactobacillus gasseri BNR17 of claim 1..].
.[.7. A culture solution of the Lactobacillus gasseri BNR17 of
claim 1..].
.[.8. A method for inhibiting weight gain comprising administering
to a subject an effective dose of the Lactobacillus gasseri BNR17
of claim 1..].
.Iadd.9. A composition comprising Lactobacillus gasseri BNR17
strain in an enteric coating, wherein the L. gasseri BNR17 strain
is the strain deposited at Korean Collection for Type Culture of
Korea Research Institute of Biotechnology and Bioscience under the
Accession number of KCTC 10902BP..Iaddend.
.Iadd.10. The composition of claim 9, which further comprises (a)
one or more selected from the group consisting of acacia gum,
calcium phosphate, alginate, tragacanth gum, calcium silicate,
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, methylcellulose, methylhydroxybenzoate,
propylhydroxybenzoate, talc, magnesium stearate, and mineral oil,
and (b) one or more selected from the group consisting of a
lactose, dextrose, sucrose, sorbitol, mannitol, starch, gelatin,
and water..Iaddend.
.Iadd.11. The composition of claim 9, which is in a form of capsule
or tablet..Iaddend.
.Iadd.12. The composition of claim 9, which further comprises one
or more additional probiotic microorganism selected from the group
consisting of Saccharomyces cerevisiae, Bacillus coagulans,
Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum,
Bifidobacterium infantis, Bifidobacterium longum, Enterococcus
faecium, Enterococcus faecalis, Lactobacillus acidophilus,
Lactobacillus alimentarius, Lactobacillus casei, Lactobacillus
curvatus, Lactobacillus delbruckii, Lactobacillus johnsonii,
Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus
helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri,
Lactobacillus sakei, Lactococcus lactis and Pediococcus
acidilactici..Iaddend.
.Iadd.13. A dry powder comprising live Lactobacillus gasseri cells
and a protectant, wherein the live Lactobacillus gasseri cells are
of the Lactobacillus gasseri BNR17 strain deposited at Korean
Collection for Type Culture of Korea Research Institute of
Biotechnology and Bioscience under the Accession number of KCTC
10902BP, and wherein the protectant comprises powdered skim milk,
whey, and/or sucrose..Iaddend.
.Iadd.14. A method of producing a product, comprising mixing a dry
powder of claim 13 with at least one substance selected from the
group a diluent, a high-fiber additive, an encapsulant, a lipid, an
enzyme and a non-pathogenic microorganism, wherein the live
Lactobacillus gasseri cells are of the Lactobacillus gasseri BNR17
strain deposited at Korean Collection for Type Culture of Korea
Research Institute of Biotechnology and Bioscience under the
Accession number of KCTC 10902BP; and wherein the protectant
comprises powdered skim milk, whey, and/or sucrose..Iaddend.
.Iadd.15. A culture comprising a culture medium and Lactobacillus
gasseri BNR17, wherein the culture is obtained by culturing the
Lactobacillus gasseri BNR17 in a culture medium suitable for
Lactobacillus gasseri BNR17; wherein the L. gasseri BNR17 strain is
the strain deposited at Korean Collection for Type Culture of Korea
Research Institute of Biotechnology and Bioscience under the
Accession number of KCTC 10902BP; wherein the culture comprises an
extracellular polysaccharide produced by the Lactobacillus gasseri
BNR17; and wherein the culturing is carried out for 12-36
hours..Iaddend.
.Iadd.16. The culture of claim 15, wherein the culture medium is an
MRS medium supplemented with 2% glucose..Iaddend.
.Iadd.17. A method for inhibiting growth of a pathogenic bacterium
in intestine of a subject in need thereof comprising administering
Lactobacillus gasseri BNR17 strain deposited at Korean Collection
for Type Culture of Korea Research Institute of Biotechnology and
Bioscience under the Accession number of KCTC 10902BP, to the
subject, wherein the pathogenic bacterium is selected from the
group consisting of E. coli, B. cereus, L. monocytogenes, P.
mirabilis, S. aureus, and S. typhimurium..Iaddend.
Description
.Iadd.SEQUENCE LISTING.Iaddend.
.Iadd.The instant application contains a Sequence Listing which has
been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety..Iaddend.
FIELD OF THE INVENTION
The present invention relates to a probiotic lactic acid bacterium,
more specifically a novel lactic acid bacterium belonging to
Lactobacillus sp. isolated from human mother's milk and having
excellent probiotic activity such as acid resistance, bile acid
resistance and antimicrobial activity and inhibitory activity
against body weight augmentation.
DESCRIPTION OF THE RELATED ART
A lactic acid bacterium shares a long history with human, which is
the microorganism that is very profitable for human health and thus
in the increasing demand. According to the recent progress of the
lactic acid bacterium studies, its applicability has been broadened
from general foods to health food and medicines. A lactic acid
bacterium is exemplified by Streptococcus sp., Pediococcus sp.,
Leuconostoc sp., Lactobacillus sp., Sporolactobacillus sp. and
Bifidobacterium sp. microorganisms.
Lactic acid bacteria inhabit in the animal's intestines where they
decompose nutrients and cellulose that the host animal has taken
and then use them as an energy source to produce lactic acid and
antibiotics in order to inhibit the growth of pathogenic bacteria
in the intestine to keep the intestine healthy. The lactic acid
bacteria have also been used for stimulation of animal growth,
improvement of feed utilization, enhancement of resistance against
disease, inhibition of the growth of pathogenic bacteria, reduction
of mortality, inhibition of the generation of toxic substances and
production of various vitamins.
However, to be effective in the intestine, the incoming lactic acid
bacteria from outside have to arrive to the intestines safe and be
attached onto the mucous membrane to be functioning. To do so,
lactic acid bacteria have to be the one that is able to adhere
directly onto the mucous membrane of the intestine, has to be less
destroyed by gastric acid when it is orally administered, has to
have strong resistance against bile acid and has to have strong
antimicrobial activity against pathogens.
When lactic acid bacteria are used for food or medicine for human
health, they are supposed to be isolated from human for better
effect. In particular, the lactic acid bacteria isolated from
mother's milk have been acknowledged to be more effective and
safer. However, the human originated lactic acid bacteria have been
mostly isolated from adult's feces or breast-feeded infant's feces.
The lactic acid bacteria isolated from mother's milk are mostly
Lactobacillus reuteri and other lactic acid bacteria have hardly
been reported.
Meanwhile, obesity is a chronic disease whose cause has not been
exactly disclosed but whose development is believed to be
attributed to the co-work of several different factors. Obesity
might cause hypertension, diabetes, cardiovascular disease,
galstone, osteoarthritis, sleep spnea syndrome, breathing disorder,
prostatic cancer, breast cancer, colon cancer, etc. The
conventional methods hired for the prevention and treatment of
obesity are largely diet-exercise therapy, surgical operation, drug
therapy, etc. The diet-exercise therapy is to encourage taking
low-calorie-low fat food and physical exercise to consume oxygen.
This method requires patience since it has to be carried out
repeatedly and persistently and that is why this method seems to be
ineffective for the general public. The surgical operation is to
eliminate body fat by surgery. This method has an advantage of
obtaining the desired results in a short time but at the same time
has disadvantages of painful surgery, doubt of the continuance of
the effect and high costs. The drug therapy needs careful attention
because it carries many side effects.
Recently, studies on polysaccharides produced by lactic acid
bacteria have been actively undergoing. The mechanism that lactic
acid bacteria produce extracellular polysaccharides is known to be
very complicated. There are huge differences in productivity and
the structure of the polysaccharides according to the kinds of
lactic acid bacteria. Polysaccharides produced by lactic acid
bacteria have been reported to have anticancer activity and immune
enhancing activity (Kitazawa, H. Int. J. Food Microbiol., 1998. 40.
169-175, Hosono, A. Biosci. Biotechnol. Biochem., 1997. 61.
312-316, Chabot, S. Lait. 2001. 81. 683-697). It is also expected
to be very safe to take the polysaccharides produced by lactic acid
bacteria because lactic acid bacteria themselves are classified as
GRAS (Generally Recognized As Safe).
SUMMARY OF THE INVENTION
The present invention relates to a lactic acid bacterium isolated
from human mother's milk, and it is an object of the present
invention to provide a lactic acid bacterium that has strong
resistance against acid, pH and bile acid and strong adherence to
intestines so as to convert low-molecular carbohydrates decomposed
by a digestive enzyme into high-molecular polysaccharides and to
excrete the polysaccharides instead of letting it be absorbed in
the body.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
To achieve the above object, the present invention provides a
Lactobacillus gasseri BNR17 strain, the lactic acid bacterium
isolated from human mother's milk.
The lactic acid bacteria strain of the present invention has
following characteristics.
{circle around (1)} Morphology--morphology after culture at
37.degree. C. for 24 hours on the Lactobacilli MRS agar plate
medium:
i. Shape, size and color of colony: round, 0.5 mm.times.2 mm,
milk-white color, smooth surface.
ii. Gram staining: positive.
iii. Type: rod type (Bacillus).
iv. Sporulation: no.
v. Mobility: no.
{circle around (2)} Physiological properties
i. Growth temperature: 25.about.45.degree. C.
ii. Growth pH: pH 4.0.about.10.0
iii. Optimum growth temperature: 37.about.40.degree. C.
iv. Optimum growth pH: pH 6.0.about.8.0
{circle around (3)} Influence of oxygen: facultative anaerobic.
{circle around (4)} Sugar availability:
Glycerol -, Ribose -, Adonitol -, Galactose +, D-Glucose +,
D-Fructose +, D-Mannose +, Mannitol -, Sorbitol -,
N-Acetylglucoside +, Esculin +, Salicin +, Cellobiose +, Maltose +,
Lactose +, Melibiose -, Saccharose +, Trehalose +, Inulin -,
Melezitose -, Raffinose -, Starch -, .beta.-Gentiobiose -,
D-Turanose +, D-Tagatose +
{circle around (5)} Acid resistance: survived at pH 2.0.
{circle around (6)} Bile acid resistance: survived at 0.3% of bile
acid.
{circle around (7)} Adherence to intestines: adhered to Caco-2
cells, the human intestinal epithelial cells.
{circle around (8)} Antibiotic resistance: resistant to Gentamycin,
Kanamycin, Streptomycin, Bacitracin, Neomycin, Nalidixic acid,
Ciprofloxacin, Polymixcin B and Trimethoprim.
sensitive to Erythromycin, Penicillin, Tetracycline, Ampicillin,
Chloramphenicol, Vancomycin and Cefoxitin, Rifampin.
{circle around (9)} Antimicrobial activity to pathogenic bacteria:
antimicrobial activity to E. coli, S. aureus, S. typhimurium, B.
cereus, L. monocytogenes, and P. mirabilis.
{circle around (10)} Presence of antimicrobial peptide: The gene
corresponding to gassericin T of bacteriocin, one of antimicrobial
peptide components of lactic acid bacteria, is detected by PCR.
{circle around (11)} Polysaccharide generation: The lactic acid
bacterium of the present invention produces approximately 520 mg/L
of polysaccharides after 24 hours of culture on the MRS medium
supplemented with 2% glucose. The polysaccharides were composed
mainly of glucose, mannose, galactose, fucose, arabinose and
D-glucosamine. The polysaccharides produced by the lactic acid
bacterium of the present invention are not decomposed by digestive
enzymes such as .alpha.-amylase and pancreatine.
Koreans have different diet habit with westerners. So, it is clear
and natural that the lactic acid bacterium isolated from Koreans
have different dietary habits with westerners. So, it is clear and
natural that the lactic acid bacterium isolated from Korean best
fits to Korean. The lactic acid bacterium isolated from Korean
mother's milk fulfills every required fundamental condition for
probiotic lactic acid bacterium, resulting in the best health
enhancing effect for Korean. The present inventors named such
lactic acid bacterium having the above characteristics as
"Lactobacillus gasseri BNR17" and deposited at Korean Collection
for Type Cultures (KCTC) of Korea Research Institute of
Biotechnology and Bioscience (KRIBB), located at #52, Oun-dong,
Yusong-ku, Taejon 305-333, Republic of Korea, on Jan. 23, 2006
(Accession No: KCTC 10902BP).
The present invention also provides a composition containing an
effective dose of the lactic acid bacterium. The composition of the
present invention can be provided in the forms of food, food
additives, animal feeds and animal feed additives.
The novel lactic acid bacterium provided by the invention,
Lactobacillus gasseri BNR17 (Accession No: KCTC 10902BP) has
excellent acid resistance, bile acid resistance and antimicrobial
activity, making it an excellent candidate as a seed for the
production of various fermented milk products and other fermented
foods. The fermented milk products herein can be exemplified by
yoghurt, calpis, cheese and butter, and the other fermented foods
herein can be exemplified by tofu, soy bean paste, Chungkukjang,
jelly and Kimchi, but not always limited thereto. The fermented
milk products and fermented foods can be easily produced by the
conventional method only with substituting the strain with the
lactic acid bacterium of the invention.
According to a preferred embodiment of the present invention,
approximately 7.7% weight gaining inhibitory effect was observed in
the experimental group rats administered with Lactobacillus gasseri
BNR17, compared with the control group rats administered with PBS
(phosphate-buffered saline)(see Table 6). In addition to the weight
gaining inhibitory effect, diet efficiency in the experimental
group was also reduced significantly, compared with the control
group. Polysaccharides included in feces of both experimental and
control groups were examined. As a result, the polysaccharide
content in feces of experimental group was higher than that of
control group (see FIG. 6). These results indicate that the
indigestible polysaccharide producing capacity of Lactobacillus
gasseri BNR17 plays a certain role in weight regulation. In the
meantime, no superficial side effects have been detected in the
experimental group rats taking Lactobacillus gasseri BNR17 and the
weight of each organ was not much different from that of control
group (see Table 7 and Table 8). Microorganism transition, one of
major concerns when human takes a microorganism, was not observed,
suggesting that the lactic acid bacterium of the present invention
is very safe for human to take (see FIG. 7).
The lactic acid bacterium food products of the present invention
can be produced as an edible form of composition either containing
Lactobacillus gasseri BNR17 alone or with any acceptable carrier.
The lactic acid bacterium of the invention can be added to the food
that does not contain any probiotic bacteria or the food that
already contains several kinds of probiotic bacteria. The
microorganism that can be co-used with the lactic acid bacterium of
the invention to produce the lactic acid bacterium food has to be
appropriate for intake by human or animals and have probiotic
activity such as inhibiting pathogenic bacteria or improving the
balance of microorganisms in the mammal's intestines, but not
always limited thereto. The probiotic microorganism is exemplified
by yeasts such as Saccharomyces, Candida, Pichia and Torulopsis;
fungi such as Aspergillus, Rhizopus, Mucor and Penicillium; and
bacteria belonging to Lactobacillus, Bifidobacterium, Leuconostoc,
Lactococcus, Bacillus, Streptococcus, Propionibacterium,
Enterococcus and Pediococcus. Preferably, the probiotic
microorganism can be selected from the group consisting of
Saccharomyces cerevisiae, Bacillus coagulans, Bacillus
licheniformis, Bacillus subtilis, Bifidobacterium bifidum,
Bifidobacterium infantis, Bifidobacterium longum, Enterococcus
faecium, Enterococcus faecalis, Lactobacillus acidophilus,
Lactobacillus alimentarius, Lactobacillus casei, Lactobacillus
curvatus, Lactobacillus delbruckii, Lactobacillus johnsonii,
Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus
helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri,
Lactobacillus sakei, Lactococcus lactis and Pediococcus
acidilactici. It is more preferred to add a probiotic microorganism
mixture having excellent probiotic activity and immune enhancing
activity as well as anticancer activity to the lactic acid
bacterium food of the invention, resulting in greater effect. A
carrier acceptable for the lactic acid bacterium food of the
invention is exemplified by a diluent, a high-fiber additive, an
encapsulant and a lipid, which have been well informed to those
skilled in the art. The lactic acid bacterium of the invention,
Lactobacillus gasseri BNR17, can be formulated as capsules, culture
suspension or dried powder.
In addition, the composition containing the lactic acid bacterium
of the invention can be prepared as animal feeds or animal feed
additives.
The animal feed additive of the invention can be prepared in dried
or liquid form and can contain other non-pathogenic microorganisms
in addition to the Lactobacillus gasseri BNR17. The addable
microorganism can be selected from the group consisting of Bacillus
subtilis producing protease, lipase and sugar converting enzyme;
lactobacillus strain having organic decomposition activity and
maintaining physical activities under anaerobic condition;
filamentous fungi such as Aspergillus oryzae (Slyter, L. L. J.
Animal Sci. 1976, 43. 910-926) contributing to the increase of milk
and weight of cattle and feed digestibility as well; and yeast such
as Saccharomyces cerevisiae (Johnson, D. E et al. J. Anim. Sci.,
1983, 56, 735-739; Williams, P. E. V. et al, 1990, 211).
The animal feed additive of the present invention can additionally
include one or more enzyme products in addition to the
Lactobacillus gasseri BNR17. The addable enzyme product can be in
dried or liquid form, which is selected from the group consisting
of lipase, phytase decomposing phytic acid into phosphate and
inositol phosphate, amylase hydrolyzing .alpha.-1,4-glycoside bond
included in starch and glycogen, phosphatase hydrolyzing organic
phosphoric acid ester, carboxymethylcellulase decomposing
cellulose, xylase decomposing xylose, maltase hydrolyzing maltose
into two glucoses and invertase hydrolyzing saccharose into
glucose-fructose.
When the lactic acid bacterium of the invention is added to the
animal feed as an additive, the proper feed raw material is
selected from the group consisting of crops, soybean protein,
peanut, green pea, sugar beet, pulp, crop byproduct, animal
intestine powder and fish powder. At this time, these materials can
be used as they are or after being processed. To process the animal
feed, for example, raw material for feed is compressed by pressure
to be discharged, but not always limited thereto. In the case of
using a protein as a raw material, extrusion is preferred.
Particularly, extrusion is to denaturate a protein by
heat-treatment, resulting in the destruction of anti-enzyme
factors. More specifically, in the case of using a soybean protein,
extrusion improves the digestibility of the protein, inactivates
anti-nutrition factors such as trypsin inhibitor, one of protease
inhibitors, and increases digestibility by a protease, resulting in
the increase of nutritional value of the protein.
The present invention further provides a pharmaceutical composition
for the prevention and treatment of obesity which contains the
effective dose of Lactobacillus gasseri BNR17 (Accession No: KCTC
10902BP).
The Lactobacillus gasseri BNR17 of the invention is generally
administered as a tablet or a capsule prepared by mixing the lactic
acid bacterium with a pharmaceutically acceptable carrier, an
excipient or another effective supplementary component.
The acceptable carrier, excipient or diluent for the pharmaceutical
composition of the invention is exemplified by lactose, dextrose,
sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate,
alginate, tragacanth gum, gelatin, calcium silicate,
microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,
syrup, methylcellulose, methylhydroxybenzoate,
propylhydroxybenzoate, talc, magnesium stearate or mineral oil. The
microorganism composition of the invention can additionally include
lubricants, wetting agents, emulsifying agents, suspension agents,
preserving agents, sweetening agents or flavors. The composition of
the invention can be prepared in the form of enteric coated
preparation in order for the composition to pass through the
stomach and reach the small intestine safely and to release
microorganism, the active ingredient, therein fast and easy,
according to the conventional method well known to those skilled in
the art.
The microorganism composition of the present invention can also be
prepared in the form of a capsule, according to the conventional
capsule production method. For example, a standard carrier is used
for the preparation of a pellet containing the freezing-dried
microorganism of the invention, which fills soft gelatin capsules.
Another example is that the microorganism of the invention is mixed
with a pharmaceutically acceptable carrier, for example soluble
gum, cellulose, silicate or oil, to prepare suspension or
dispersing solution, which fills soft gelatin capsules.
The pharmaceutical composition of the present invention can be
provided as a unit drug form for oral administration as an enteric
coated preparation. "Enteric coating" herein indicates that a drug
is not decomposed by gastric acid and maintained as being coated
but is decomposed in the small intestine to release active
ingredients therein, which includes every kind of pharmaceutically
acceptable coatings. "Enteric coating" of the invention is
maintained at least two hours in the artificial gastric juice such
as HCl solution (pH 1) at 36-38.degree. C. but the coating is
preferably decomposed within 30 minutes in the artificial
intestinal juice such as KH.sub.2PO.sub.4 buffer solution (pH
6.8).
Enteric coating of the invention is performed, in which one core is
coated by 16-30 mg, preferably 16-20 mg or less than mg of the
composition. The preferable thickness of the coating is 5-100 .mu.m
and more preferably 20-80 .mu.m for the best results. Materials for
the enteric coating can be selected among the well-informed high
molecular substances. Those high molecular substances are described
in numbers of references (L. Lachman, et al., The Theory and
Practice of Industrial Pharmacy, 3.sup.rd edition, 1986, pp.
365.about.373; H. Sucker, et al., Pharmazeutische Technologie,
Thieme, 1991, pp. 355-359; Hagers Handbuch der pharmazeutischen
Praxis, 4.sup.th edition, Vol. 7, pp. 739-742, and 766-778,
(SpringerVerlag, 1971); and Remington's Pharmaceutical Sciences,
13.sup.th edition, pp. 1689.about.1691 (Mack Publ., Co., 1970)),
which can be exemplified by cellulose ester derivatives, cellulose
ether, methylacrylate copolymer of acrylic resin and copolymer of
maleic acid and phthalic acid derivatives.
The enteric coating of the invention can be performed by the
conventional method which is spraying the coating solution onto a
core. The acceptable solvent for the enteric coating is selected
from the group consisting of alcohol such as ethanol, ketone such
as acetone, halogenized hydrocarbon such as CH.sub.2Cl.sub.2 and a
mixture thereof. A softener such as di-n-butylphthalate or
triacetine can be added to the coating solution at the ratio of
1:0.05-0.3 (coating material:softener). It is preferred to spray
serially and the amount of spry is determined by considering the
coating conditions. Spraying pressure can be regulated and
generally 1-1.5 bar is considered to give best results.
The "pharmaceutically effective dosage" of the invention indicates
the minimum amount of the microorganism of the invention that is
able to reduce low-sugar carbohydrates to be absorbed into the
intestines of mammals. The dosage of the microorganism, which is
delivered to the human body by the composition of the invention,
can be regulated according to the administration pathway and
subjects.
The composition of the invention can be administered to a subject
at least once a day, everyday. Unit dosage indicates the unit
separated physically to be appropriate for unit administration to a
subject, either human or other mammals, and each unit contains a
required amount of acceptable carrier and a required amount of the
microorganism of the invention for the treatment effect. The unit
dosage for oral administration of the composition of the invention
is preferably 0.1-10 g and more preferably 0.5-5 g. The
pharmaceutically effective dosage of the microorganism of the
invention is 0.1-10 g/day. However, the dosage might vary according
to the weight of a patient, the severity of obesity, and effective
supplementary ingredients and microorganisms. The one day dosage
can be divided into several sub-units so that they can be
administered serially if necessary. Thus, the dosage of the
composition of the invention cannot limit the spirit and scope of
the invention in any way.
The regular administration of the composition of the invention
results in the interruption of the absorption of saccharides inside
the human body by releasing microorganisms to compete and form
microflora, which interrupts the absorption, and further involves
in the convert of monosaccharides such as carbohydrate into
polysaccharides so as to inhibit the absorption thereof. In
addition, dietary fiber produced by the microorganism provides
preferable conditions for useful enterobacteria to grow with
stimulating intestinal motility. Therefore, the composition of the
invention can be effectively used for the prevention and treatment
of obesity.
To maximize the weight reducing effect or obesity preventive effect
of the pharmaceutical composition, any weight reducing agent known
to those skilled in the art can be additionally included in the
composition by a proper amount. The amount can be determined by
those skilled in the art after multiple tests. The effective
ingredient as an additive, the weight reducing agent, is preferably
selected from the group consisting of conjugated linoleic acid,
polydextrose, inulin, guar gum, arabic gum, L-carteine, grape seed
extract, fructooligosaccharide, xylooligosaccharide, raffinose,
gluconic acid, champignon, polyanthocyanidine, lactulose, lactitol,
lactosucrose, Angelica gigas extract, Hovenia dulcis extract and
tangerine peel extract, but not always limited thereto.
The present invention also provides a culture solution prepared by
culturing Lactobacillus gasseri BNR17 (Accession No: KCTC 10902BP).
The medium used to prepare the culture solution is not limited, and
any medium that contains a medium for microorganism culture can be
used. The culture solution of the invention can additionally
contain any additive if necessary for a specific use. For example,
to maximize the weight reducing effect, any weight reducing agent
well known to those skilled in the art can be added to the culture
solution and at this time the content of the agent can be
determined by those skilled in the art after examining the
effective dose range through repeated tests.
The present invention also provides a bacteriocin peptide produced
by the lactic acid bacterium of the invention and a gene encoding
the same. The present inventors named the bacteriocin peptide as
"gassericin BNR17" which was confirmed to have the nucleotide
sequence represented by SEQ. ID. NO: 5. The nucleotide sequence of
the gassericin BNR17 was compared with that of the conventional
antimicrobial peptide, gassericin T (NCBI Blast Search No.
AB029612, SEQ. ID. NO: 6), and as a result, the gassericin BNR17
had approximately 98% homology with the gassericin T.
The present invention further provides a recombinant vector
containing the gassericin BNR17 gene.
The recombinant vector of the present invention can be prepared by
inserting the gene having the nucleotide sequence represented by
SEQ. ID. NO: 5 into a general expression vector for E. coli. The
mother vector for the construction of the recombinant vector is not
limited to a specific one, and almost every microorganism
expression vector can be used but an E. coli expression vector is
preferred.
The present invention also provides a transformant transformed with
the recombinant vector.
The transformant of the invention can be easily generated by
introducing the above recombinant vector into a random host cell.
The host cell herein can be selected from the group consisting of
eukaryotic or prokaryotic cells and multicellular animal originated
cell lines, but not always limited thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
FIG. 1 is a diagram showing the sequence comparison between
Lactobacillus gasseri BNR17 of the invention (upper sequence in
figure, SEQ. ID. NO: 1) and Lactobacillus gasseri KC26 (NCBI
GENBANK Accession No: AF243156, lower sequence in figure, SEQ. ID.
NO: 2) 16s rRNAs.
FIG. 2 is a microphotograph showing the enteric adherence of
Lactobacillus gasseri BNR17 of the invention.
FIG. 3 is a set of photographs showing that Lactobacillus gasseri
BNR17 of the invention has antimicrobial activity against various
pathogenic bacteria.
FIG. 4 is a graph showing the glucose consumption and
polysaccharide production according to the growth of Lactobacillus
gasseri BNR17 of the invention. ; cell growth, .diamond-solid.;
glucose concentration, ; EPS (polysaccharide) concentration
FIG. 5 is a graph showing the amount of feces of a rat taking
Lactobacillus gasseri BNR17 of the invention.
FIG. 6 is a graph showing the EPS (polysaccharide) concentration in
the feces of a rat taking Lactobacillus gasseri BNR17 of the
invention.
FIG. 7 is a set of electrophoresis photographs showing the RAPD-PCR
profiles of colonies isolated from other organs than the small
intestine of a rat taking Lactobacillus gasseri BNR17 of the
invention, in which primers represented by SEQ. ID. NO: 7 (A), NO:
8 (B) and NO: 9 (C) were used.
Lane 1; Lb. gasseri BNR17, Lanes 2-5; colonies isolated from other
organs than the small intestine of a rat taking Lb. gasseri
BNR17,
M; DNA size marker.
EXAMPLES
Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on
consideration of this disclosure, may make modifications and
improvements within the spirit and scope of the present
invention.
Example 1
Lactic Acid Bacterium Isolation from Human Mother's Milk
Human mother's milk was taken from a woman delivered of a baby not
more than two weeks ago. Then, the mother's milk was diluted with
PBS and the undiluted milk and diluted milk were distributed on a
lactobacillus selection medium respectively. The medium was
cultured at 37.degree. C. for 2-3 days and the colonies generated
therein were sorted by morphology and color. The isolated colonies
were Gram-stained and observed under a microscope to select those
colonies that were Gram-positive and had rod-shaped structure. The
selected colonies were cultured in MRS liquid medium (pH 6.8) at
37.degree. C. for 24 hours. Colonies in the culture solution under
the pH lower than 4.5 were selected. The colonies were cultured in
MRS medium (pH 2.0) for 2 hours, followed by further culture in MRS
medium supplemented with 0.3% oxgall for 9 hours. The survived
lactobacillus strain that exhibited acid resistance and bile acid
resistance was isolated and identified by 16S rRNA sequencing. As a
result, the strain was confirmed to belong Lactobacillus gasseri
species (SEQ. ID. NO: 1, FIG. 1) and named as "Lactobacillus
gasseri BNR17".
Example 2
Sugar Utilization of the Isolated Lactic Acid Bacterium
Sugar utilization of Lactobacillus gasseri BNR17 of the invention
isolated above was investigated by comparing with other standard
strains using API50CHL kit (Biomerieux, France) and the results are
shown in Table 1. In Table 1, 5314 indicates Lactobacillus gasseri
CECT5714; 5315 indicates Lactobacillus gasseri CECT5715; 11413
indicates Lactobacillus gasseri LMG11413; 18194 indicates
Lactobacillus gasseri LMG18194; 4479 indicates Lactobacillus
gasseri CECT4479; 18176 indicates Lactobacillus gasseri LMG 18176;
and 13047 indicates Lactobacillus gasseri LMG13047.
TABLE-US-00001 TABLE 1 BNR17 5714 5715 11413 18194 4479 18176 13047
Glycerol 0 0 0 0 0 0 0 0 Erythritol 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0
0 0 4 0 0 0 0 0 Ribose 0 0 0 0 0 0 0 0 D-Xylose 0 0 0 0 0 0 0 0
L-Xylose 0 0 0 0 0 0 0 0 Adonitol 0 0 0 0 0 0 0 0 .beta.-Methyl- 0
0 0 0 0 0 0 0 xyloside Galactose 5 5 5 5 5 5 5 5 D-Glucose 5 5 5 5
5 5 5 5 D-Fructose 5 5 5 5 5 5 5 5 D-mannose 5 5 5 5 5 5 5 5 0 0 0
5 0 0 0 0 Rhamnose 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 Inositol 0 0 0 0
0 0 0 0 0 0 3 5 0 0 0 0 Sorbitol 0 0 0 0 0 0 0 0 .alpha.-Methyl-D-
0 0 0 0 0 0 0 0 mannoside .alpha.-Methyl-D- 0 0 0 0 0 0 0 0
glucoside N-Acetlyglu- 5 4 5 5 5 5 5 5 cosamine 5 5 5 0 0 5 5 0 5 5
5 1 1 5 5 4 Esculine 5 5 5 5 5 5 5 5 Salicine 5 5 5 5 5 5 5 3
Cellobiose 5 5 5 5 5 5 5 5 Maltose 5 5 5 5 5 5 5 5 5 0 4 5 5 5 5 0
0 0 0 0 0 0 5 0 Saccharose 5 5 5 5 5 5 5 5 Trehalose 5 5 5 5 5 5 5
5 Inuline 0 0 0 0 0 0 0 0 Melezilose 0 0 0 0 0 0 0 0 D-Raffmose 0 0
0 0 0 0 5 0 5 3 5 1 1 5 3 3 0 0 0 4 4 0 0 0 Xylitol 0 0 0 0 0 0 0 0
b-Gentobiose 5 5 5 5 5 5 5 5 5 0 0 0 0 0 0 0 D-Lycose 0 0 0 0 0 0 0
0 5 5 5 3 3 5 5 5 D-Fucose 0 0 0 0 0 0 0 0 L-Fucose 0 0 0 0 0 0 0 0
D-Arabinol 0 0 0 0 0 0 0 0
As shown in Table 1, compared with other lactobacillus strains, the
Lactobacillus gasseri BNR17 of the invention was distinguishable in
sugar utilization from others (shown in thick Italics).
Example 3
Enzyme Activity of the Isolated Lactic Acid Bacterium
The enzyme activity of the Lactobacillus gasseri BNR17 isolated in
Example 1 was compared with those of other standard strains using
APIZYM kit (Biomerieux, France) and the results are shown in Table
2. In Table 2, 13134 indicates Lactobacillus gasseri LMG13134.
TABLE-US-00002 TABLE 2 BNR17 11413 13047 13134 18176 18194 4479
5714 5715 Control - - - - - - - - - Alkaline phosphates - - - - - -
- - - Esterase (C4) 1 2 1 1 1 1 1 1 1 Esterase lipase (C8) - - - 1
- - - - - Lipase (C4) - - - - - - - - - Leucine + + + + + + + + +
arylamidase 1 1 1 3 2 - - - 1 1 1 1 4 2 3 4 1 1 Trypsin - - - - - -
- - - .alpha.-chymotrypsin - - 1 - - - - - - + 1 2 + - 1 2 1 1 + 1
+ + + - + + + + + - - + - + + + + 4 1 - + + - - - - - - 1 1 + - 1 2
1 - 1 3 + + 3 4 + 1 + 1 - + - - - 1 .alpha.-mannosidase - - - - - -
- - .alpha.-fiteesidase - - - - - - - - -
As shown in Table 2, the Lactobacillus gasseri BNR17 of the
invention was distinguishable in enzyme activity from other strains
(shown in thick Italics).
Example 4
Acid Resistance and Bile Acid Resistance
To investigate acid resistance and bile acid resistance of the
strain of the invention, Lactobacillus gasseri BNR17 was inoculated
in 4 ml of MRS liquid medium and cultured at 37.degree. C. for
18-20 hours. Some of the culture solution was reinoculated in
another MRS medium with regulated pH of 2.0 at the concentration of
10.sup.7 CFU/ml and cultured at 37.degree. C. for 2 hours. The
number of live cells was counted using MRS agar plate. The culture
solution tested for acid resistance was used again for
centrifugation. The cells were recovered and inoculated in MRS
liquid medium (pH 6.8) supplemented with 0.3% oxgall, followed by
culture at 37.degree. C. for 9 hours. The number of live cells was
also counted using MRS agar plate. The results are shown in Table
3.
TABLE-US-00003 TABLE 3 Before Treatment at 0.3% expall treatment pH
2.0 treatment BNR17 3.1 .times. 10.sup.7 2.1 .times. 10.sup.7 1.5
.times. 10.sup.7
As a result, even after the treatment with strong acid (pH 2.0),
the Lactobacillus gasseri BNR17 exhibited high survival rate, and
so did in the medium supplemented with 0.3% oxgall.
Example 5
Enteric Adherence
The strain of the invention was inoculated on the plate on which a
human intestinal epithelial cell line CaCo-2 was cultured in
PRMI1640 (Gibco) at the concentration of 10.sup.7 CFU/ml. The
strain was cultured at 37.degree. C. for one hour, followed by
washing three times with PBS to eliminate non-adhered cells. The
sample was fixed with methanol and then stained with crystal
violet, followed by observation under a microscope. As a result,
the Lactobacillus gasseri BNR17 of the invention was confirmed to
be very well adhered on CaCo-2 cells (FIG. 2).
Example 6
Antimicrobial Activity Against Pathogenic Bacteria
E. coli, S. aureus, S. typhimurium, B. cereus, L. monocytogenes and
P. mirabilis were cultured at 37.degree. C. for 18 hours in BHI
liquid medium (Difco) and then inoculated in 6 of 5 ml BHI agar
media (agar content: 0.7%) respectively at the concentration of
10.sup.5 CFU/ml. These media were overlapped on 6 plates with BHI
agar media (agar content: 1.5%) fixed thereon. After hardening
those 6 plate media, a well of 4 mm in diameter was made in each
medium, in which 40 .mu.l of the supernatant (2.times.) of lactic
acid bacterium culture solution cultured at 37.degree. C. for 24
was added, followed by culture at 37.degree. C. for 5 hours.
As a result, a clear growth inhibition ring was observed around the
well, suggesting that the strain of the invention has antimicrobial
activity against various pathogenic bacteria (Table 4 and FIG.
3).
TABLE-US-00004 TABLE 4 Diameter of growth Pathogenic bacteria
inhibition ring (mm) E. coli KCTC1039 16 B. cereus KCTC1526 16 L.
monocytogenes KCTC3710 12 P mirabilis KCTC2510 14 S. aureus
KCTC1928 6 S. typhimurium KCTC2421 18
Example 7
Antibiotic Resistance
Lactobacillus gasseri BNR17 culture solution was smeared on MRS
agar plate by using a swab, on which a disc containing
erythromycin, penicillin, gentamycin, kanamycin, streptomycin,
bacitracin, chloramphenicol, vancomycin, tetracycline, ampicillin,
cefoxitin, rifampin, neomycin, nalidixic acid, ciprofloxacin,
polymixcin B or trimethoprim was placed, followed by culture at
37.degree. C. for 24 hours.
As a result, Lactobacillus gasseri BNR17 of the invention was
confirmed to have resistance against gentamycin, streptomycin and
trimethoprim.
Example 8
Detection of the Antimicrobial Peptide Gene
Bacteriocin gene was investigated by PCR performed by using the
Lactobacillus gasseri BNR17 genomic DNA as a template and primers
represented by SEQ. ID. NO: 3 and NO: 4 which were specific to the
nucleotide sequence of a gene of bacteriocin known as an
antimicrobial peptide produced by Lactobacillus gasseri
species.
As a result, the PCR product corresponding to gassericin was
confirmed and represented by SEQ. ID. NO: 5. The nucleotide
sequence was compared with that of gassericin T (NCBI Blast Search
No. AB029612) represented by SEQ. ID. NO: 6 and confirmed to have
approximately 98% homology.
Example 9
.beta.-glucuronidase Activity
.beta.-glucuronidase produced by enterobacteria has been known as
one of oncogenic enzymes and thus the strain that has this enzyme
activity is considered as a harmful strain. To investigate whether
the Lactobacillus gasseri BNR17 of the invention has
.beta.-glucuronidase activity or not, the enzyme activity of
Lactobacillus gasseri BNR17 was tested using API ZYM kit
(Biomerieux, France).
As a result, the strain of the invention was confirmed not to have
.beta.-glucuronidase activity, suggesting that the strain was safe
(Table 5).
TABLE-US-00005 TABLE 5 Enzyme Activity .alpha.-galactosidase
Positive .beta.-galactosidase Positive .beta.-glucuronidase
Negative .alpha.-glucosidase Negative .beta.-glucosidase
Positive
Example 10
Glucose Consumption and Polysaccharide Production
Lactobacillus gasseri BNR17 was inoculated in MRS medium (Difco)
prepared by adding 2% glucose (w/v) at the concentration of
10.sup.6 cfu/ml and cultured for 96 hours, during which the cell
number was measured stepwise and at the same time glucose
consumption and extracellular polysaccharide (EPS) production were
measured as well.
As a result, the highest level of Lactobacillus gasseri BNR17 was
observed on the 12.sup.th hour of culture and since then the level
had been decreased. Glucose concentration was rapidly reduced after
7 hours of culture and no changes of glucose concentration were
detected after 36 hours, suggesting that most of glucose was
consumed within 36 hours from the culture started. EPS production
was maximized on the 24.sup.th hour (the highest level: 520 mg/k)
and was slightly reduced on the 36.sup.th hour but increased again
thereafter. It was presumed to be attributed to autolysis of the
cells causing various polysaccharides in the cells to be released
into the culture solution (FIG. 4).
Example 11
Decomposition of the Polysaccharide Produced by Lactobacillus
Gasseri BNR17 by a Digestive Enzyme
100 mg of each .alpha.-amylase (Sigma) and pancreatin (Sigma) was
dissolved in 0.05 M phosphate buffer (pH 7.0). 50 .mu.l of the
above enzyme solution and 150 .mu.l of 0.05 M phosphate buffer (pH
7.0) were added to 200 .mu.l of polysaccharide (EPS) solution
extracted from the supernatant of Lactobacillus gasseri BNR17
culture solution, followed by reaction at 37.degree. C. for one
hour. The reaction mixture was heated at 100.degree. C. for 15
minutes to inactivate enzymes therein, followed by cooling at room
temperature. Glucose concentration was measured with a glucose kit
(Sigma).
As a result, glucose was not detected in the polysaccharide
solution before the treatment of each digestive enzyme, while 3.70
mg/l and 19.1 mg/l of glucose were respectively detected after the
treatment of pancreatin and .alpha.-amylase. This result indicates
that the polysaccharide produced by Lactobacillus gasseri BNR17 was
hardly decomposed by a digestive enzyme.
Example 12
Weight Gaining Inhibitory Effect of Lactobacillus Gasseri BNR17
8 week old male SD rats were grouped into two. One group was orally
administered with PBS only (pH 7.4) and the other group was orally
administered with PBS suspended with 10.sup.9 CFU/ml of
Lactobacillus gasseri BNR17, everyday for 8 weeks. Changes of
weights, food intakes, and blood chemical values such as
cholesterol level were measured once a week. The amounts of feces
and EPS in feces were also measured to investigate the relation of
weight gaining inhibitory effect of Lactobacillus gasseri BNR17 and
polysaccharide production capacity thereof. 8 weeks later, all the
test animals were sacrificed and dissected to extract the liver,
kidney, spleen, MLN (mesenteric lymph node), which were measured
their weights. Some of each organ extracted was homogenized and
smeared on LBS agar, a lactobacillus selection medium, which was
then cultured and RAPD (random amplified polymorphic DNA)-PCR
profiles of the generated colonies were investigated. The result
was compared with the RAPD-PCR profile of Lactobacillus gasseri
BNR17 to investigate whether the strain was transferred to other
organs.
As a result, approximately 179.1% weight increase was observed for
8 weeks in the control group orally administered with PBS only,
while approximately 171.6% weight increase was observed in the
experimental group orally administered with Lactobacillus gasseri
BNR17 (Table 6). The experimental group also exhibited lower rates
of one-day weight increase and food efficiency ratio than the
control.
TABLE-US-00006 TABLE 6 Weight Food Weight (g) gaining efficiency
Group Initial weight Final weight* (g/day) ratio** Control 221.20
.+-. 3.759 393.73 .+-. 4.860 3.081 0.131 .+-. 0.078 BNR17 223.66
.+-. 10.077 380.85 .+-. 21.517 2.807 0.115 .+-. 0.067
In Table 6, food efficiency ratio (FER) indicates weight gaining (g
day)/food intake (g day). *P<0.05, **P<0.05.
The results of measuring the amounts of feces and EPS in feces of
both the control and the experimental groups are shown in FIG. 5
and FIG. 6. The amounts of feces were not much different between
the control and the experimental groups, but the ESP amount was
significantly increased in the experimental group administered with
Lactobacillus gasseri BNR17. This result indicates that
Lactobacillus gasseri BNR17 converts sugar components taken inside
body into indigestible polysaccharides so as to release the
polysaccharides out of the body, resulting in the decrease of in
vivo absorption rate and inhibition of weight gaining.
To examine safety of the strain for human administration, blood
chemical values and organ weights were measured. Each levels and
values were similar in the control and the experimental groups,
suggesting that the strain did not cause side effects (Table 7 and
Table 8).
TABLE-US-00007 TABLE 7 Cholesterol Glucose HDL LDL Total protein
Triglyceride Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) (OL) (mg/dL)
Control 99.7 .+-. 11.7 88.9 .+-. 12.1 38.5 .+-. 3.5 21.5 .+-. 1.6
8.6 .+-. 0.3 115.9 .+-. 11.0 BNR17 96.3 .+-. 6.3 79.2 .+-. 4. 1
40.8 .+-. 1.8 20.8 .+-. 3.1 9.0 .+-. 0.3 132.6 .+-. 5.5
TABLE-US-00008 TABLE 8 Group Liver Kidney Spleen Control 0.029 .+-.
0.001 0.007 .+-. 0.000 0.002 .+-. 0.000 BNR17 0.027 .+-. 0.003
0.007 .+-. 0.001 0.002 .+-. 0.000
In Table 8, each number indicates organ weight (g)/rat weight
(g).
To investigate whether the strain was transferred to other organs,
RAPD-PCR profiles of colonies of each organ tissue cultured on LBS
agar plate were investigated by using primers p1, p2 and OPL5
respectively represented by SEQ. ID. NO: 7-NO: 9. PCR using the
primers p1 and p2 was performed as follows; 94.degree. C. (2
minutes), 36.degree. C. (5 minutes), 72.degree. C. (5 minutes)--4
cycles/94.degree. C. (1 minute), 36.degree. C. (1 minute),
72.degree. C. (2 minutes)--36 cycles. PCR using the primer OPL5 was
performed as follows; 94.degree. C. (2 minutes)--1 cycle/94.degree.
C. (40 seconds), 45.degree. C. (1 minute), 72.degree. C. (1
minute)--2 cycles/94.degree. C. (40 seconds), 52.degree. C. (1
minute), 72.degree. C. (3 minutes)--30 cycles/70.degree. C. (5
minutes)--1 cycle.
As a result, no colonies exhibited similar profiles to BNR17 (FIG.
7). Thus, BNR17 was confirmed to be safe strain which is not
transferred to other organs except the small intestine when it is
taken.
Manufacturing Example 1
Preparation of Fermented Milk
Raw milk in which milk solid non fat content was regulated by 8-20%
using powdered skim milk was sterilized at 72-75.degree. C. for 15
seconds. The sterilized raw milk was cooled down to the proper
temperature, to which Lactobacillus gasseri BNR17 of the invention
was inoculated at the concentration of 10.sup.6 cfu/ml, followed by
culture until pH reached 4-5. Upon completion of the culture, the
culture solution was cooled down. In the meantime, 0.1-50 weight %
of fruit juice concentrate, 0.1-20 weight % of dietary fiber,
0.5-30 weight % of glucose, 0.1-15 weight % of oligosaccharide,
0.01-10 weight % of calcium and 0.001-5 weight % of vitamin were
all dissolved to prepare syrup. The syrup was sterilized, cooled
down, and mixed with the above culture solution, followed by
stirring for homogenization. The resultant mixture was packed,
resulting in the preparation of fermented milk. Flavor, physical
property, and taste of the fermented milk product were tested, and
the results were satisfactory.
Manufacturing Example 2
Preparation of Lactic Acid Bacteria Powder
Lactobacillus gasseri BNR17 of the invention was inoculated into
MRS liquid medium at the concentration of 10.sup.6 cfu/ml, followed
by pH-control fermentation at 37.degree. C. for 18-24 hours.
pH-control was performed by using 30 volume % NaOH as a
neutralizing agent to pH 5.7.+-.0.2. Upon completion of the
culture, centrifugation was performed at 4.degree. C. with
10,000.times.g to recover cells. A protectant supplemented with 5
weight % of skim milk, 2.5 weight % of whey, and 5 weight % of
sucrose (for the total weight of the composition) was prepared.
Equal amounts of the recovered cells and the protectant were mixed,
followed by pulverization by using a freeze dryer. The produced
Lactobacillus gasseri BNR17 dried powder contained over
1.times.10.sup.11 cfu/g live cells. The protectant can additionally
include 10 weight % of trehalose, 10 weight % of maltodextrine and
7.5 weight % of lactose.
Manufacturing Example 3
Preparation of Lactic Acid Bacteria Products
Lactic acid bacteria products such as lactic acid bacteria foods
and digestives were prepared from the lactic acid bacteria powder
produced in Manufacturing Example 2. 10 weight % of
oligosaccharide, 20 weight % of anhydrous glucose, 5 weight % of
crystalline fructose, 2 weight % of vitamin C, 5 weight % of fruit
powder flavor, 5 weight % of aloe, 15 weight % of dietary fiber,
and 18 weight % of Psyllium Husk were added to 20 weight % of
Lactobacillus gasseri BNR17 dried powder, and the mixture was
packed in sticks or bottles. The live cells in the lactic acid
bacteria product prepared thereby were more than 5.times.10.sup.8
cfu/g.
Manufacturing Example 4
Preparation of a Composition for Feed Additive
A composition for feed additive containing Lactobacillus gasseri
BNR17 was prepared by the following compositions shown in Table
9.
TABLE-US-00009 TABLE 9 Component ratio of the composition for feed
additive (weight %) Lacto- Non- bacillus Enzyme pathogenic gasseri
prepa- micro- Amino BNR17 ration organism acid Others Manufacturing
100 -- -- -- -- Example <4-1> Manufacturing 90 10 -- -- --
Example <4-2> Manufacturing 80 10 10 -- -- Example
<4-3> Manufacturing 70 10 10 10 -- Example <4-4>
Manufacturing 60 15 15 8 2 Example <4-5> Manufacturing 50 20
15 8 2 Example <4-6>
The enzyme preparation used herein was a mixture of phytase,
cellulase, xylase, maltase and invertase, and the non-pathogenic
microorganism was Aspergillus oryzae.
INDUSTRIAL APPLICABILITY
As explained hereinbefore, the Lactobacillus gasseri BNR17 of the
invention has wide growth temperature and pH ranges allowed. And,
the strain of the invention not only has excellent acid resistance,
bile acid resistance and enteric adsorption capacity but also
strong antimicrobial activity against pathogenic microorganisms, in
addition to weight gaining inhibitory effect. Therefore, the strain
of the invention can be effectively used for the production of
fermented milk and other fermented products and be very useful as
an additive for animal feed as well.
SEQUENCE LISTING
Sequence listing is attached herewith.
SEQUENCE LISTINGS
1
91840DNALactobacillus gasseri BNR17misc_feature(181)..(181)n is a,
c, g, or t 1gctgactcct ataaaggtta tcccaccggc tttgggtgtt acagactctc
atggtgtgac 60gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc
gcgattacta 120gcgattccag cttcgtgtag gcgagttgca gcctacagtc
cgaactgaga acggctttca 180nagatccgct tgccttcgca ggttcgcttc
tcgttgtacc gtccattgta gcacgtgtgt 240agcccaggtc ataaggggca
tgatgacttg acgtcatccc caccttcctc cggtttgtca 300ccggcagtct
cattagagtg cccaacttaa tgatggcaac taatgacaag ggttgcgctc
360gttgcgggac ttaacccaac atctcacgac acgagctgac gacagccatg
caccacctgt 420ctcagcgtcc ccgaagggaa ctcctaatct cttaggtttg
cactggatgt caagacctgg 480taaggttctt cgcgttgctt cgaattaaac
cacatgctcc accgcttgtg cgggcccccg 540tcaattcctt tgagtttcaa
ccttgcggtc gtactcccca ggcggagtgc ttaatgcgtt 600agctgcagca
ctgagaggcg gaaacctccc aacacttagc actcatcgtt tacggcatgg
660actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc
gtcagttgca 720gaccagagag ccgccttcgc cactggtgtt cttccatata
tctacgcatt ccaccgctac 780acatggagtt ccactctcct cttctgcact
caagttcaac agtttctgat gcaattctcc 8402840DNALactobacillus gasseri
KC26 2gctgactcct ataaaggtta tcccaccggc tttgggtgtt acagactctc
atggtgtgac 60gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc
gcgattacta 120gcgattccag cttcgtgtag gcgagttgca gcctacagtc
cgaactgaga acggctttca 180gagatccgct tgccttcgca ggttcgcttc
tcgttgtacc gtccattgta gcacgtgtgt 240agcccaggtc ataaggggca
tgatgacttg acgtcatccc caccttcctc cggtttgtca 300ccggcagtct
cattagagtg cccaacttaa tgatggcaac taatgacaag ggttgcgctc
360gttgcgggac ttaacccaac atctcacgac acgagctgac gacagccatg
caccacctgt 420ctcagcgtcc ccgaagggaa ctcctaatct cttaggtttg
cactggatgt caagacctgg 480taaggttctt cgcgttgctt cgaattaaac
cacatgctcc accgcttgtg cgggcccccg 540tcaattcctt tgagtttcaa
ccttgcggtc gtactcccca ggcggagtgc ttaatgcgtt 600agctgcagca
ctgagaggcg gaaacctccc aacacttagc actcatcgtt tacggcatgg
660actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc
gtcagttgca 720gaccagagag ccgccttcgc cactggtgtt cttccatata
tctacgcatt ccaccgctac 780acatggagtt ccactctcct cttctgcact
caagttcaac agtttctgat gcaattctcc 840320DNAArtificial
SequencegaT-950 forward primer, specific to Lactobacillus gasseri
3ggagtaggtg gagcgacagt 20420DNAArtificial SequencegaT-1075 reverse
primer, specific to Lactobacillus gasseri 4tccaccagta gctgccgtta
205110DNAArtificial Sequencegassericin BNR17 gene originated from
Lactobacillus gasseri BNR17 5tgccgttacg ccagcccatg ctattggaac
atagtgtgct ccaacagagc cacaagcagg 60accgcaaact gcatttccaa gagcccgtcc
agcgactgtc gctccaccta 1106110DNAArtificial Sequencegassericin T
gene originated from Lactobacillus gasseri and disclosed in NCBI
Blast Search No. AB029612 6tgccgttacg ccagcccatg ctattggaac
atagtgtgct ccaacaaagc cacaagcagg 60accgcaaact gcatttccaa gagcccatcc
agcgactgtc gctccaccta 110710DNAArtificial Sequenceprimer p1, random
sequence according to RAPD-PCR technique 7agcagcgtgg
10810DNAArtificial Sequenceprimer p2, random sequence according to
RAPD-PCR technique 8ggcatgacct 10910DNAArtificial Sequenceprimer
OPL5, random sequence according to RAPD-PCR technique 9acgcaggcac
10
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