U.S. patent application number 14/670099 was filed with the patent office on 2015-07-16 for novel lactobacillus plantarum isolated from leaves of camellia sinensis.
The applicant listed for this patent is AMOREPACIFIC CORPORATION. Invention is credited to Jun Cheol CHO, Jin Oh CHUNG, Il Young KWACK, Bum Jin LEE, Tae-Hun PARK, Kye Ho SHIN, Se Jin YOU.
Application Number | 20150197722 14/670099 |
Document ID | / |
Family ID | 47296634 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197722 |
Kind Code |
A1 |
KWACK; Il Young ; et
al. |
July 16, 2015 |
NOVEL LACTOBACILLUS PLANTARUM ISOLATED FROM LEAVES OF CAMELLIA
SINENSIS
Abstract
Disclosed are novel Lactobacillus plantarum strains as well as a
composition containing the novel Lactobacillus plantarum strains or
a culture thereof.
Inventors: |
KWACK; Il Young; (Yongin-si,
KR) ; YOU; Se Jin; (Yongin-si, KR) ; PARK;
Tae-Hun; (Yongin-si, KR) ; LEE; Bum Jin;
(Yongin-si, KR) ; SHIN; Kye Ho; (Yongin-si,
KR) ; CHUNG; Jin Oh; (Yongin-si, KR) ; CHO;
Jun Cheol; (Yongin-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMOREPACIFIC CORPORATION |
Seoul |
|
KR |
|
|
Family ID: |
47296634 |
Appl. No.: |
14/670099 |
Filed: |
March 26, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14124483 |
Dec 6, 2013 |
|
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PCT/KR2012/004569 |
Jun 8, 2012 |
|
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14670099 |
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Current U.S.
Class: |
435/252.9 |
Current CPC
Class: |
C12P 7/56 20130101; A61K
2035/115 20130101; A23Y 2220/67 20130101; A61K 36/82 20130101; A61P
1/12 20180101; C12N 1/20 20130101; A23L 29/065 20160801; A61P 1/04
20180101; C12R 1/25 20130101; A61P 1/10 20180101; A61K 35/747
20130101 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12R 1/25 20060101 C12R001/25 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2011 |
KR |
10-2011-0056465 |
Jun 10, 2011 |
KR |
10-2011-0056466 |
Jun 10, 2011 |
KR |
10-2011-0056468 |
Jun 10, 2011 |
KR |
10-2011-0056469 |
Claims
1. A Lactobacillus plantarum strain, wherein the Lactobacillus
plantarum strain is a Lactobacillus plantarum APsulloc 331266
(Accession No.: KCCM11181P) strain, or a composition comprising the
strain.
2. The strain according to claim 1, wherein the strain is isolated
from tea tree (Camellia sinensis) leaves.
3. The strain according to claim 1, wherein the strain has acid
resistance.
4. The strain according to claim 3, wherein the strain has acid
resistance at pH 2 to pH 4 for 0.5 hour to 5 hours.
5. The strain according to claim 1, wherein the strain has bile
acid resistance.
6. The strain according to claim 1, wherein the strain has lactic
acid producing ability.
7. The strain according to claim 6, wherein the strain has lactic
acid producing ability, which produces D-type lactic acid in an
amount of 70% or less based on the produced lactic acid.
8. A composition comprising the strain according to claim 1 or a
culture solution thereof.
9. The composition according to claim 8, which is a fermented food
composition.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/124,483, filed on Dec. 6, 2012, which claims priority
to PCT Application No. PCT/KR2012/004569, filed on Jun. 8, 2012,
which claims priority to Korean Patent Application No.
10-2011-0056465, filed on Jun. 10, 2011, Korean Patent Application
No. 10-2011-0056466, filed on Jun. 10, 2011, Korean Patent
Application No. 10-2011-0056468, filed on Jun. 10, 2011, and Korean
Patent Application No. 10-2011-0056469, filed on Jun. 10, 2011, and
all the benefits accruing therefrom under 35 U.S.C. .sctn.119, the
contents of which in its entirety are herein incorporated by
reference.
TECHNICAL FIELD
[0002] The present disclosure relates to a novel Lactobacillus
plantarum isolated from tea tree (Camellia sinensis) leaves.
BACKGROUND ART
[0003] Tea for drinking is one obtained by deactivating oxygenase
present in shoots or leaves of theaceous Camellia sinensis and
removing water therefrom. It contains caffeine, tannin, flavonoids,
essential oils and the like as well as vitamins, and has been
widely used at various fields such as a food field.
DISCLOSURE
Technical Problem
[0004] The present disclosure is directed to providing a novel
Lactobacillus plantarum strain. Further, it is directed to
providing a composition including the novel Lactobacillus plantarum
strain or a culture solution thereof.
Technical Solution
[0005] In one aspect, there is provided a Lactobacillus plantarum
APsulloc 331261 strain.
[0006] In one aspect, there is provided a Lactobacillus plantarum
APsulloc 331263 strain.
[0007] In one aspect, there is provided a Lactobacillus plantarum
APsulloc 331266 strain.
[0008] In one aspect, there is provided a Lactobacillus plantarum
APsulloc 331269 strain.
[0009] In another aspect, there is provided a composition including
at least one of the Lactobacillus plantarum APsulloc 331261 strain,
the APsulloc 331263 strain, the APsulloc 331266 strain and the
APsulloc 331269 strain, or a culture solution thereof.
Advantageous Effects
[0010] The novel Lactobacillus plantarum strain according to the
present disclosure is excellent in acid resistance, and therefore,
it is also viable in the stomach when it is taken by a food, and
further, it may have higher intestinal deliverity. The
Lactobacillus plantarum strain according to the present disclosure
is excellent in bile acid resistance, and therefore, it is superior
to intestinal fix. Further, the Lactobacillus plantarum strain
according to the present disclosure is excellent in antibacterial
activity, and therefore, it is superior to harmful bacteria
inhibitory effect. The Lactobacillus plantarum strain according to
the present disclosure has lower D-lactic acid ratio in the
produced lactic acid than the existing Lactobacillus plantarum.
Accordingly, even adults or infants, who are susceptible to lactic
acid, may freely intake the strain. The Lactobacillus plantarum
strain according to the present disclosure produces fewer amount of
lactic acid than the existing Lactobacillus plantarum, and
therefore, when foods are fermented by using thereof, the foods may
have gentle flavor. Thus, the novel Lactobacillus plantarum strain
according to the present disclosure may be widely used in various
fields, for example, a food field.
BEST MODE
[0011] Lactic acid bacteria are bacteria producing lactic acid by
degrading sugar such as glucose. The lactic acid produced by lactic
acid fermentation of lactic acid bacteria may prevent growth of
pathogens and harmful bacteria, and this characteristic is used for
producing foods such as dairy foods, kimchis, brewed foods and the
like. Further, the lactic acid bacteria live in the intestine of
mammals and inhibit abnormal fermentation by various germs, and
therefore, those are important bacteria useful as a medicine for
intestinal disorders.
[0012] Lactobacillus plantarum is a strain belongs to the lactic
acid bacteria, and it is known to mainly grow when kimchi is much
fermented and tastes sour. Optical isomers of the produced lactic
acid are D-type and L-type. The Lactobacillus plantarum may be
widely used to various foods need fermentation. Accordingly, if a
Lactobacillus plantarum having excellent acid resistance, bile acid
resistance and antibacterial activity is developed, it may be
usefully used.
[0013] Hereinafter, the present disclosure now will be described in
detail.
[0014] One aspect of the present disclosure provides a
Lactobacillus plantarum APsulloc 331261 (Accession No.: KCCM11179P)
strain. One aspect of the present disclosure provides a
Lactobacillus plantarum APsulloc 331263 (Accession No.: KCCM11180P)
strain. One aspect of the present disclosure provides a
Lactobacillus plantarum APsulloc 331266 (Accession No.: KCCM11181P)
strain. One aspect of the present disclosure provides a
Lactobacillus plantarum APsulloc 331269 (Accession No.: KCCM11182P)
strain. At least one of the Lactobacillus plantarum APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269 according to the present disclosure is a strain isolated
from tea tree (Camellia sinensis) leaves, and belongs to
Lactobacillus plantarum. Specifically, the Lactobacillus plantarum
strain according to the present disclosure may be isolated by a
method containing: a step of salting the tea tree leaves in salt of
5 to 15 wt %, based on the weight of the tea tree leaves; a step of
mixing the salted tea tree leaves with a sugar solution, for
example, 0.1% to 3% fructooligosaccharide, followed by culturing at
25 to 35.degree. C. for 1 to 5 days; and a step of collecting the
solution cultured to less than pH 5, followed by culturing at
anaerobic condition of 25 to 35.degree. C. for 1 to 5 days.
[0015] At least one of the Lactobacillus plantarum APsulloc 331261,
the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269
according to one aspect of the present disclosure has excellent
acid resistance. When intaking lactic acid bacteria, for example,
Lactobacillus plantarum as a probiotic, it is preferred to have
higher intestinal deliverity, in order to display characteristic
effects of the lactic acid bacteria. In order to enhance the
intestinal deliverity, viability in the stomach, where pH is low
due to gastric acid secretion, should be high. It is known that pH
of the empty stomach may be about 1.2 to 2, but when taking foods,
pH may be about 2 to 3. The Lactobacillus plantarum strain
according to the present disclosure may have excellent acid
resistance at more than pH 2 to 4, specifically more than pH 2 to
3.5 or pH 2.5 to 4, more specifically pH 2.5 to 3.5, further more
specifically about pH 2.5 to 3. On the other hand, when intaking
foods, it is known that the average retention time of the foods may
be about 1 to 3 hours. The Lactobacillus plantarum strain according
to the present disclosure may have excellent acid resistance for
0.5 to 5 hours, specifically for 1 to 4 hours, at more than pH 2 to
pH 4, specifically more than pH 2 to pH 3.5 or pH 2.5 to 4, more
specifically pH 2.5 to 3.5, further more specifically pH 2.5 to 3.
Thus, the Lactobacillus plantarum strain according to the present
disclosure, which is viable even at lower pH of the stomach during
the time stayed in the stomach, may have high intestinal deliverity
when it is taken as a food.
[0016] The Lactobacillus plantarum strain according to one aspect
of the present disclosure has excellent bile acid resistance. The
foods passed through the stomach are delivered into the intestine,
and at this time, the secreted bile acid helps food digestion. It
is known that the strain having high bile acid resistance has good
intestinal fix. The Lactobacillus plantarum strain according to one
aspect of the present disclosure having excellent bile acid
resistance is superior to the intestinal fix.
[0017] The Lactobacillus plantarum strain according to one aspect
of the present disclosure has lactic acid producing ability. In
general, the lactic acid produced by lactic acid bacteria may be
L-type and D-type. Of them, the metabolic rate of the D-lactic acid
in the body is lower than the L-lactic acid. Accordingly, the
higher blood D-lactic acid concentration may cause lactic acid
toxicosis. Thus, it is preferred that adults or infants, who are
susceptible to lactic acid, do not take lactic acid bacteria, which
largely produces D-lactic acid, if possible.
[0018] The Lactobacillus plantarum strain according to another
aspect of the present disclosure may produce lactic acid containing
D-type in an amount of 75% or less, specifically 70% or less, more
specifically 65% or less.
[0019] The Lactobacillus plantarum strain according to further
another aspect of the present disclosure may produce lactic acid in
an amount of 17.5 g/L or less, specifically 17 g/L or less, more
specifically 16.5 g/L or less, further more specifically 15 g/L or
less, further more specifically 14.5 g/L or less, further more
specifically 14 g/L or less, further more specifically 13.5 g/L or
less.
[0020] Like this, because the Lactobacillus plantarum strain
according to the present disclosure produces lactic acid containing
D-type with lower rate than the existing Lactobacillus plantarum,
adults or infants, who are susceptible to lactic acid, may freely
take it. Further, because its lactic acid yield is lower than the
existing Lactobacillus plantarum, when fermenting foods by using
thereof, the foods may have gentler flavor.
[0021] One aspect of the present disclosure provides an extract or
a culture solution of at least one of the Lactobacillus plantarum
APsulloc 331261 strain, the APsulloc 331263 strain, the APsulloc
331266 strain and the APsulloc 331269 strain. Another aspect of the
present disclosure provides a composition containing at least one
of the Lactobacillus plantarum APsulloc 331261 strain, the APsulloc
331263 strain, the APsulloc 331266 strain and the APsulloc 331269
strain, an extract thereof, or a culture solution thereof.
[0022] One aspect of the present disclosure provides a food
composition containing at least one selected from the Lactobacillus
plantarum APsulloc 331261 strain, the APsulloc 331263 strain, the
APsulloc 331266 strain and the APsulloc 331269 strain, its extract,
or its culture solution.
[0023] The food composition may be a health food composition, and
also may be a fermented food composition, which needs fermentation,
for example, teas, dairy goods, kimchis, brewed foods.
[0024] The formulation of the food composition is not particularly
limited, but for example, it may be formulated into tablet, pill,
hard or soft capsule, granule, drink, caramel, diet bar, tea bag
and the like. The health food composition may further comprise, in
addition to the active ingredient, commonly used other ingredients,
which may be suitably selected by those skilled in the art
depending on the formulation or purpose of the composition. The
addition of the other ingredients may give a synergic effect.
[0025] Determination of the dose of the active ingredient is within
the level of those skilled in the art. For example, its daily dose
may be Lactobacillus plantarum about 10.sup.5 to 10.sup.13 CFU/day,
more specifically about 10.sup.6 to 10.sup.10 CFU/day, but is not
limited thereto, and may be varied with various factors including
the age, physical condition, complication, etc. of a subject to be
treated.
[0026] One aspect of the present disclosure provides a cosmetic
composition containing at least one of the Lactobacillus plantarum
APsulloc 331261 strain, the APsulloc 331263 strain, the APsulloc
331266 strain and the APsulloc 331269 strain, an extract thereof,
or a culture solution thereof. The cosmetic composition may be
provided as any formulation suitable for topical application. For
example, it may be provided as a formulation of oil in water
emulsion, water in oil emulation, suspension, solid, gel, powder,
paste, foam or aerosol composition. The composition of the said
formulation may prepared by a conventional method in the art.
[0027] The cosmetic composition may further include other
ingredients, which may not adversely affect a main desired effect,
preferably may provide a synergic effect to the main effect. The
cosmetic composition according to the present disclosure may
include a material selected from the group consisting of vitamin,
polymer peptide, polysaccharide and spingolipid. Further, the
cosmetic composition according to the present disclosure may
include moisturizing agent, emollient, surfactant, UV absorbing
agent, preservative, disinfecting agent, antioxidant, pH modifier,
organic and inorganic dye, aromatic, cooling agent or
antiperspirant. The amount of the ingredients may be suitable
selected in such a manner that they may not adversely affect the
purpose and effect of the present disclosure, and the amount may be
0.01 to 5 wt %, specifically 0.01 to 3 wt %, based on the total
weight of the composition.
[0028] One aspect of the present disclosure provides a
pharmaceutical composition containing at least one of the
Lactobacillus plantarum APsulloc 331261 strain, the APsulloc 331263
strain, the APsulloc 331266 strain and the APsulloc 331269 strain,
an extract thereof, or a culture solution thereof. The
pharmaceutical composition may be used for preventing or treating
intestinal disorder such as irritable bowel syndrome, constipation
and diarrhea.
[0029] The pharmaceutical composition according to one aspect of
the present disclosure may be administered orally or parenterally,
e.g., rectally, topically, transdermally, intravenously,
intramuscularly, intra-abdominally, subcutaneously, etc. Examples
of formulations for oral administration include tablet, pill, soft
or hard capsule, granule, powder, fine granule, solution, emulsion,
pellet and the like, but not limited thereto. Examples for
formulations for parenteral administration include solution,
suspension, lotion, gel, injectable solution, drop, suppository,
patch or spray, but not limited thereto. The formulation may be
easily formulated according to conventional methods, and
surfactants, excipients, wetting agent, emulsifier, suspending
agent, salt or buffer for osmotic pressure control, colorant,
flavoring agent, stabilizer, preservative, preserved agent or other
conventional additives may be suitably used for the
formulation.
[0030] Active ingredient of the pharmaceutical composition
according to one aspect of the present disclosure may be varied
with the age, sex and body weight of a subject to be treated,
pathological condition be treated, severity of the pathological
condition, administration route and the judgment of a prescriber.
Determination of the dose considering these factors is within the
level of those skilled in the art. For example, the daily dose may
be 0.1 mg/kg/day to 5000 mg/kg/day, specifically 50 mg/kg/day to
500 mg/kg/day, but not limited thereto.
[0031] The Lactobacillus plantarum APsulloc 331261, the APsulloc
331263, the APsulloc 331266 and the APsulloc 331269 are deposited
at Korean Culture Center of Microorganisms (KCCM, address: 361-221,
Yurim B/D. hongje-1-dong, Seodaemun-gu, Seoul, Republic of Korea)
on Mar. 28, 2011 under Accession Nos.: KCCM11179P, KCCM11180P,
KCCM11181P and KCCM11182P.
(1) Lactobacillus plantarum APsulloc 331261
Depository Institution Name: Korean Culture Center of
Microorganisms
Accession No.: KCCM11179P
Deposition Date: 2011.03.28
[0032] (2) Lactobacillus plantarum APsulloc 331263
Depository Institution Name: Korean Culture Center of
Microorganisms
Accession No.: KCCM11180P
Deposition Date: 2011.03.28
[0033] (3) Lactobacillus plantarum APsulloc 331266
Depository Institution Name: Korean Culture Center of
Microorganisms
Accession No.: KCCM11181P
Deposition Date: 2011.03.28
[0034] (4) Lactobacillus plantarum APsulloc 331269
Depository Institution Name: Korean Culture Center of
Microorganisms
Accession No.: KCCM11182P
Deposition Date: 2011.03.28
[0035] Hereinafter, isolation methods, identification methods and
characteristics of the novel Lactobacillus plantarum strains
according to the present disclosure now will be described in detail
with reference to the examples (and experiments). However, the
following examples (and experiments) are for illustrative purposes
only and not intended to limit the scope of this disclosure.
Example 1
Isolation of Lactobacillus Plantarum Strain
[0036] Tea tree leaves 200 g are washed twice with distilled water
to remove impurities. Moisture is cleared off from the washed tea
tree leaves. Then, the tea tree leaves are mixed with table salt of
8 wt %, based on the weight of the tea tree leaves, and then stored
at room temperature for 3 hours. The salted tea tree leaves are
mixed with 1% fructooligosaccharide solution 1000 mL, and then
incubated in an incubator at 32.degree. C. for 3 days. 3 days
later, whether pH of the cultured solution is lowered to less than
5 is checked, and in the case of less than pH 5, the cultured
solution is collected and incubated in Difco Lactobacilli MRS
Agar.RTM. medium. At this time, the incubation is conducted in a
32.degree. C., anaerobic chamber for 2 days, and then white
colonies are collected.
[0037] Through the method described above, Lactobacillus plantarum
APsulloc 331261, APsulloc 331263, APsulloc 331266 and APsulloc
331269 are isolated from tea tree leaves, respectively.
Example 2
Identification of Lactobacillus Plantarum Strain
(1) Strain Culture
[0038] The APsulloc 331261 isolated in Example 1 is streaked on a
MRS agar plate, and cultured at 37.degree. C. for 2 days. The
obtained single colony is inoculated to MRS broth 10 mL, and then
cultured at 37.degree. C. overnight to prepare a Lactobacillus
plantarum strain culture solution. The method described above is
repeated to the APsulloc 331263, the APsulloc 331266 and the
APsulloc 331269, respectively, to prepare Lactobacillus plantarum
strain culture solutions.
(2) Analysis of Sugar Fermentation Pattern of Lactobacillus
Plantarum Strain
[0039] The APsulloc 331261 strain culture solution prepared as
described in (1) is inoculated to MRS broth 10 mL to the
concentration of 0.5% and cultured at 37.degree. C. overnight. The
culture solution is centrifuged at 8,000 rpm for 5 min, supernatant
is removed, and then only bacteria are collected. Then, 0.85%
saline buffer 2 mL is added to the bacteria and suspended. Later
process is conducted by using API 50CHL kit (Biomerieux) according
to a manufacturer's protocol. Specific process is as follows.
[0040] First of all, while gradually adding the strain suspension
to API suspension medium 5 mL, the amount of suspension needed to
make cloudiness of about McFarland Standard 2 (Biomerieux) is
measured. Twice of the measured amount of the suspension is added
to API 50CHL medium 10 mL, and then shaken for mixing. The above
mixture is added to cupules containing different substrate, one
drop of mineral oil is put thereto, and then the mixture is
cultured at 37.degree. C. for 2 days to analyze sugar fermentation
pattern. The method described above is repeated to culture
solutions of the APsulloc 331263, the APsulloc 331266 and the
APsulloc 331269 strain to analyze sugar fermentation pattern.
[0041] The results of sugar fermentation patterns of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269, compared with Lactobacillus plantarum strain (KCTC3108) as
a standard strain, and the results of identification of the
APsulloc 331261, the APsulloc 331263, the APsulloc 331266 and the
APsulloc 331269 by using the above results are as shown in the
following Tables. Table 1: APsulloc 331261, Table 2: APsulloc
331263, Table 3: APsulloc 331266 and Table 4: APsulloc 331269.
TABLE-US-00001 TABLE 1 APsulloc APsulloc KCTC3108 331261 KCTC3108
331261 Substrate 24 h 48 h 24 h 48 h substrate 24 h 48 h 24 h 48 h
Control - - - - Esculin + + + + Glycerol - - - - Salicin + + + +
Erythritol - - - - Cellobiose ? + + + D-Arabinose - - - - Maltose +
+ + + L-Arabinose + + + + Lactose + + + + Ribose + + + + Melibiose
+ + + + D-Xylose - - - - D-Saccharose + + + + (Sucrose) L-xylose -
- - - Trehalose + + + + Adonitol - - - - Inulin - - - -
.beta.-Methyl- - - - - Melezitose + + + + D-xylose Galactose + + +
+ Raffinose - - + + Glucose + + + + Amidon (Starch) - - - -
Fructose + + + + Glycogen - - - - Mannose + + + + Xylitol - - - -
Sorbose - - - - Gentiobiose - - + + Rhamnose - - - - D-Turanose + +
+ + Dulcitol - - - - D-Lyxose - - - - Inositol - - - - D-Tagatose -
- - - Mannitol + + + + D-Fucose - - - - Sorbitol + + + + L-Fucose -
- - - .alpha.-Methyl-D- ? + - - D-Arabitol ? - ? - mannoside
.alpha.-Methyl-D- - - - - L-Arabitol - - - - glucoside N-Acetyl- +
+ + + Gluconic acid ? + ? + glucosamine Amygdalin + + + +
2-Ketogluconate - - - - Arbutin + + + + 5-Ketogluconate - - - -
TABLE-US-00002 TABLE 2 APsulloc APsulloc KCTC3108 331263 KCTC3108
331263 Substrate 24 h 48 h 24 h 48 h Substrate 24 h 48 h 24 h 48 h
Control - - - - Esculin + + + + Glycerol - - - - Salicin + + + +
Erythritol - - - - Cellobiose ? + + + D-Arabinose - - - - Maltose +
+ + + L-Arabinose + + + + Lactose + + + + Ribose + + + + Melibiose
+ + + + D-Xylose - - - - D-Saccharose + + + + (Sucrose) L-Xylose -
- - - Trehalose + + + + Adonitol - - - - Inulin - - - -
.beta.-Methyl- - - - - Melezitose + + + + D-xylose Galactose + + +
+ Raffinose - - ? + Glucose + + + + Amidon (Starch) - - - -
Fructose + + + + Glycogen - - - - Mannose + + + + Xylitol - - - -
Sorbose - - - - Gentiobiose - - + + Rhamnose - - - - D-Turanose + +
- - Dulcitol - - - - D-Lyxose - - - - Inositol - - - - D-Tagatose -
- - - Mannitol + + + + D-Fucose - - - - Sorbitol + + + + L-Fucose -
- - - .alpha.-Methyl-D- ? + - - D-Arabitol ? - - - mannoside
.alpha.-Methyl-D- - - - - L-Arabitol - - - - glucoside N-Acetyl- +
+ + + Gluconic acid ? + + + glucosamine Amygdalin + + + +
2-Ketogluconate - - - - Arbutin + + + + 5-Ketogluconate - - - -
TABLE-US-00003 TABLE 3 APsulloc APsulloc KCTC3108 331266 KCTC3108
331266 Substrate 24 h 48 h 24 h 48 h Substrate 24 h 48 h 24 h 48 h
Control - - - - Esculin + + + + Glycerol - - - - Salicin + + + +
Erythritol - - - - Cellobiose ? + + + D-Arabinose - - - - Maltose +
+ + + L-Arabinose + + + + Lactose + + + + Ribose + + + + Melibiose
+ + + + D-Xylose - - - - D-Saccharose + + + + (Sucrose) L-Xylose -
- - - Trehalose + + + + Adonitol - - - - Inulin - - - -
.beta.-Methyl- - - - - Melezitose + + + + D-xylose Galactose + + +
+ Raffinose - - + + Glucose + + + + Amidon (Starch) - - - -
Fructose + + + + Glycogen - - - - Mannose + + + + Xylitol - - - -
Sorbose - - - - Gentiobiose - - + + Rhamnose - - - - D-Turanose + +
+ + Dulcitol - - - - D-Lyxose - - - - Inositol - - - - D-Tagatose -
- - - Mannitol + + + + D-Fucose - - - - Sorbitol + + + + L-Fucose -
- - - .alpha.-Methyl-D- ? + - - D-Arabitol ? - - - Mannoside
.alpha.-Methyl-D- - - - - L-Arabitol - - - - glucoside N-Acetyl- +
+ + + Gluconic acid ? + + + glucosamine Amygdalin + + + +
2-Ketogluconate - - - - Arbutin + + + + 5-Ketogluconate - - - -
TABLE-US-00004 TABLE 4 APsulloc APsulloc KCTC3108 331269 KCTC3108
331269 Substrate 24 h 48 h 24 h 48 h Substrate 24 h 48 h 24 h 48 h
Control - - - - Esculin + + + + Glycerol - - - - Salicin + + + +
Erythritol - - - - Cellobiose ? + + + D-Arabinose - - - - Maltose +
+ + + L-Arabinose + + - - Lactose + + + + Ribose + + + + Melibiose
+ + + + D-Xylose - - + + D-Saccharose + + + + (Sucrose) L-Xylose -
- - - Trehalose + + + + Adonitol - - - - Inulin - - - -
.beta.-Methyl- - - - - Melezitose + + + + D-xylose Galactose + + +
+ Raffinose - - + + Glucose + + + + Amidon (Starch) - - - -
Fructose + + + + Glycogen - - - - Mannose + + + + Xylitol - - - -
Sorbose - - - - Gentiobiose - - + + Rhamnose - - - - D-Turanose + +
+ + Dulcitol - - - - D-Lyxose - - - - Inositol - - - - D-Tagatose -
- - - Mannitol + + + + D-Fucose - - - - Sorbitol + + + + L-Fucose -
- - - .alpha.-Methyl-D- ? + ? + D-Arabitol ? - - - mannoside
.alpha.-Methyl-D- - - - - L-Arabitol - - - - glucoside N-Acetyl- +
+ + + Gluconic acid ? + ? + glucosamine Amygdalin + + + +
2-Ketogluconate - - - - Arbutin + + + + 5-Ketogluconate - - - - +:
Substrate is degraded, -: Substrate is not degraded, ?: Unable to
determine
TABLE-US-00005 TABLE 5 Strain Name % Index T Index KCTC
Lactobacillus plantarum 99.9 0.8 3108 Lactobacillus pentosus 0.1
0.29 APsulloc Lactobacillus plantarum 99.4 0.99 331261
Lactobacillus pentosus 0.4 0.71 APsulloc Lactobacillus plantarum
98.9 0.97 331263 Lactobacillus pentosus 0.7 0.71 APsulloc
Lactobacillus plantarum 99.4 0.99 331266 Lactobacillus pentosus 0.4
0.71 APsulloc Lactobacillus plantarum 99.4 0.79 331269
Lactobacillus pentosus 0.5 0.51
[0042] As can be seen from the above, all of the APsulloc 331261,
the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269
show the consistency (% index) to the Lactobacillus plantarum of
99% or more. Accordingly, it is confirmed that those strains are
belong to the Lactobacillus plantarum.
[0043] Further, compared with the standard strain (KCTC3108), the
APsulloc 331261 is different in use of .alpha.-methyl-mannoside and
raffinose, the APsulloc 331263 is different in use of
.alpha.-methyl-mannoside, raffinose and D-turanose, the APsulloc
331266 is different in use of .alpha.-methyl-mannoside and
raffinose, and the APsulloc 331269 is different in use of
L-arabinose and raffinose. Accordingly, it is confirmed that all of
them are different strains from the standard strain.
(3) Analysis of Enzyme Activity Pattern of Lactobacillus Plantarum
Strain
[0044] The APsulloc 331261 strain culture solution prepared as
described in (1) is inoculated to MRS broth 10 mL to the
concentration of 0.5% and cultured at 37.degree. C. overnight. The
culture solution is centrifuged at 8,000 rpm for 5 min, supernatant
is removed, and then only bacteria are collected. Then, 0.85%
saline buffer 2 mL is added to the bacteria and suspended. Later
process is conducted by using API ZYM kit (Biomerieux) according to
a manufacturer's protocol. Specific process is as follows.
[0045] First of all, while gradually adding the strain suspension
to API suspension medium 5 mL, the amount of suspension needed to
make cloudiness of about McFarland Standard 2 (Biomerieux) is
measured. Twice of the measured amount of the suspension is added
to API 50CHL medium 10 mL, and then shaken for mixing. The above
mixture 65 .mu.l is added the each cupule, and cultured at
37.degree. C. for 4 hours. Each one drop of ZYM A reagent and ZYM B
reagent is put into each cupule, and 5 min later, scored from 0 to
5 according to the color intensity, and then the score 3 or more is
decided as positive.
[0046] The method described above is repeated to the APsulloc
331263, the APsulloc 331266 and the APsulloc 331269 strain culture
solution to analyze enzyme activity patter.
[0047] The results of enzyme activity patterns of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269, compared with Lactobacillus plantarum strain (KCTC3108) as
a standard strain are as shown in the following Tables. Table 6:
APsulloc 331261, Table 7: APsulloc 331263, Table 8: APsulloc 331266
and Table 9: APsulloc 331269.
TABLE-US-00006 TABLE 6 KCTC3108 APsulloc 331261 Enzyme Score Result
Score Result Control 0 - 0 - Alkaline phosphatase 0 - 1 - Esterase
1 - 2 - Esterase lipase 1 - 2 - Lipase 0 - 2 - Leucine arylamidase
5 + 4 + Valine arylamidase 4 + 4 + Cystine arylamidase 1 - 2 -
Trypsin 0 - 0 - .alpha.-Chymotrypsin 0 - 1 - Acid phosphatease 1 -
3 + Naphthol-AS-BI-phosphohydrolase 1 - 3 + .alpha.-Galactosidase 1
- 3 + .beta.-Galactosidase 5 + 5 + .beta.-Glucuronidase 1 - 2 -
.beta.-Glucosidase 3 + 5 + N-Acetyl-.beta.-glucosaminidase 0 - 4 +
.alpha.-Mannosidase 0 - 1 - .alpha.-Fucosidase 0 - 1 -
TABLE-US-00007 TABLE 7 KCTC3108 APsulloc 331263 Enzyme Score Result
Score Result Control 0 - 0 - Alkaline phosphatase 0 - 1 - Esterase
1 - 2 - Esterase lipase 1 - 2 - Lipase 0 - 1 - Leucine arylamidase
5 + 4 + Valine arylamidase 4 + 4 + Cystine arylamidase 1 - 2 -
Trypsin 0 - 0 - .alpha.-Chymotrypsin 0 - 1 - Acid phosphatease 1 -
3 + Naphthol-AS-BI-phosphohydrolase 1 - 3 + .alpha.-galactosidase 1
- 3 + .beta.-galactosidase 5 + 5 + .beta.-glucuronidase 1 - 2 -
.alpha.-glucosidase 3 + 2 - .beta.-glucosidase 3 + 5 +
N-acetyl-.beta.-glucosaminidase 0 - 4 + .alpha.-Mannosidase 0 - 1 -
.alpha.-Fucosidase 0 - 1 -
TABLE-US-00008 TABLE 8 KCTC3108 APsulloc 331266 Enzyme Score Result
Score Result Control 0 - 0 - Alkaline phosphatase 0 - 1 - Esterase
1 - 1 - Esterase lipase 1 - 1 - Lipase 0 - 1 - Leucine arylamidase
5 + 4 + Valine arylamidase 4 + 3 + Cystine arylamidase 1 - 2 -
Trypsin 0 - 1 - .alpha.-Chymotrypsin 0 - 1 - Acid phosphatease 1 -
3 + Naphthol-AS-BI-phosphohydrolase 1 - 3 + .alpha.-galactosidase 1
- 3 + .beta.-galactosidase 5 + 5 + .beta.-glucuronidase 1 - 2 -
.beta.-glucosidase 3 + 5 + N-acetyl-.beta.-glucosaminidase 0 - 4 +
.alpha.-Mannosidase 0 - 1 + .alpha.-Fucosidase 0 - 0 -
TABLE-US-00009 TABLE 9 KCTC3108 APsulloc 331269 Enzyme Score Result
Score Result Control 0 - 0 - Alkaline phosphatase 0 - 1 - Esterase
1 - 1 - Esterase lipase 1 - 1 - Lipase 0 - 1 - Leucine arylamidase
5 + 4 + Valine arylamidase 4 + 3 + Cystine arylamidase 1 - 2 -
Trypsin 0 - 1 - .alpha.-Chymotrypsin 0 - 1 - Acid phosphatease 1 -
3 + Naphthol-AS-BI-phosphohydrolase 1 - 3 + .alpha.-galactosidase 1
- 3 + .beta.-galactosidase 5 + 5 + .beta.-glucuronidase 1 - 2 -
.beta.-glucosidase 3 + 5 + N-acetyl-.beta.-glucosaminidase 0 - 4 +
.alpha.-Mannosidase 0 - 1 - .alpha.-Fucosidase 0 - 0 -
[0048] As can be seen from the above, all of the APsulloc 331261,
the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269
have different enzyme activity intensities of acid phosphatease,
naphthol-AS-BI-phosphohydrolase, .alpha.-galactosidase and
N-acetyl-.beta.-glucosaminidase from the standard strain
(KCTC3108). Accordingly, it is confirmed that all of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269 are different strains from the standard strain.
[0049] Further, the APsulloc 331261, the APsulloc 331263, the
APsulloc 331266 and the APsulloc 331269 are decided as negative
against the .beta.-glucuronidase activity known as one of
representative oncogenic enzymes, which can induce cancer by
modifying procarcinogen to carcinogen in the intestine. In
addition, the APsulloc 331263 inhibits the .alpha.-glucosidase
activity. Accordingly, it is confirmed that all of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269 are acceptable to be used to a food composition.
(4) Evaluation of Antibiotic Resistance of Lactobacillus Plantarum
Strain
[0050] Sterilized MRS agar 20 mL is added to a petri dish
(Diameter: 100 mm), and cooled in a clean bench to prepare a
medium. The APsulloc 331261 strain culture solution prepared in (1)
is inoculated to MRS broth 10 mL to the concentration of 0.5%, and
then cultured at 37.degree. C. for 6 hours. The resulting solution
is diluted to have absorbance of about 0.08 to 0.13 at 625 nm. A
sterilized cotton swab is fully soaked in the diluted solution, and
then streaked evenly on the prepared MRS agar plate overall. An
antibiotic sensitivity disk is dropped on the plate with a proper
distance. After culturing at 37.degree. C. for 24 hours, diameter
of a clear zone is measured.
[0051] The method described above is repeated to the APsulloc
331263, the APsulloc 331266 and the APsulloc 331269 strain culture
solutions to evaluate antibiotic resistance.
[0052] Concentration of the antibiotic sensitivity disk and
evaluation standard are as follows.
TABLE-US-00010 TABLE 10 Antibiotics Zone Diameter Analysis
Ingredient Resistant Interme- Susceptible Name Concentration (R)
diate (I) (S) Ampicillin 10 .mu.g .ltoreq.13 14-16 .gtoreq.17
Ceftazidime 30 .mu.g .ltoreq.14 15-17 .gtoreq.18 Chloramphenicol 30
.mu.g .ltoreq.12 13-17 .gtoreq.18 Ciprofloxacin 5 .mu.g .ltoreq.15
16-20 .gtoreq.21 Clindamycin 2 .mu.g .ltoreq.14 15-20 .gtoreq.21
Erythromycin 15 ug .ltoreq.13 14-22 .gtoreq.23 Gentamycin 120 .mu.g
.ltoreq.6 7-9 .gtoreq.10 Imipenem 10 .mu.g .ltoreq.13 14-15
.gtoreq.16 Streptomycin 10 .mu.g .ltoreq.11 12-14 .gtoreq.15
Neomycin 30 .mu.g .ltoreq.12 13-16 .gtoreq.17 Nitrofurantoin 300
.mu.g .ltoreq.14 15-16 .gtoreq.17 Penicillin 10 U .ltoreq.14 --
.gtoreq.15 Polymyxin B 300 U .ltoreq.8 9-11 .gtoreq.12 Tetracycline
30 .mu.g .ltoreq.14 15-18 .gtoreq.19 Trimethoprim 5 .mu.g
.ltoreq.10 11-15 .gtoreq.16 Vancomycin 30 .mu.g .ltoreq.14 15-16
.gtoreq.17
[0053] The results of antibiotic resistance evaluation of the
APsulloc 331261, the APsulloc 331263, the APsulloc 331266 and the
APsulloc 331269, compared with Lactobacillus plantarum strain
(KCTC3108) as a standard strain, are as follows. Table 11: APsulloc
331261 and APsulloc 331263, and Table 12: APsulloc 331266 and
APsulloc 331269.
TABLE-US-00011 TABLE 11 KCTC3108 APsulloc 331261 APsulloc 331263
Zone Zone Zone Antibiotics (mm) Result (mm) Result (mm) Result
Ampicillin 29 S 27 S 31 S Ceftazidime 23 S 16 I 15 I
Chloramphenicol 24 S 24 S 25 S Ciprofloxacin - R - R - R
Clindamycin 12 R 29 S 8 R Erythromycin 26 S 25 S 27 S Gentamycin 20
S 19 S 19 S Imipenem 39 S 39 S 42 S Streptomycin - R - R - R
Neomycin 11 R 9 R 9 R Nitrofurantoin - R 27 S 29 S Penicillin 24 S
18 S 19 S Polymyxin B - R - R - R Tetracycline 17 I 17 I 17 I
Trimethoprim - R - R - R Vancomycin - R - R - R
TABLE-US-00012 TABLE 12 KCTC3108 APsulloc 331266 APsulloc 331269
Zone Zone Zone Antibiotics (mm) Result (mm) Result (mm) Result
Ampicillin 29 S 21 S 21 S Ceftazidime 23 S 10 R 10 I
Chloramphenicol 24 S 22 S 22 S Ciprofloxacin - R - R - R
Clindamycin 12 R 9 R 9 S Erythromycin 26 S 28 S 28 S Gentamycin 20
S 18 S 18 S Imipenem 39 S 39 S 39 S Streptomycin - R - R - R
Neomycin 11 R 9 R 9 R Nitrofurantoin - R 27 S 27 S Penicillin 24 S
12 R 12 R Polymyxin B - R - R - R Tetracycline 17 I 17 I 17 I
Trimethoprim - R - R - R Vancomycin - R - R - R R: Resistant, I:
Intermediate, S: Susceptible
[0054] As can be seen from the above, compared with the standard
strain (KCTC3108), the APsulloc 331261 is different in antibiotic
resistance pattern of ceftazidime, clindamycin and nitrofurantoin,
and the APsulloc 331263 is different in antibiotic resistance
pattern of ceftazidime and nitrofurantoin. Compared with the
standard strain (KCTC3108), the APsulloc 331266 is different in
antibiotic resistance pattern of ceftazidime, nitrofurantoin and
penicillin, and the APsulloc 331269 is different in antibiotic
resistance pattern of ceftazidime, clindamycin, nitrofurantoin and
penicillin. Accordingly, it is confirmed that all of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269 are different strains from the standard strain.
[0055] When analyzing sugar fermentation pattern, enzyme activity
pattern and antibiotic resistance pattern overall, it is confirmed
that all of the APsulloc 331261, the APsulloc 331263, the APsulloc
331266 and the APsulloc 331269 are belong to the Lactobacillus
plantarum, and are different strains from the Lactobacillus
plantarum standard strain (KCTC3108).
Test Example 1
Acid Resistance Evaluation
(1) Acid Resistance Evaluation Depending on pH
[0056] The APsulloc 331261 strain culture solution is inoculated to
MRS broth 10 mL to the concentration of 0.5%, and cultured at
37.degree. C. overnight. pH is controlled to 2.0, 2.5, 3.0, 3.5,
respectively, with HCl. The culture solution 50 .mu.l is inoculated
to sterilized MRS broth 5 mL, and then cultured at 37.degree. C.
for 1 hour. After 1 hour, the culture solution is diluted with
peptone saline buffer solution to measure the number of bacteria
per mL. After measuring the number of bacteria in the culture
solution, viability is calculated by considering the value
multiplying the number by 0.01 as control, and considering the
number of control bacteria as 100%.
[0057] The method described above is repeated to the APsulloc
331263, the APsulloc 331266 and the APsulloc 331269 strain culture
solutions to evaluate acid resistance depending on pH.
[0058] The results of comparing acid resistance of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269 with the Lactobacillus plantarum strain (KCTC3108) as a
standard strain are shown in the following Tables. Table 13:
APsulloc 331261 and APsulloc 331263, and Table 14: APsulloc 331266
and APsulloc 331269.
TABLE-US-00013 TABLE 13 KCTC3108 APsulloc 331261 APsulloc 331263
Viability Viability Viability pH cfu/ml (%) cfu/ml (%) cfu/ml (%)
Control 2.9 .times. 10.sup.7 100 3.8 .times. 10.sup.7 100 1.9
.times. 10.sup.7 100 2.0 3.0 .times. 10.sup.1 0 1.0 .times.
10.sup.1 0 <10.sup.1 0 2.5 2.5 .times. 10.sup.7 87.4 3.3 .times.
10.sup.7 87.7 1.4 .times. 10.sup.7 76.6 3.0 3.2 .times. 10.sup.7
110.3 3.7 .times. 10.sup.7 110.3 1.8 .times. 10.sup.7 94.7 3.5 2.8
.times. 10.sup.7 97.7 4.0 .times. 10.sup.7 105.3 1.8 .times.
10.sup.7 93.4
TABLE-US-00014 TABLE 14 KCTC3108 APsulloc 331266 APsulloc 331269
Viability Viability Viability pH cfu/ml (%) cfu/ml (%) cfu/ml (%)
Control 2.9 .times. 10.sup.7 100 5.0 .times. 10.sup.7 100 5.0
.times. 10.sup.7 100 2.0 3.0 .times. 10.sup.1 0 <10.sup.1 0 3.6
.times. 10.sup.2 0 2.5 2.5 .times. 10.sup.7 87.4 6.0 .times.
10.sup.7 118.2 5.6 .times. 10.sup.7 130.7 3.0 3.2 .times. 10.sup.7
110.3 -- -- 6.5 .times. 10.sup.7 112.7 3.5 2.8 .times. 10.sup.7
97.7 5.8 .times. 10.sup.7 114.2 6.2 .times. 10.sup.7 124.0
[0059] As can be seen from the above, all of the APsulloc 331261,
the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269
show higher viability at about pH 2.5 to 3.5 than the standard
strain. Namely, it is confirmed that the above Lactobacillus
plantarum strains have excellent acid resistance. When considering
pH of the stomach with foods is about 2 to 3, it is confirmed that
the Lactobacillus plantarum strains may have higher viability in
the stomach when contained in the food composition.
(2) Acid Resistance Evaluation Depending on Time
[0060] The APsulloc 331261 culture solution is inoculated to MRS
broth 10 mL to the concentration of 0.5%, and cultured at
37.degree. C. overnight. The culture solution 50 .mu.l, which is
controlled to pH 2.5 with HCl, is inoculated to sterilized MRS
broth 5 mL, and cultured at 37.degree. C. for 3 hours. After each 1
hour and 3 hours, the culture solution is diluted with peptone
saline buffer solution, and then the number of bacteria per mL is
measured. After measuring the number of bacteria in the culture
solution, viability is calculated by considering the value
multiplying the number by 0.01 as control, and considering the
number of control bacteria as 100%.
[0061] The method described above is repeated to the APsulloc
331263, the APsulloc 331266 and the APsulloc 331269 strain culture
solutions to evaluate acid resistance depending on time.
[0062] The results of comparing acid resistance of the APsulloc
331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc
331269 with the Lactobacillus plantarum strain (KCTC3108) as a
standard strain are shown in the following Tables. Table 15:
APsulloc 331261 and APsulloc 331263, and Table 16: APsulloc 331266
and APsulloc 331269.
TABLE-US-00015 TABLE 15 KCTC3108 APsulloc 331261 APsulloc 331263
Time Viability Viability Viability (hr) cfu/ml (%) cfu/ml (%)
cfu/ml (%) 0 3.3 .times. 10.sup.7 100 6.8 .times. 10.sup.7 100 3.4
.times. 10.sup.7 100 1 2.9 .times. 10.sup.7 86.9 6.6 .times.
10.sup.7 98 2.9 .times. 10.sup.7 87.2 3 2.2 .times. 10.sup.7 67.3
6.4 .times. 10.sup.7 94.6 2.7 .times. 10.sup.7 79.9
TABLE-US-00016 TABLE 16 KCTC3108 APsulloc 331266 APsulloc 331269
Time Viability Viability Viability (hr) cfu/ml (%) cfu/ml (%)
cfu/ml (%) 0 3.3 .times. 10.sup.7 100 6.2 .times. 10.sup.7 100 7.1
.times. 10.sup.7 100 1 2.9 .times. 10.sup.7 86.9 5.7 .times.
10.sup.7 91.4 7.6 .times. 10.sup.7 107.1 3 2.2 .times. 10.sup.7
67.3 5.8 .times. 10.sup.7 92.5 5.6 .times. 10.sup.7 78.8
[0063] A can be seen from the above, it is confirmed that all of
the APsulloc 331261, the APsulloc 331263, the APsulloc 331266 and
the APsulloc 331269 have higher viability at pH 2.5 even after 1
hour and 3 hours, respectively, than the standard strain. Namely,
it is confirmed that all of the above Lactobacillus plantarum
strains have excellent acid resistance for a long time. When
considering average retention time of the intaken foods in the
stomach is about 1 to 3 hours, it is confirmed that the
Lactobacillus plantarum strains may have higher viability during
the time stayed in the stomach when contained in the food
composition.
[0064] Thus, all of the APsulloc 331261, the APsulloc 331263, the
APsulloc 331266 and the APsulloc 331269 are viable even at lower pH
of the stomach during the time stayed in the stomach. Accordingly,
when the strains are intaken as a food, those are viable in the
stomach, and further, have high intestinal deliverity.
Test Example 2
Bile Acid Resistance Evaluation
[0065] The APsulloc 331261 culture solution is inoculated to MRS
broth 10 mL to the concentration of 0.5%, and cultured at
37.degree. C. overnight. Ox gall is added thereto to the
concentration of 0.3% and 0.5%, respectively, to prepare a MRS agar
plate. MRS agar where the ox gall is not added is used as control.
After diluting the strain culture solution and streaking on MRS
agar medium, the plate is cultured at 37.degree. C. for 2 days. The
results of calculating the number of each colony and the viability
(%) of the APsulloc 331261 when considering the number of control
bacteria as 100% are shown in the following Tables. The method
described above is repeated to the APsulloc 331263, the APsulloc
331266 and the APsulloc 331269 strain culture solutions to evaluate
bile acid resistance, and then the results thereof are shown in the
following Tables. Table 17: APsulloc 331261 and APsulloc 331266,
and Table 18: APsulloc 331263 and APsulloc 331269.
TABLE-US-00017 TABLE 17 Ox Gall APsulloc 331261 APsulloc 331266
Concentration (%) cfu/ml Viability (%) cfu/ml Viability (%) 0 3.3
.times. 10.sup.9 100 3.3 .times. 10.sup.9 100 0.3 3.2 .times.
10.sup.9 96.5 3.2 .times. 10.sup.9 96.8 0.5 3.0 .times. 10.sup.9 89
3.0 .times. 10.sup.9 85
TABLE-US-00018 TABLE 18 Ox Gall KCTC3108 APsulloc 331263 APsulloc
331269 Concentration Viability Viability Viability (%) cfu/ml (%)
cfu/ml (%) cfu/ml (%) 0 3.3 .times. 10.sup.9 100 3.3 .times.
10.sup.9 100 3.3 .times. 10.sup.9 100 0.3 3.2 .times. 10.sup.9 97
3.2 .times. 10.sup.9 111.9 3.2 .times. 10.sup.9 98.1 0.5 3.0
.times. 10.sup.9 91.9 3.0 .times. 10.sup.9 106.9 3.0 .times.
10.sup.9 95.3
[0066] As can be seen from the above, all of the APsulloc 331261,
the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269
show excellent bile acid resistance. It is known that the strain
having excellent bile acid resistance is also excellent in
intestinal fix. Accordingly, it is confirmed that all of the
APsulloc 331261, the APsulloc 331263, the APsulloc 331266 and the
APsulloc 331269 have excellent intestinal fix and intestinal
deliverity.
Test Example 3
Lactic Acid Producing Ability Evaluation
[0067] The APsulloc 331261 strain culture solution is inoculated to
MRS broth 10 mL to the concentration of 0.5% and cultured at
37.degree. C. overnight. The culture solution is centrifuged at
8,000 rpm for 15 min, only supernatant is collected. The collected
supernatant is treated at 80.degree. C. for 15 min to stop enzyme
reaction. The heat treated supernatant is diluted 100 times with
distilled water. Later process is conducted by using D-lactic
acid/L-lactic acid UV method kit (R-biopharm) according to a
manufacturer's protocol. Specific process is as follows
[0068] Kit solution 1 (glycylglycine buffer/L-glutamate) 1 mL,
solution 2 (NAD solution) 0.2 mL and GPT suspension solution 3 0.02
mL are sequentially added to a cuvette. The supernatant prepared
above 0.1 mL is added to the cuvette. Deionized water 1 mL is added
to control, and deionized water 0.9 mL is added to the sample
followed by well mixing thereof. After 5 min, absorbance (A1) is
measured at 340 nm. D-LDH solution 4 0.02 mL is added thereto, and
mixed well followed by reacting for 30 min. Absorbance (A2) is
measured at 340 nm. L-LDH solution 5 0.02 mL is added thereto, and
mixed well followed by reacting for 30 min. Absorbance (A3) is
measured at 340 nm. The concentrations of D-lactic acid and
L-lactic acid in samples are calculated according to a calculating
method. The results compared with the Lactobacillus plantarum
strain (KCTC3108) as a standard strain are shown in the following
Table.
[0069] The method described above is repeated to the APsulloc
331263, the APsulloc 331266 and the APsulloc 331269 strain culture
solutions to evaluate lactic acid producing ability, and the
results thereof are shown in the following Tables. Table 19:
APsulloc 331261 and APsulloc 331263, and Table 20: APsulloc 331266
and APsulloc 331269.
TABLE-US-00019 TABLE 19 KCTC3108 APsulloc 331261 APsulloc 331263
Lactic Conc. Ratio Conc. Ratio Conc. Ratio acid (g/L) (%) (g/L) (%)
(g/L) (%) D-type 11.3 72 8.9 62 9.2 69 L-type 4.3 28 5.5 38 4.2 31
Total 15.6 100 14.4 100 13.4 100
TABLE-US-00020 TABLE 20 APsulloc 331266 APsulloc 331269 Lactic
KCTC3108 Conc. Ratio Conc. Ratio acid Ratio (%) (g/L) (%) (g/L) (%)
D-type 72 10.2 62 11.7 69 L-type 28 6.3 38 5.2 31 Total 100 16.5
100 16.9 100
[0070] As can be seen from the above, all of the APsulloc 331263,
the APsulloc 331266 and the APsulloc 331269 produce D-lactic acid
and L-lactic acid. Further, the total lactic acid yields of them
are smaller than the standard strain, and the ratio of the D-lactic
acid to the produced lactic acid is low. Accordingly, foods
containing the Lactobacillus plantarum strains may be freely taken
to adults or infants, who are susceptible to lactic acid, and when
foods are fermented by using the Lactobacillus plantarum strains,
the foods may have gentler flavor.
Test Example 4
Antibacterial Activity Evaluation
(1) APsulloc 331261
[0071] The APsulloc 331261 culture solution is inoculated to MRS
broth 10 mL to the concentration of 0.5%, and cultured at
37.degree. C. overnight. Sterilized MRS agar 15 mL is aliquoted to
a petri dish to prepare a medium, and then each 1 .mu.l of the
APsulloc 331261 culture solution is spotted thereon, and cultured
at 37.degree. C. for 24 hours.
[0072] On the other hand, Shigella flexneri is streaked on Tryptic
soy agar, and cultured at 37.degree. C. overnight. Then a colony is
inoculated to BHI broth and cultured overnight. BHI soft agar (agar
1%) is sterilized and cooled to about 45 to 50.degree. C., and then
Shigella flexneri culture solution 1% is inoculated thereto.
[0073] The Shigella flexneri culture solution 10 mL is overlaid on
the APsulloc331261 culture solution and hardened. After culturing
at 37.degree. C. for 24 hours, size of a clear zone is measured.
The results compared with the Lactobacillus plantarum strain
(KCTC3108) as a standard strain are shown in the following
Table.
TABLE-US-00021 TABLE 21 Test Strain Clear Zone Diameter (mm) KCTC
No. Strain KCTC3108 APsulloc 331261 2008 Shigella flexneri 26
27
[0074] As can be seen from the above, it is confirmed that the
APsulloc 331261 is more excellent in antibacterial effect against
the Shigella flexneri than the standard strain.
(2) APsulloc 331266
[0075] The APsulloc 331266 culture solution is inoculated to MRS
broth 10 mL to the concentration of 0.5%, and cultured at
37.degree. C. overnight.
[0076] On the other hand, Listeria monocytogens and Bacillus cereus
are streaked on Tryptic soy agar, respectively, and cultured at
37.degree. C. overnight. Then a colony is inoculated to BHI broth
and cultured overnight.
[0077] BHI soft agar (agar 1%) is sterilized and cooled to about 45
to 50.degree. C., and then Listeria monocytogens and Bacillus
cereus culture solutions 1% are inoculated thereto, respectively.
15 mL of the resulting solutions are aliquoted to a petri dish,
respectively, and cooled about 1 hour to prepare a medium. 5 mL of
sterilized MRS soft agar (agar 1%) is overlaid on the medium
prepared above and hardened. Each 5 .mu.l of the APsulloc 331266
culture solution is spotted on the hardened medium and dried. After
culturing at 37.degree. C. for 24 hours, size of a clear zone is
measured. The results compared with the Lactobacillus plantarum
strain (KCTC3108) as a standard strain are shown in the following
Table.
TABLE-US-00022 TABLE 22 Test Strain Clear Zone Diameter (mm) KCTC
no. strain KCTC3108 APsulloc 331266 3710 Listeria monocytogens 16.8
19.3 3624 Bacillus cereus 10.3 12.3
[0078] As can be seen from the above, it is confirmed that the
APsulloc 331266 is more excellent in antibacterial effect against
the Listeria monocytogens and the Bacillus cereus than the standard
strain.
(3) APsulloc 331269
[0079] The APsulloc 331269 culture solution is inoculated to MRS
broth 10 mL to the concentration of 0.5%, and cultured at
37.degree. C. overnight. The culture solution is centrifuged at
8000 rpm for 5 min, and only supernatant is collected. The
supernatant 10 mL is prepared by sterilizing with a 0.22 .mu.l
syringe filter.
[0080] On the other hand, Salmonella typhimurium and Staphylococcus
aureus are streaked on Tryptic soy agar, respectively, and cultured
at 37.degree. C. overnight. Then a colony is inoculated to BHI
broth and cultured overnight.
[0081] BHI agar is sterilized, and 20 mL thereof is put into a
petri dish and cooled to prepare a medium. Salmonella typhimurium
and Staphylococcus aureus culture solutions are diluted 50 times
with sterilized physiological saline solution, respectively, and
then streaked evenly on the medium prepared above with a sterilized
cotton swab. A sterilized paper disk is put thereon, and the
supernatant of the APsulloc 331269 culture solution prepared above
100 .mu.l is dropped on the paper disk. The medium is stored at
room temperature for about 3 hours for absorption, and then
cultured at 37.degree. C. for 24 hours. Size of a clear zone is
measured, and the results compared with the Lactobacillus plantarum
strain (KCTC3108) as a standard strain are shown in the following
Table.
TABLE-US-00023 TABLE 23 Test Strain Clear Zone Diameter (mm) KCTC
No. Strain KCTC3108 APsulloc 331269 2514 Salmonella typhimurium 8.8
9.3 1621 Staphylococcus aureus 10 10.8
[0082] As can be seen from the above, it is confirmed that the
APsulloc 331269 is more excellent in antibacterial effect against
the Salmonella typhimurium and the Staphylococcus aureus than the
standard strain.
* * * * *