U.S. patent application number 16/858640 was filed with the patent office on 2020-08-13 for method for preparing microbial preparation and microbial preparation produced by the same.
The applicant listed for this patent is RNA INC.. Invention is credited to Dae Kyun Chung, Boram Jeon, Bong Jun Jung, Hangeun Kim, Hye Rim Kim, Seong Jae Kim, Seung Su Lee, Yoon Doo Lee, Jae Yeon Park.
Application Number | 20200255795 16/858640 |
Document ID | / |
Family ID | 55630954 |
Filed Date | 2020-08-13 |
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United States Patent
Application |
20200255795 |
Kind Code |
A1 |
Chung; Dae Kyun ; et
al. |
August 13, 2020 |
METHOD FOR PREPARING MICROBIAL PREPARATION AND MICROBIAL
PREPARATION PRODUCED BY THE SAME
Abstract
The present disclosure relates to a method for preparing an
aglycone or hydrolyzed glycoside converted from a glycoside and,
specifically, to a method for preparing an aglycone or hydrolyzed
glycoside from a glycoside by converting a glycoside into an
aglycone form or hydrolyzed glycoside by using a microorganism
producing .beta.-glycosidase, and then recovering the aglycone or
hydrolyzed glycoside accumulated in the cells of the
microorganism.
Inventors: |
Chung; Dae Kyun;
(Gyeonggi-do, KR) ; Kim; Hangeun; (Gyeonggi-do,
KR) ; Lee; Seung Su; (Gyeonggi-do, KR) ; Jung;
Bong Jun; (Gyeonggi-do, KR) ; Kim; Hye Rim;
(Daegu, KR) ; Lee; Yoon Doo; (Gyeonggi-do, KR)
; Park; Jae Yeon; (Gyeonggi-do, KR) ; Jeon;
Boram; (Gyeonggi-do, KR) ; Kim; Seong Jae;
(Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RNA INC. |
Gyeonggi-do |
|
KR |
|
|
Family ID: |
55630954 |
Appl. No.: |
16/858640 |
Filed: |
April 26, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15515918 |
Mar 30, 2017 |
10676708 |
|
|
PCT/KR2015/010325 |
Sep 30, 2015 |
|
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16858640 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/20 20130101; A23L
11/07 20160801; A61K 2800/70 20130101; A61Q 19/08 20130101; A61K
8/99 20130101; A23G 3/366 20130101; A23L 19/09 20160801; C12R 1/25
20130101; C12R 1/225 20130101; A23V 2002/00 20130101; C12N 9/2445
20130101; C12P 1/04 20130101; A61Q 19/00 20130101; C12Y 302/01021
20130101; A61K 2800/85 20130101; A23Y 2220/67 20130101; A61Q 19/007
20130101; A61P 17/00 20180101; A23L 33/135 20160801; C12P 23/00
20130101; A23C 9/1234 20130101 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12P 1/04 20060101 C12P001/04; C12P 23/00 20060101
C12P023/00; C12N 9/42 20060101 C12N009/42; A61Q 19/08 20060101
A61Q019/08; A61Q 19/00 20060101 A61Q019/00; A61K 8/99 20060101
A61K008/99; A23G 3/36 20060101 A23G003/36; A23C 9/123 20060101
A23C009/123; A23L 11/00 20060101 A23L011/00; A23L 33/135 20060101
A23L033/135; A23L 19/00 20060101 A23L019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2014 |
KR |
10-2014-0131390 |
Claims
1. A microorganism or a lysate thereof, wherein the microorganism
is a .beta.-glycosidase-expressing microorganism and contains
intracellularly accumulated converted ginsenoside or hydrolyzed
glycoside.
2. The microorganism or lysate of claim 1, wherein said
intracellularly accumulated converted ginsenoside or hydrolyzed
glycoside comprises one selected from the group consisting of rg3,
rc and f2.
3. The microorganism or lysate of claim 1, wherein said
intracellularly accumulated converted ginsenoside or hydrolyzed
glycoside comprises one ginseng extracts comprise one or more of a
ginsenoside selected from the group consisting of rg1 and rb1.
4. The microorganism or lysate of claim 1, wherein said
intracellularly accumulated converted ginsenoside or hydrolyzed
glycoside comprises one or more selected from the group consisting
of genistein, daidzein, glycitein, sapogenin,
3,4-Hydroxyphenylactic acid, 4-HPA, m-coumaric acid, p-coumaric
acid, O-beta-D-glucuroniodes, stilbenoids, rutin, quercetin,
hesparetin, baicalein, wogonin, mogroside IIIE, mendelonitrile,
benzaldehyde, and coumarin-derived compounds.
5. The microorganism or lysate of claim 1, wherein said
intracellularly accumulated converted ginsenoside or hydrolyzed
glycoside comprises one or more of rg3, rc, and f2.
6. The microorganism or lysate of claim 1, wherein the
microorganism comprises one or more selected from the group
consisting of lactic acid bacteria, bifidus, yeast,
corynebacterium, aspergillus and clostridium.
7. The microorganism or lysate of claim 1, wherein the
microorganism comprises one or more selected from the group
consisting of generally recognized as safe (GRAS) microorganisms,
bifidus, yeast, Bacillus licheniformis, S. thermophilus, L. casei,
Streptomyces sp. Bifidobacteria, Lactobacillus delbrueckii Rh2,
Sporosarcina sp., Saccharomyces cerevisiae, Pyrococcus furiosus,
Lactobacillus plantarum, Aspergillus ochraceus, Aactobacillus
delbrueckii Rh2, Pseudomonas sp., Aspergillus niger; Pseudomonas
fluorescens, Bifidobacterium adolescentis, Aspergillus sojae,
Cunninghamella blakesleeana, Cifidobacteria and Lactobacillus,
Lactic acid bacteria, Bifidobacterium pseudocatenulatum,
Penicillium melinii, Eubacterium ramulus, Clostridium orbiscindens,
Aspergillus awamori, Lactobacillus brevis, Aspergillus parasiticus
speare BGB, Aspergillus aculeatus, and Aspergillus niger.
8. The microorganism or lysate of claim 1, wherein the
microorganism comprises a lactic acid bacterium.
9. The microorganism or lysate of claim 1, wherein the
microorganism comprises one or more selected from the group
consisting of L. plantarum, L. sakei, L. rhamnosus GG; L.
delbrueckii, L. acidophilus, L. johnsonii, L. casei and L.
gasseri.
10. The microorganism or lysate of claim 1, wherein the
microorganism comprises Lactobacillus plantarum K8 (accession
number: KCTC 10887BP).
11. A lysate of the microorganism of claim 1.
12. An antioxidant, intestinal regulation, cosmetic, probiotic, or
food composition, comprising the microorganism or lysate of claim
1.
13. A cosmetic composition comprising the microorganism or lysate
of claim 1 for enhancement of skin function of skin moisturizing,
skin color, skin elasticity, wrinkles or dermis density.
14. A probiotic composition comprising the microorganism of claim
1, in a dose form with a viable cell count in a range of from
10.sup.8 to 10.sup.10 CFU/day.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional under 35 USC .sctn. 120 of U.S. patent
application Ser. No. 15/515,918 filed Mar. 30, 2017 in the names of
Dae Kyun Chung, et al. for Method for Preparing Microbial
Preparation and Microbial Preparation Produced by the Same, which
in turn is a U.S. national phase under 35 USC .sctn. 371 of
International Patent Application PCT/KR2015/010325 filed Sep. 30,
2015, which in turn claims priority of Korean Patent Application
10-2014-0131390 filed Sep. 30, 2014. The disclosures of U.S. patent
application Ser. No. 15/515,918, International Patent Application
PCT/KR2015/010325, and Korean Patent Application 10-2014-0131390
are hereby incorporated herein by reference, in their respective
entireties, for all purposes.
TECHNICAL FIELD
[0002] The present disclosure relates to a method for preparing a
microbial preparation containing an aglycone or hydrolyzed
glycoside in the cells and, specifically, to a method for preparing
a microorganism which converting a glycoside into an aglycone form
or hydrolyzed glycoside by using a microorganism producing
.beta.-glycosidase, and having the aglycone or hydrolyzed glycoside
accumulated in the cells.
DESCRIPTION OF THE RELATED ART
[0003] Lactic acid bacteria are one of the most beneficial
microorganisms available to humans and means a bacterial that
decomposes carbohydrates such as glucose or lactose during
fermentation to produce lactic acid. In 1858, the presence of
lactic acid bacteria was discovered by the French microbiologist
Louis Pasteur, and the Ilija Mecnikov, a Russian immunologist, was
awarded the Nobel Prize for physiology in 1908, declared "the
eternal youth hypothesis by the fermented milk" in his later years,
and then claimed that the longevity cause of the Bulgarian people
was in the lactic acid bacteria fermented milk, and from the claim,
full-scale researches have begun and more than 300 kinds of lactic
acid bacteria have been discovered so far. The lactic acid bacteria
live in the intestines of the most mammals including the humans,
and particularly, more than 100 trillion of 100 kinds of bacteria
combining healthy and harmful bacteria are present in the
intestines of the human body. Since the lactic acid bacteria
produce the lactic acid as a metabolite to acidify the intestines,
the lactic acid bacteria are known as bacteria which serve to
inhibit proliferation of harmful bacteria and reduce the production
of ammonia or carcinogens by abnormal fermentation, that is,
performs the intestinal regulation.
[0004] It is known that the physiological activity of the lactic
acid bacteria is that they mainly produce organic acids to lower
the pH of the intestines, which inhibits the proliferation of
harmful bacteria in the intestines and maintain the normal
intestinal microbial flora. They also act to convert a glycoside
isoflavone into an aglycone which is easily absorbed in the body by
producing .beta.-glucosidase.
[0005] Particularly, in the case of isoflavone, there are four
forms of a glycoside to which sugar is coupled, an aglycone in
which the sugar is removed, acetylglucoside, and malonylglucoside,
and among them, the aglycone isoflavone is hydrolyzed by
.beta.-glucosidase in the intestines to be converted into an
aglycone as an active aglycone and absorbed. However, isoflavone
included in general foods is present as a glycoside form to which
the sugar is coupled, and isoflavone taken from the diet needs to
be converted into an aglycone form by the intestinal microorganism
so as to be absorbed. Accordingly, the microorganism having
activity of .beta.-glycosidase may play a very important role in
bioavailability of the isoflavone.
[0006] As a method of extracting isoflavone in the related art,
there is a method of extracting powder of a plant including
isoflavone with water, acidifying the extract, separating the
extract from the sludge to pass through a column filled with an
absorbent resin, absorbing isoflavone in the extract on the resin,
passing an alcoholic solvent through the column, and recovering the
isoflavone absorbed on the resin. As another method, there is a
method of extracting isoflavone including a first step of binding
and extracting isoflavone to cyclodextrin, a derivative thereof, or
a polymer thereof by adding a plant or a plant processing product
including isoflavone in the cyclodextrin, the derivative thereof,
or the polymer thereof and a second step of isolating a binder of
isoflavone and cyclodextrin from the extract by injecting a
coagulant in the extract formed in the first step and the
extraction. Particularly, in order to extract the aglycone
isoflavone from the native beans, various mechanical and chemical
methods are used, and there is a method of extracting isoflavone by
using ultrasonic waves and then purifying the extract by liquid
chromatography. However, the extraction method comprises many steps
and includes problems such as a decrease in production rate, an
increase in production cost, and possibility of contamination
during a purifying process.
[0007] Bioconversion of glycoside isoflavone to aglycone isoflavone
by microorganisms has been studied (Kuo et al., Appl Microbiol
Biotechnol 2006.73:314-320; Lee et al., J Agric Food Chem 2013.
61:12101-12110), and above all, bioconversion of glycoside
isoflavone to aglycone isoflavone by lactic acid bacteria is
possible and researches therefor have been actively conducted
(Cavallini et al., Alim. Nutr. 2010. 21:175-182). For example, Ali
and his colleagues examined effects on conversion into aglycone as
an active form of metabolism of isoflavone glycoside by lactic acid
bacteria and fat and cholesterol metabolism. According to the
result, the lactic acid bacteria did not induce enhancement of
efficacy of the isoflavone. The reason is that bioconversion
efficiency by lactic acid bacteria is low (Ali, et al., J Nutr
Biochem. 2004. 15:583-90). According to another report, conversion
into aglycone by lactic acid bacteria bifidobacterium was 73 to 74%
or less when soy milk was not added. However, in the case of adding
the soy milk, the conversion rate increased to 84 to 85% (2007 11.
Newhope 360.com).
[0008] Meanwhile, saponin is a glycoside of steroid, steroid
alkaroid, or triterpene, and a general term of substances that
dissolve in water and exhibit a foaming action like soap. The
saponin is an ingredient for enhancing human immunity and
specially, saponin included in ginseng is famous. In the ginseng,
32 types of various saponins are much included. Particularly, it is
known that saponin included only in red ginseng obtained by
processing the ginseng has a considerable effect in the treatment
of adult diseases when administered for a long time. Ginsenoside
may inhibit the growth of all kind of cancer cells. Even in the
experiment related to cataract, the ginsenoside exhibited an
excellent effect. Ginsenoside (ginseng saponin) is a compound that
has carbohydrates (glycones) and non-carbohydrate residues
(aglycones) in the same molecule. The carbohydrate residue binds to
the non-carbohydrate residue through an acetal linkage at the
1-carbon position. The non-sugar component is referred to as an
aglycone and the sugar component is referred to as a glycone. When
the carbohydrate site is glucose, it is referred to as a glucoside.
Accordingly, by the hydrolysis of ginsenoside which is the
glycoside of ginseng, the ginsenoside is decomposed into the
glycone and sapogenin as the aglycone.
[0009] Microorganisms that hydrolyze the isoflavone glycoside
include Bifidobacterium longum, Lactobacillus bulgaricus,
Aspergilus niger, and Sachcaroplyspora erythraea, and these
microorganisms produce commonly .beta.-glycosidase. In addition,
researches for developing microorganisms that produce high-potency
.beta.-glycosidase capable of efficiently converting glycoside
isoflavones into aglycone isoflavones have been continued.
[0010] However, the techniques in the related art include many
complicated steps and thus there are problems such as a decrease in
production rate, an increase in production cost, and possibility of
contamination during a purifying process.
[0011] Under these technical backgrounds, the inventors of the
present application have confirmed that conventional problems may
be improved by converting glycosides into aglycones or hydrolyzed
glycoside by using a microorganism producing .beta.-glycosidase and
then accumulating the converted aglycones or hydrolyzed glycoside
in the microorganism and using a microorganism containing the
aglycones or hydrolyzed glycoside thereof as a raw material, and
completed the present disclosure.
SUMMARY
[0012] An object to be achieved by the present disclosure is to
provide a method of preparing a microorganism capable of preparing
an aglycone or hydrolyzed glycoside converted from a glycoside in a
high concentration by using a microorganism producing
.beta.-glycosidase.
[0013] Another object to be achieved by the present disclosure is
to provide a microorganism or a lysate containing an aglycone or
hydrolyzed glycoside thereof and a use thereof, in which the
aglycone or hydrolyzed glycoside prepared by the method is
accumulated in cells.
[0014] According to an aspect of the present disclosure, there is
provided a method of preparing a microorganism in which an aglycone
or hydrolyzed glycoside is accumulated in cells. The method
comprises accumulating an aglycone or hydrolyzed glycoside
converted from a glycoside in the microorganism by incubating the
microorganism expressing .beta.-glycosidase in a medium containing
glycoside or reacting with a reactant containing glycoside.
[0015] According to another aspect of the present disclosure, there
is provided a microorganism or a lysate thereof which is prepared
by the method and in which an aglycone or hydrolyzed glycoside is
accumulated in cells.
[0016] According to yet another aspect of the present disclosure,
there is provided a composition comprising the microorganism or the
lysate thereof.
[0017] According to still another aspect of the present disclosure,
there is provided a food comprising the microorganism or the lysate
thereof.
[0018] According to still yet another aspect of the present
disclosure, there is provided a composition for improving a skin
function comprising the microorganism or the lysate thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0020] FIG. 1 illustrates a result of conversion into soybean
aglycone using Lactobacillus plantarum K8 by a preparing method
according to the present disclosure. Conversion of a glycoside
isoflavone into an aglycone isoflavone by Lactobacillus plantarum
K8. In FIG. 1, a raw material represents a culture medium
containing soybean extracts, STD represents an isoflavone standard;
and a L. plantarum K8-LTA extract represents a lysate lysed by
using a microfluidizer after Lactobacillus plantarum K8 is
incubated in a culture medium containing a soybean extract.
[0021] FIG. 2 illustrates a result of conversion into a soybean
aglycone by using Lactobacillus plantarum K8, Lactobacillus
delbrueckii, Lactobacillus rhamnosus GG, and Lactobacillus sakei by
the preparing method according to the present disclosure.
Comparison of production of aglycones of Lactobacillus including
Lactobacillus plantarum K8. In FIG. 2, STD represents an isoflavone
standard; delbrueckii represents Lactobacillus delbrueckii; GG
represents Lactobacillus rhamnosus GG; K8 represents Lactobacillus
plantarum K8; and sakei represents Lactobacillus sakei.
[0022] FIG. 3 illustrates a measured quantity of a soybean aglycone
detected in a preparing process of Lactobacillus plantarum K8
lysates by the preparing method according to the present
disclosure. In FIG. 3, gg represents Lactobacillus rhamnosus GG,
del represents Lactobacillus delbrueckii, and k8 represents
Lactobacillus plantarum K8.
[0023] FIG. 4 is a graph illustrating saponin converted from
ginsenoside by using Lactobacillus plantarum K8 by the preparing
method according to the present disclosure. In FIG. 4, rg1, rg3(s),
rh2, rb1, rc, rd, rb2, and f2 are ginsenoside compounds. The above
names are classified according to a molecular structure as kinds of
saponin. In FIG. 4, washing represents a wash solution after
incubating Lactobacillus; Ginseng ext represents a ginseng extract;
and k8 represents Lactobacillus plantarum K8.
[0024] FIG. 5 is a diagram illustrating an absorption prediction
mechanism of hydrolyzed saponin by Lactobacillus plantarum K8.
[0025] FIG. 6 illustrates a result of measuring detection amounts
of low molecular ginsenoside. An intake amount represents an amount
of a ginseng extract fed to mice and an absorption amount
represents an amount of a ginsenoside detected from the blood of
the mice.
[0026] FIG. 7 is a result illustrating an absorption rate of a low
molecular ginsenoside. The absorption rate is a result of
converting an absorbed amount in the intake amount to a
percentage.
[0027] FIG. 8 is a table showing a preparation process according to
the present disclosure.
[0028] FIG. 9 is a table showing sugar availability of
Lactobacillus plantarum k8.
[0029] In the related art, there have been attempts to convert a
glycoside into an aglycone or hydrolyzed glycoside using a
microorganism, but it is not yet reported that the converted
aglycone or hydrolyzed glycoside is included in the cell of the
microorganism. Accordingly, results of bioconversion into the
aglycone or hydrolyzed glycoside by the microorganism according to
the present disclosure and accumulation in the cells of the
microorganism are new. The inventors of the present application
confirmed that a concentration of the aglycone or hydrolyzed
glycoside accumulated in the cells of the microorganism was more
than 1.5 times, more than two times higher than that of the
aglycone or hydrolyzed glycoside in a microbial culture medium to
produce the aglycone or hydrolyzed glycoside in a high
concentration.
[0030] As an action mechanism of probiotics, remodeling of an
intestinal environment, inhibition of pathogens, inhibition of
proinflammatory factors, an effect on epithelial differentiation,
strengthening of an intestinal barrier effect, and the like are
known. The actions of the probiotics on suppression of colorectal
cancer have various biochemical pathways, and inhibit occurrence of
colorectal cancer through a cell cycle, reactive oxygen species,
apoptosis, production of specific bacterial enzymes, and a host
metabolism. Further, lactic acid bacteria protect a body from
diseases through an immune activation action that activates an
immune system. In the villi of the intestinal surface, the lactic
acid bacteria interact with immune cells such as leukocytes or
lymphocytes and promotes the activity of immunity. The lactic acid
bacteria induce the expression of human beta defensin (hBD2) in
human colon cancer cells to regulate the intestinal immunity.
Further, production of IL-8 which is inflammatory cytokine and
inhibition of expression of Hsp70 are performed by exposure of
lactic acid bacteria in Caco-2 cells which is colon cancer cells.
As a result, it can be seen that the lactic acid bacteria is
directly involved in the regulation of an inflammatory response by
regulating cytokines. Further, the lactic acid bacteria in the
intestine regulate an autoimmune response and a toll-like receptor
(TLR). This means that the effect of the lactic acid bacteria on
regulation of intestinal health is associated with TLR
signaling.
[0031] Lactobacillus plantarum K8 absorbs ginsenoside in a
glycoside form and then decomposes the glycoside form into
hydrolyzed saponin by using .beta.-glycosidase. The sugars are used
in the metabolism of the lactic acid bacteria, whereas since the
hydrolyzed saponin is an unnecessary substance for the metabolism
of the lactic acid bacteria, the hydrolyzed saponin is accumulated
in the body and then released outside the cells over time. The
Lactobacillus plantarum K8 containing the hydrolyzed saponin
exhibited a body absorption rate 1,300 times or more higher than a
case of taking ginsenoside (a saponin extract) which is not
modified in a test using an animal (Example 7). The high absorption
rate is because the Lactobacillus plantarum K8 serves as a capsule
to prevent breakage of saponin and safely transfer the saponin to
the intestines. Further, it is expected that the Lactobacillus
plantarum K8 containing the hydrolyzed saponin is selectively
up-taken by a tissue resident macrophage in the intestines and then
moves to lamina propria or lymph node to release the hydrolyzed
saponin to the blood (see FIG. 5).
[0032] Based thereon, an aspect of the present disclosure relates
to a method of preparing a microorganism in which an aglycone or
hydrolyzed glycoside is accumulated in cells. The method comprises
accumulating an aglycone or hydrolyzed glycoside converted from a
glycoside in the microorganism by incubating the microorganism
expressing .beta.-glycosidase in a glycoside-contained medium or
reacting with a glycoside-contained reactant.
[0033] The microorganism used in the present disclosure represents
production activity of the aglycone or hydrolyzed glycoside.
Particularly, the microorganism has ability of converting a
glycoside into an aglycone form or hydrolyzed glycoside through the
activity of .beta.-glycosidase. Under an anaerobic condition, the
microorganism has ability of converting glycoside forms genistin
and diadzin to aglycone forms genistein and diadzein. In the case
of lysing the microorganism, the aglycones accumulated in the cells
may be detected (see FIG. 1).
[0034] The microorganism is not particularly limited as long as the
microorganism is microorganisms producing .beta.-glycosidase, but
may be one or more selected from the group consisting of lactic
acid bacteria, lactococcus, corynebacterium, generally recognized
as safe (GRAS) microorganisms, bifidus, yeast, bacillus,
aspergillus and clostridium. The lactic acid bacteria may be one or
more selected from the group consisting of L. plantarum, L. sakei,
L. rhamnosus GG, L. delbrueckii, L. acidophilus, L. johnsonii, L.
casei, L. gasseri, and Leuconostoc mesenteroid.
[0035] The inventors of the present application isolated and
identified L. plantarum K8 that produces .beta.-glycosidase from
Kimchi which is the Korean traditional fermented food. First, the
kimchi and the kimchi liquid were diluted by 10 times in stages by
using sterile physiological saline and then the undiluted solution
and the diluted solution were spreaded on a lactobacillus selective
agar (LBS agar, Difco). Colonies exhibited after incubating for 2
to 3 days at 37.degree. C. were isolated again purely according to
a type and a color. Gram staining and microscopic observation of
the isolated colonies were performed and then only gram-positive
and rod-shaped colonies were screened, and while these colonies
were incubated in a lactobacilli MRS liquid medium (Difco) at pH
6.8 and 37.degree. C. for 24 hrs, the colonies in which pH of the
culture solution is decreased to 4.5 or less were re-screened.
Thereafter, when the colonies were incubated for 2 hrs in a MRS
medium at pH 2.0 and then incubated for 9 hrs in the MRS medium
added with 0.3% of oxgall, the viable acid-resistant and
biliary-resistant lactobacillus strains were isolated. The isolated
strains were identified through a biochemical test using an API CHL
50 kit and 16S rRNA sequencing. As the identified result, it was
confirmed that the strains were strains belonging to Lactobacillus
plantarum species and the strains were called "Lactobacillus
plantarum K8" (accession number: KCTC 10887BP).
[0036] In yet another example, the microorganism producing the
.beta.-glycosidase may be one or more selected from the group
consisting of generally recognized as safe (GRAS) microorganisms,
Bifidus, Yeast, Bacillus licheniformis, S. thermophilus, L. casei,
Streptomyces sp. Bifidobacteria, Lactobacillus delbrueckii Rh2,
Sporosarcina sp., Saccharomyces cerevisiae, Pyrococcus furiosus,
Lactobacillus plantarum, Aspergillus ochraceus, Lactobacillus
delbrueckii Rh2, Pseudomonas sp., Aspergillus niger, Pseudomonas
fluorescens, Bifidobacterium adolescentis, Aspergillus sojae,
Cunninghamella blakesleeana, Bifidobacteria and Lactobacillus,
Lactic acid bacteria, Bifidobacterium pseudocatenulatum,
Penicillium melinii, Eubacterium ramulus, Clostridium orbiscindens,
Aspergillus awamori, Lactobacillus brevis, Aspergillus parasiticus
speare BGB, Aspergillus aculeatus, and Aspergillus niger.
[0037] In order to prepare lactic acid bacteria comprising aglycone
isoflavone or hydrolyzed saponin, Lactobacillus plantarum K8 was
incubated in a medium added with a soybean extract or glycoside
saponin and lysed by using a microfluidizer, and then the lysed
Lactobacillus plantarum K8 was fractioned through a solvent system
fraction. With respect to an EtOAc fraction with isoflavone or
sapogenin, the Lactobacillus plantarum K8 was prepared in a
concentration of 2,000 ppm and then an analysis was performed by
using LC-MS/MS. It can be confirmed that after lysing, the contents
of daidzein and genistein as aglycone isoflavones and the content
of Rg3 as a hydrolyzed saponin are increased.
[0038] The Lactobacillus plantarum K8 was lysed by using a
sonicator or a microfluidizer and lyophilized to prepare a
`Lactobacillus plantarum K8-LTA` lysate. Specifically, the step (a)
may comprise the steps of static culture for microbial activation,
shaking culture, and feeding culture in sequence. In this case, the
microorganism may be static-cultured for 12 hrs at 37.degree. C.
and cultured with shaking under a condition of 6 rpm for 12 hrs at
37.degree. C. A medium (for example, an MRS medium) composed of
carbon sources, nitrogen sources, vitamins and minerals may be used
for incubating the microorganism.
[0039] In some cases, before the step (a), a step of pre-treating
the plant comprising the glycoside with a protease may be further
included. The protease may be one or more selected from the group
consisting of for example, promod, alkalase, neutrase, and
papain.
[0040] In detail, detailed preparing processes are described in the
following `Preparing Process Table` and FIG. 8.
[0041] [Preparing Process Table] Membrane Cell-Recycling Reactor
(MCR) Preparing Process
[0042] 1) Sterilization of fermentation line: A line flowing into a
fermentation pipe is sterilized by using the steam.
[0043] 2) Co-sterilization of fermentation tank and feeding tank:
The fermentation tank and the feeding tank are sterilized for 15 to
20 minutes under a condition of 121.+-.1.degree. C. and 1.2 to 1.5
kgf/cm.sup.2 by injecting the steam into the tank by using a
sparger, sampling, and a lower steam.
[0044] 3) Weighting of raw materials and sterilization of medium:
Raw materials of a culture medium are accurately weighted according
to the standard of the product, respectively, and then injected
into both the fermentation tank and the feeding tank and sterilized
according to a condition 2) after adjusting the amounts according
to a capacity.
[0045] 4) Cooling: Cooling is performed to 37.degree. C. after
sterilization.
[0046] 5) Bacterial seed activation: The frozen lactobacillus are
rapidly thawed in a 37.degree. C. thermostatic bath and then
inoculated in a sterilized culture medium of 1.5 L and
static-cultured for 12 hours.
[0047] 6) Main incubation: The bacterial seed diluted with 1/100 is
incubated in a sterilized main culture medium of 30 L.
[0048] 7) Feeding incubation: The incubation conditions are
frequently checked so as to be maintained at all times. When an
optical density (OD) value reaches a predetermined value, a used
medium is removed by circulating the membrane and a new medium is
injected to the feeding tank by a discharged amount. The bacterial
cells are incubated by continuously removing and injecting a medium
of about 200 L.
[0049] 8) Concentration adjustment: A drain line is sterilized with
steam for 30 minutes, and then a solution flows through the drain
line and centrifuged, and diluted with an appropriate amount of
reverse osmosis distilled water to adjust the concentration.
[0050] 9) collection of live cells and product packaging or lysing
of bacterial cells: The concentration-adjusted bacterial cells are
lysed by a microfluidizer.
[0051] 10) steriliztion: The lysed bacterial cells are maintained
for 30 minutes at 80.degree. C. and the medium solution is
sterilized and cooled again. According to a purpose, the
steriliztion may be excluded.
[0052] 11) Collection and packaging of bacterial cells: The cooled
solution is contained and packaged in a polyethylene (PE) bottle by
using a tank drain line.
[0053] In yet another example, the microorganism is inoculated and
then incubated in a medium, and transferred to a minimal medium
comprising a glycoside, and may be additionally cultured for 1 to
36 hrs, for example, 4 to 24 hrs. According to Example 6 of the
present disclosure, when the microorganism is incubated by using
the minimal medium comprising the glycoside, it is confirmed that
the accumulation of the aglycone or hydrolyzed glycoside in the
cells may be increased compared with a control group in which the
same amount of cells is incubated in the MRS medium comprising the
glycoside saponin.
[0054] In one example, the glycoside may be originated from one
selected from the group consisting of ginseng, wild ginseng,
soybean, Codonopsis lanceolata, buckwheat, bellflower, water
parsley, mung beans, garlic, onion, ginkgo, and kudzu.
[0055] The glycoside may be an isoflavone glycoside, a saponin
glycoside, a phenol glycoside (phenol), a cynophore glycoside (a
nitrile glycoside), an anthraquinone glycoside, a cardiac
glycoside, a bitter glycoside (amara), a coumarin glycoside
(coumarin), a sulfur glycoside (a rioglycoside and a sulfur), or an
flavonoid glycoside (flavonoid). For example, the method according
to the present disclosure may be converting one or more glycosides
selected from the group consisting of daidzin, genistin, glycitin,
saponin, procyanidin, naringenin, quercetin, rutinose, hesparidin,
baicalin, wogonoside, mogroside V, amygdalin, and 3-phenyl coumarin
into one or more hydrolyzed glycosides selected from the group
consisting of genistein, daidzein, glycitein, hydrolyzed saponin,
3,4-Hydroxyphenylactic acid, 4-HPA, m-coumaric acid, p-coumaric
acid, O-beta-D-glucuroniodes, stilbenoids, rutin, quercetin,
hesparetin, baicalein, wogonin, mogroside IIIE, mendelonitrile,
benzaldehyde, and coumarin-derived compounds.
[0056] The aglycones or hydrolyzed glycoside produced by the
preparing method may be accumulated in the cells. Based thereon,
another aspect of the present disclosure relates to a microorganism
or a lysate thereof in which an aglycone or hydrolyzed glycoside
produced by the preparing method is accumulated in cells. It is
confirmed that the concentration of the aglycone or hydrolyzed
glycoside accumulated in the cells is two times higher than that of
the aglycone or hydrolyzed glycoside in a microbial culture
medium.
[0057] The lysate may be prepared by using a physical method. For
example, the microorganism may be lysed 4 to 9 times by using bead
mills, presses, a sonicator, or a microfluidizer and then
lyophilized and powdered. If necessary, the lysed bacterial cells
are maintained for 30 minutes at 80.degree. C. and the medium
solution may be sterilized and cooled again.
[0058] Yet another aspect of the present disclosure relates to a
composition comprising the microorganism or the lysate thereof.
[0059] The microorganism or the lysate thereof may be used as an
antioxidant composition, a composition for intestinal regulation, a
raw material composition for cosmetics, and a probiotic
composition. Particularly, the microorganism or the lysate thereof
may be used as a feed composition, a composition for food addition,
and other fermented products. The lysed cell wall fractions of the
microorganism or the lysate thereof according to the present
disclosure, lived microbes, dead microbes, dried microbes or
cultures may be included as an active ingredient, and excipients or
carriers may be additionally included.
[0060] The culture comprises a culture solution itself incubated in
a liquid medium, a filtrate (a centrifuged supernatant) obtained by
filtering or centrifuging the culture solution to remove the
strain, and a cell lysate obtained by ultrasonicating or lysozyming
the culture solution. The content of the microorganism or the
lysate thereof in the composition of the present disclosure may
vary according to a use and a formulation of the composition.
[0061] The composition according to the present disclosure may be
prepared and administered by various formulations and methods. For
example, the microorganism or the lysate thereof or the culture is
combined with a carrier or a flavoring which is generally used in a
pharmaceutical field and then may be prepared and administered in
forms such as tablets, troches, capsules, elixirs, syrups, powders,
suspensions, or granules. As the carriers, binders, lubricants,
disintegrants, excipients, solubilizers, dispersants, stabilizers,
suspending agents, and the like may be used. The administering
method may use an oral, parenteral, or applying method, and a dose
may be appropriately selected according to the absorption, the
inactivity rate, and the excretion rate of active ingredients in
the body, and age, gender, status, and the like of persons to be
administered. Preferably, the composition may be administered with
an effective dose of 10 mg/day, and in the case of a probiotic
composition, the viable cell count in the composition may be
10.sup.8 to 10.sup.10 CFU/day.
[0062] The antioxidant composition may be used for removing active
oxygen, and the intestinal regulation composition or the probiotic
composition promotes the internal absorption of isoflavone to
prevent or improve dyspepsia or diarrhea.
[0063] Further, the feed composition may be prepared in forms such
as fermented feed, compound feed, a pellet form, silage, and the
like. The fermented feed may be prepared by fermenting organic
materials by adding the microorganism or the lysate thereof of the
present disclosure and various other known microorganisms or
enzymes, and the compound feed may be prepared by combining various
kinds of regular feeds and the lactic acid bacteria of the present
disclosure. The pellet form feed may be prepared by formulating the
fermented feed or the compound feed by a pellet machine and the
silage may be prepared by fermenting green feed with the lactic
acid bacteria according to the present disclosure.
[0064] Further, the microorganism or the lysate thereof or the
culture thereof may be used as food additives for foods such as
baby foods, kimchi, beverages, dairy products, and the like.
[0065] Further, the present disclosure relates to food comprising
the microorganism or the lysate thereof.
[0066] The microorganism or the lysate thereof of the present
disclosure or the culture thereof may be used as a cosmetic
composition, and specifically, may used for various uses such as
basic cosmetics, cosmetics, hair cosmetics, whitening cosmetics,
wrinkle cosmetics, and anti-aging cosmetics.
[0067] Furthermore, the present disclosure provides a composition
for improving a skin function comprising the microorganism or the
lysate thereof. The improvement of the skin function may mean
improve skin moisturizing, skin color, skin elasticity, wrinkles or
dermis density.
[0068] Further, the microorganism or the lysate thereof according
to the present disclosure may be used as a starter for preparing
fermented products. The fermented products include fermented meat
products such as ham and sausage, fermented raw food products,
fermented milk products, fermented soybean liquids, kimchi, and the
like. The fermented products may be prepared according to general
methods known in the art. For example, the fermented raw food
products may be prepared by combining the microorganism or the
lysate thereof with grain powder such as brown rice and adlay,
vegetable powder, and mushroom powder or prepared by fermenting the
grain powder with the microorganism or the lysate thereof or two to
five types of mixed lactic acid bacteria comprising the
microorganism or the lysate thereof at an appropriate temperature
and then appropriately combining the vegetable and mushroom powder
to have excellent nutritional balance and palatability. Further,
the fermented milk products may be prepared by fermenting raw milk
or skimmed milk powder with the lactic acid bacteria according to
the present disclosure or two to five types of mixed lactic acid
bacteria comprising the same at an appropriate temperature, and the
fermented soybean liquid may be prepared by fermenting a soybean
liquid with the lactic acid bacteria according to the present
disclosure or two to five types of mixed lactic acid bacteria
comprising the same at an appropriate temperature.
[0069] Hereinafter, the present disclosure will be described in
detail by Examples. However, the following Examples just exemplify
the present disclosure, and the contents of the present disclosure
are not limited to the following Examples.
EXAMPLES
Example 1: Biochemical Characteristic Test of Lactobacillus
plantarum K8
[0070] In order to evaluate the antioxidant activity of
Lactobacillus plantarum K8 isolated and identified from kimchi
which was the Korean traditional fermented food, an API test
(Biomerieux, France) was performed. Biochemical characteristics and
sugar availability of the lactic acid bacteria were as listed in
Table 1 below and in FIG. 9.
TABLE-US-00001 TABLE 1 characteristics of Lactobacillus plantarum
k8 Characters Results Type of bacteria Bar Type Optimum growth
temperature 37.degree. C. Motility No Gram-Staining + Activity to
Catalase - Influence of oxygen Oblivious anaerobic
In the table of FIG. 9: Abbreviation: MDX,
Methyl-.beta.D-xylopyranoside; MDM,
Methyl-.alpha.D-mannopyranoside; MDG,
Methyl-.alpha.D-glucopyranoside; NAG, N-acetylglucosamine; ESC,
Esculin ferric citrate; GNT, Potassium gluconate; 2KG, Potassium
2-ketogluconate; 5KG, Potassium 5-ketogluconate.
[0071] In the table of FIG. 9: In 24-hour measurement, negative was
exhibited, but in 48-hr measurement, when the color was slightly
changed, it was represented by `?`.
Example 2: Production of Aglycone of Lactobacillus plantarum K8
[0072] Lactobacillus plantarum k8 was incubated in a medium added
with soybean extracts and lysed by using a microfluidizer, and then
the lysed Lactobacillus plantarum K8 was fractioned by a solvent
fraction system. With respect to an EtOAc fraction with isoflavone,
the Lactobacillus plantarum K8 lysate sample was prepared in a
concentration of 2000 ppm and then an analysis was performed by
using LC-MS/MS. As a result, it was confirmed that in the case of
soybean raw materials, the contents of daidzin and genistin as
glycoside isoflavones were high, whereas after incubation with
Lactobacillus plantarum K8, the contents of daidzein and genistein
as aglycone isoflavones were increased. On the contrary, the
contents of the glycoside isoflavones were reduced in the media
after incubation with Lactobacillus plantarum K8 (see FIG. 1).
Before the Lactobacillus plantarum K8 according to the present
disclosure was inoculated, in the case of including a pre-treatment
process of stirring for 8 hrs at 60.degree. C. by adding 1 ml/L of
alkalase, 1 ml/L of neutrase and 0.2 g/L of papain, the
intracellular accumulation rate after the conversion into the
aglycone was increased by about 10% (Table 2).
TABLE-US-00002 TABLE 2 Intracellular accumulation rate after the
conversion into the aglycone by enzyme pre-treatment daidzein
(ng/mL) genistein (ng/mL) No treatment of enzyme* 8,672.46
18,286.58 pre-treatment of enzyme 9,539.24 20,115.24
Example 3: Evaluation of Production Activity of Aglycone
[0073] In order to determine conversion ability of the aglycone
isoflavone of the lactic acid bacteria and accumulation ability of
the aglycone isoflavone in the cells, L. delbrueckii, L. rhamnosus
GG, L. plantarum K8, and L. sakei were inoculated in a culture
medium including soybean extracts and incubated for 12 hrs at
37.degree. C., and then the cells were lysed by using a
microfluidizer. In the lysed cells, the content of isoflavone was
measured by LC/MS-MS and illustrated in FIG. 2. As a result, the
aglycones of daidzein and genistein were detected in 4 types of
lactobacillus (see FIG. 2). The result means that the glycoside
isoflavone may be bioconverted into the aglycone isoflavone by all
kinds of lactobacillus or microorganisms secreting
.beta.-glycosidase, and further, the result exhibits that the
converted aglycone isoflavone may be accumulated in the cells.
Example 4: Production Activity of Aglycone Isoflavone for Each
Process of Lactic Acid Bacteria
[0074] Experiments for measuring the isoflavone content during the
production of the lactic acid bacteria lysate were performed. L.
rhamnosus GG, L. delbrueckii, and L. plantarum K8 were incubated in
a culture medium including soybean extracts for 12 hrs at
37.degree. C. and then washed three times by using sterilized
water. The isoflavone contents of the wash liquid and the
lactobacillus lysates were measured by using the LC/MS-MS,
respectively. As a result, the glycoside isoflavones were not
detected from the three-time wash liquid and the lactobacillus
lysates. Meanwhile, in the case of the aglycone isoflavones, a
small amount was detected from the wash liquid and the aglycone
isoflavone was detected from the lactobacillus lysates in a high
concentration (see FIG. 3). In another experiment, the contents of
glycoside and aglycone isoflavones were measured in Lactobacillus
plantarum K8 culture media, culture supernatant (sup.), wash
1-time, 2-time and 3-time, lysates, lysates supernatant (sup.), and
lysates precipitate (ppt.). As a result, it was confirmed that in
the lysates, the lysates sup., and the lysates ppt., the content of
isoflavone, particularly, aglycone isoflavone was largely increased
(see Table 3). In the result, the aglycone isoflavone may not be
smeared in the surface of lactic acid bacteria or caused by the
residual culture solution, and thus, the result means that the
aglycone isoflavone is accumulated in the lactic acid bacteria.
TABLE-US-00003 TABLE 3 Comparison of aglycone isoflavone amount
according to process of preparation daidzin genistin daidzein
genistein No. Name (ng/mL) (ng/mL) (ng/mL) (ng/mL) 1 Media 3,689.6
2,385.1 4,394.1 1,857.2 2 Culture sup. 398.3 372.3 1,139.2 1,796.8
3 Wash 1 N.D.* 2,431.7 3,163.1 1,588.3 4 Wash 2 N.D. 673.2 6,552.7
8,727.3 5 Wash 3 N.D. 1,800.0 6,638.2 4,601.0 6 Lysates N.D. N.D.
36,545.1 101,626.4 7 Lysates sup. 3,823 1,2276.5 12,272.1 8,785.7 8
Lysates ppt. N.D. 291.1 37,907.4 72,453.7 *N.D.: not determined
Example 5: Conversion of Saponin by Lactic Acid Bacteria
[0075] In order to determine conversion of ginseng saponin into
hydrolyzed saponin and intracellular accumulation, Lactobacillus
plantarum K8 was incubated in a culture solution added with ginseng
extracts and then the Lactobacillus plantarum K8 was lysed by using
a microfluidizer. After lysing, ginsenoside compounds were analyzed
by LC/MS-MS. As a result, it was confirmed that Lactobacillus
plantarum K8 uptook in ginsenoside in the same context as the
result of isoflavone (see FIG. 4, comparison of area values of
washing and lysates). That is, peaks which were not exhibited in
the wash liquid and the ginseng extracts were exhibited in the
lactic acid bacteria lysates, and it can be seen that the peaks are
transformed by enzymes of the lactic acid bacteria (see FIG. 4,
represented by a dotted box). The key point of ginsenoside
conversion is the conversion of ginsenoside which has a large
amount and low activity into ginsenoside of Rg3, Rc, and F2 which
have a small amount, but excellent activity. In the experimental
result, it can be confirmed that the ginsenoside is converted into
Rg3 which is a kind of high-grade saponin by Lactobacillus
plantarum K8.
Example 6: Increase in Intracellular Accumulation of Aglycone Using
Minimal Medium
[0076] Lactobacillus plantarum K8 was inoculated in 5 mL of an MRS
medium and then static-incubated for 24 hrs at 37.degree. C. and
transferred to 50 mL of the MRS medium and additionally incubated
for 12 hrs. The incubated cells were transferred to 1 L of the MRS
medium and incubated for 15 hrs, and then washed with PBS three
times. The washed cells were inoculated in 1 L of a M9 minimal
medium including saponin glycoside and a MRS medium including
saponin glycoside and lysozyme (0.5 g/L) by the same number and
then static-incubated for 6 hrs. In yet another experiment, the
cells were transferred to a MRS medium including saponin glycoside
and incubated for 6 hrs at 42.degree. C. As a control group, the
same amount of cells was incubated in the MRS medium including
saponin glycoside for 6 hrs. The incubated cells were washed and
then lysed by using a microfluidizer, and the ginsenoside compounds
were analyzed by LC/MS-MS. In the case of cells using the M9
minimal medium, the hydrolyzed saponin compound is 3.84 times or
more greater than that of the control group (see Table 4).
TABLE-US-00004 TABLE 4 Comparison in detected amount of hydrolyzed
saponin depending on culture conditions of Lactobacillus plantarum
K8 total area BuOH fr. (mg) relative ratio MRS (Lysozyme) 7545 6.6
0.32 MRS (Heat shock) N.D. 5.6 -- minimal medium (M9) 40762 12.4 mg
3.84 MRS 17779 7.4 mg 1
Example 7: Detection of Ginsenoside from Blood of Mice
[0077] The content of ginsenoside was measured in the blood
isolated from mice uptaking ginseng extracts and functional
hydrolyzed saponin-containing Lactobacillus plantarum K8 for 3
days. In the case of the ginseng extracts, the ginsenoside of about
150 ng of 1,085,630 ng of a total taking amount was absorbed.
Meanwhile, the amount of the ginseng extracts taken in by the
hydrolyzed saponin-containing Lactobacillus plantarum K8 was 11,653
ng and a total content of the ginsenoside detected from the blood
was about 2160 ng (see FIG. 6). When converting the contents into a
percentage, in the case of the ginseng extracts, an absorption rate
of 0.014% was exhibited, but the absorption rate of the ginsenoside
by the hydrolyzed saponin-containing Lactobacillus plantarum K8
reaches about 20% (see FIG. 7).
[0078] Particularly, in the mice taking the hydrolyzed
saponin-containing Lactobacillus plantarum K8, a large amount of F2
and Rg3 ginsenoside are absorbed, and it is known that the F2 and
Rg3 have the most excellent functionality even in the
ginsenoside.
[0079] Although a large amount of ginseng extracts is taken, there
is no large meaning in the taking amount due to low absorption
rate. Meanwhile, in the case of the hydrolyzed saponin-containing
Lactobacillus plantarum K8, the absorption rate is about 1300 times
or more greater than that in the case where the ginseng extracts
are directly taken. That is, when 100 g of the ginseng extracts is
directly taken, 99% or more of the taking amount is not absorbed
but discarded, but 20 g of the hydrolyzed saponin-containing
Lactobacillus plantarum K8 may be absorbed. The result means that
low-molecular ginsenoside in the hydrolyzed saponin-containing
Lactobacillus plantarum K8 is naturally protected by the lactic
acid bacteria during the digestive process of mice.
Example 8: Intracellular Accumulation of Aglycone Saponin Using
Yeast
[0080] Saccharomyces cerevisiae was inoculated in 5 ml of a YPD
medium and shaking-incubated for 24 hrs at 37.degree. C., and then
transferred to 500 ml of a YPD medium added with ginseng extracts
(1 g/L) and then additionally incubated for 12 hrs. After
centrifuging, a supernatant was separately refrigerated and a
pellet was washed 3 times with sterile distilled water. The washed
yeast was lysed three times by using a microfluidizer and then
hydrolyzed saponin compounds were analyzed through LC/MS-MS. In the
present experiment, a total of six groups of samples prepared for
analysis were divided into a supernatant after incubation (1), wash
1-time (2), wash 2-time (3), wash 3-time (4), before lysing (5),
and after lysing (6). The result is listed in Table 5 below.
TABLE-US-00005 TABLE 5 Preparation of Saccharomyces cerevisiae
comprising hydrolyzed saponin Sample Hydrolyzed saponin (ng/mL)
Culture medium (1) 675 Washing 1 (2) 175 Washing 2 (3) 165 Washing
3 (4) 245 Before lysis (5) 45 After lysis (6) 1,222
Example 9: Clinical Trial Result on Candy Including Lactobacillus
plantarum K8 Lysate
[0081] In order to evaluate efficacy of human skin moisturizing and
skin color improvement for Lactobacillus plantarum K8 lysates
(Lactobacillus plantarum K8-LTA), clinical trials were performed.
The present research was performed based on the request contents of
the client and guidelines for the human application test
(established by the Korea Food and Drug Administration in December,
2011), human application tests of health functional foods (Korea
Food & Drug Administration, in December, 2008) and guideline
for good clinical practice (GCP), and the approval of the Clinical
Study Deliberation Committee of Kyung Hee University (Study No.
KHUSBC 2012-011) was obtained.
[0082] 41 female subjects aged 25 to 60 who had dry skin and whose
skin color began to be dull or had already been dull were selected
and on an empty stomach, the products (Table 25) were taken in with
2 tablets in the morning and 2 tablets in the evening. A test
product (candy including 2.1% Lactobacillus plantarum K8-LTA) and a
control product (candy without including 2.1% Lactobacillus
plantarum K8-LTA) were allocated double-blind randomly to the
selected subjects and administered for 8 weeks, and thereafter, at
respective time points of before using the test product, in 4 weeks
after using, and in 8 weeks after using, a skin improvement effect
was evaluated by performing skin color visual evaluation, skin
moisture, dead skin quantity, TEWL, skin color (L* value) and
questionnaire evaluation, safety evaluation, dietary survey, and
weight (including BMI) survey. Further, before the product
administration and in 8 weeks after the product administration, the
blood analysis was performed. It was confirmed that the candy
including Lactobacillus plantarum K8-LTA included the aglycone
isoflavone through an analysis of LC/MS-MS.
[0083] 1. Visual Evaluation Result
[0084] As compared with before administering the product, skin
color (brightness) was significantly improved (decreased)
statistically in both the control group and the test group in 4
weeks and 8 weeks after the product administration. In comparison
between the groups, in 4 weeks and 8 weeks after the product
administration, there is no statistically significant difference
between the groups (p<0.05).
[0085] 2. Device Evaluation
[0086] 2-1. Skin Moisture Amount
[0087] As compared with before the product administration, in 4
weeks after the product administration, the skin moisture at the
face and the forearm was statistically significantly increased in
both test groups, and in 8 weeks after the product administration,
the skin moisture at the face was statistically significantly
increased in both the test group and the control group, and the
skin moisture at the forearm was statistically significantly
increased in the test group. In comparison between the groups, in 4
weeks and 8 weeks after the product administration at the face and
in 4 weeks after the product administration at the forearm, as
compared with the control group, in the test group, the skin
moisture was statistically significantly increased (p<0.05)
(Table 6).
TABLE-US-00006 TABLE 6 Changes in moisture amount between groups
.DELTA. Mean .+-. S.D. Site Weeks Group (A.U) p-value.dagger. Face
4 Control 0.64 .+-. 0.48 0.000***{circumflex over ( )} Test 6.47
.+-. 1.14 8 Control 2.39 .+-. 0.61 0.007** Test 8.10 .+-. 1.86
Forearm 4 Control 0.49 .+-. 0.44 0.030* Test 2.22 .+-. 0.62 8
Control 1.35 .+-. 1.04 0.068{circumflex over ( )} Test 4.14 .+-.
0.84 .dagger.p-value: Independent t-test (*p < 0.05, **p <
0.01, ***p < 0.001) .dagger.p-value{circumflex over ( )}:
Mann-Whitney test
[0088] 2-2. Transepidermal Water Loss (TEWL)
[0089] As compared with before the product administration, in 8
weeks after the product administration, at the face and the
forearm, TEWL was statistically significantly decreased in both
test groups. In comparison between the groups, in 8 weeks after the
product administration, at the forearm, the TEWL was statistically
significantly decreased in the test group as compared with the
control group (p<0.05) (Table 7).
TABLE-US-00007 TABLE 7 Comparison of Transepidermal water loss
between groups .DELTA. Mean .+-. S.D. Site Group Weeks (g/m.sup.2h)
p-value.dagger. IF.degree.(%) Face Control 0 13.02 .+-. 2.15 -- --
4 13.21 .+-. 2.12 0.392 .tangle-solidup.1.46 8 12.76 .+-. 2.08
0.330 1.20 Test 0 12.98 .+-. 1.83 -- -- 4 12.90 .+-. 2.05 0.837
0.62 8 11.93 .+-. 1.62 0.008** 8.09 Forearm Control 0 6.89 .+-.
1.16 -- -- 4 6.90 .+-. 1.16 0.936 .tangle-solidup.0.15 8 6.61 .+-.
1.31 0.187 4.06 Test 0 7.56 .+-. 1.04 -- -- 4 7.35 .+-. 1.35 0.303
2.78 8 6.50 .+-. 1.33 0.002**{circumflex over ( )} 14.02
.dagger.p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p
< 0.001) .dagger.p-value{circumflex over ( )}: Wilcoxon singled
rank test. .degree.Intensification factor(IF): (W X - W 0)/W 0
.times. 100, calculated by mean value
[0090] 2-3. Dead Skin Quantity
[0091] As compared with before the product administration, in 4
weeks and 8 weeks after the product administration, at the face,
the dead skin quantity was statistically significantly decreased in
the test group and at the forearm, the dead skin quantity was
statistically significantly decreased in both the control group and
the test group. In comparison between the groups, at both the face
and the forearm, in 4 weeks and 8 weeks after the product
administration, as compared with the control group, in the test
group, the dead skin quantity was statistically significantly
decreased (p<0.05) (Table 8).
TABLE-US-00008 TABLE 8 Chages in dead skin quantity between groups
Site Weeks Group .DELTA. Mean .+-. S.D. (pixels) p-value.dagger.
Face 4 Control 272.35 .+-. 3,795.82 0.002** 4 Test -14,696.67 .+-.
2,306.89 0.002** 8 Control -7,373.50 .+-. 4,395.46 0.000*** 8 Test
-38,263.71 .+-. 3,653.16 0.000*** Forearm 4 Control -29,518.20 .+-.
4,504.67 0.007**{circumflex over ( )} 4 Test -48,557.33 .+-.
5,394.31 0.007**{circumflex over ( )} 8 Control -35,369.05 .+-.
5,180.61 0.000*** 8 Test -72,941.62 .+-. 5,427.48 0.000***
.dagger.p-value: Independent t-test (*p < 0.05, **p < 0.01,
***p < 0.001) .dagger.p-value{circumflex over ( )}: Mann-Whitney
test
[0092] 2-4. Skin Color Brightness (L*Value)
[0093] As compared with before the product administration, at the
cheek portion, in 4 weeks after the product administration, the
skin color brightness was significantly increased in the test
group, and in 8 weeks after the product administration, the skin
color brightness was significantly increased in both the control
group and the test group. At the pigmented portion, in 4 weeks and
8 weeks after the product administration, the skin color brightness
(L*value) was significantly increased in both the control group and
the test group (p<0.05). In comparison between the groups, in 4
weeks and 8 weeks after the product administration at the cheek
portion and in 8 weeks after the product administration at the
pigmented portion, as compared with the control group, in the test
group, the skin color brightness (L*value) was significantly
increased (p<0.05) (Table 9).
TABLE-US-00009 TABLE 9 Comparison of Skin color brightness
(L*value) between groups depending on time .DELTA.Mean difference
.+-. Site Weeks Group Std. Error (L*value) p-value.dagger. Face
(cheek) 4 weeks Control group 0.25 .+-. 0.12 0.007**{circumflex
over ( )} Test group 0.81 .+-. 0.32 8 weeks Control group 0.48 .+-.
0.15 0.000***{circumflex over ( )} Test group 2.20 .+-. 0.29 Dark
spot 4 weeks Control group 0.97 .+-. 0.26 0.215{circumflex over (
)} Test group 1.15 .+-. 0.18 8 weeks Control group 1.60 .+-. 0.30
0.004** Test group 2.79 .+-. 0.26 .dagger.p-value: Independent
t-test (*p < 0.05, **p < 0.01, ***p < 0.001 Significant
difference between two groups) .dagger.p-value{circumflex over (
)}: Mann-Whitney test
[0094] 2-5. Blood Analysis
[0095] As the analysis result, as compared with before the product
administration, in 8 weeks after the product administration, in the
control group, there was a significant difference in T. Protein,
Albumin, and Creatinine items and in the test group, there was a
significant difference in T. Protein, Creatinine, and Pulse items,
but an average value of parameters was in a normal range
(p<0.05). In comparison between the groups, in 8 weeks after the
product administration, there was no significant difference between
the two groups in all parameters (Table 10).
TABLE-US-00010 TABLE 10 Result of blood analysis depending on types
of items .DELTA.Mean difference .+-. Items Groups Std. Error
(L*value) p-value.dagger. T. Protein Control group -0.25 .+-. 0.06
0.743 T. Protein Test group -0.28 .+-. 0.07 0.743 Albumin Control
group -0.10 .+-. 0.03 0.224 Albumin Test group -0.02 .+-. 0.05
0.224 T. Bil Control group -0.06 .+-. 0.04 0.599 T. Bil Test group
-0.10 .+-. 0.06 0.599 SGOT Control group 0.10 .+-. 1.71
0.743{circumflex over ( )} SGOT Test group 0.57 .+-. 0.84
0.743{circumflex over ( )} SGPT Control group 2.45 .+-. 1.50 0.617
SGPT Test group 1.48 .+-. 1.23 0.617 T. Chol Control group -1.25
.+-. 3.14 0.607 T. Chol Test group -3.57 .+-. 3.19 0.607
Triglyceride Control group 1.70 .+-. 15.20 0.754{circumflex over (
)} Triglyceride Test group -13.24 .+-. 8.07 0.754{circumflex over (
)} Blood glucose level Control group -2.70 .+-. 2.12 0.530 (Before
eating) Blood glucose level Test group -0.62 .+-. 2.49 0.530
(Before eating) BUN (Urea nitrogen) Control group 0.59 .+-. 0.66
0.879 BUN (Urea nitrogen) Test group 0.44 .+-. 0.69 0.879
Creatinine Control group 0.06 .+-. 0.02 0.758{circumflex over ( )}
Creatinine Test group 0.08 .+-. 0.02 0.758{circumflex over ( )}
Hemoglobin Control group 0.10 .+-. 0.11 0.875{circumflex over ( )}
Hemoglobin Test group 0.03 .+-. 0.16 0.875{circumflex over ( )}
Systolic blood Control group 1.35 .+-. 2.37 0.343 pressure Systolic
blood Test group 4.76 .+-. 2.64 0.343 pressure Diastolic pressure
Control group -1.30 .+-. 1.12 0.117 Diastolic pressure Test group
2.19 .+-. 1.83 0.117 Pulse Control group 2.80 .+-. 1.86
0.290{circumflex over ( )} Pulse Test group 5.38 .+-. 1.80
0.290{circumflex over ( )} .dagger.p-value: Independent t-test (*p
< 0.05, **p < 0.01, ***p < 0.001 Significant difference
between two groups) .dagger.p-value{circumflex over ( )}:
Mann-Whitney test
[0096] 2-6. Diet Survey and Weight
[0097] As the analysis result, as compared with before the product
administration, in 8 weeks after the product administration, in the
test group, the weight was significantly increased, but in 4 weeks
and 8 weeks after the product administration, there was no
significant difference in diet survey, weight, and BMI index
(p<0.05). In comparison between the groups, in 4 weeks and 8
weeks after the product administration, in both the control group
and the test group, there was no significant difference in diet
survey, weight, and BMI index (p<0.05) (Table 11).
TABLE-US-00011 TABLE 11 Diet survey and changes in weight between
groups depending on time (n = 41) .DELTA.Mean difference .+-. Items
Weeks Groups Std. Error (L*value) p-value.dagger. Diet survey 4
weeks Control group 0.01 .+-. 0.02 0.213{circumflex over ( )} Diet
survey 4 weeks Test group -0.01 .+-. 0.03 0.213{circumflex over (
)} Diet survey 8 weeks Control group 0.03 .+-. 0.02
0.168{circumflex over ( )} Diet survey 8 weeks Test group -0.01
.+-. 0.02 0.168{circumflex over ( )} weight 4 weeks Control group
0.31 .+-. 0.15 0.794 weight 4 weeks Test group 0.24 .+-. 0.20 0.794
weight 8 weeks Control group 0.55 .+-. 0.27 0.676{circumflex over (
)} weight 8 weeks Test group 0.67 .+-. 0.25 0.676{circumflex over (
)} BMI index 4 weeks Control group 0.05 .+-. 0.07 0.574 BMI index 4
weeks Test group 0.11 .+-. 0.09 0.574 BMI index 8 weeks Control
group 0.22 .+-. 0.12 0.947 BMI index 8 weeks Test group 0.21 .+-.
0.11 0.947 .dagger.p-value: Independent t-test (*p < 0.05, **p
< 0.01, ***p < 0.001 Significant difference between two
groups) .dagger.p-value{circumflex over ( )}: Mann-Whitney test
[0098] 3. Questionnaire Evaluation Result
[0099] 3-1. Questionnaire Evaluation Result on Efficacy
[0100] In items of `moisturizing skin`, `softening`, `reduced
pulling`, `reduced dead skin`, and `healthy skin`, in 4 weeks and 8
weeks after the product administration, 60% and 70% or more of the
subjects answered that the items were positively improved in both
the control product and the test product. In particular, in 4 weeks
after the product administration, items of `reduced pulling` and
`ordered skin texture` were improved 10% or more in the test
product as compared with the control group (Table 12).
TABLE-US-00012 TABLE 12 Questionnaire evaluation result on efficacy
depending on time (n = 41) Control group Test group (n = 20) (n =
21) Positive Satis- Positive Satis- Items responses faction
responses faction Items Week (N) (%) (N) (%) Level of improving 4 W
9 45.00% 8 38.10 in skin moisturizing.dagger. 8 W 13 65.00% 11
52.38 Level of improving 4 W 8 40.00% 8 38.10 in skin color.dagger.
8 W 11 55.00% 9 42.86 1. moisturizing 4 W 14 70.00% 14 66.67 skin 8
W 16 80.00% 16 76.19 2. softening 4 W 17 85.00% 15 71.43 8 W 18
90.00% 18 85.71 3. reduced pulling 4 W 13 65.00% 17 80.95 8 W 15
75.00% 16 76.19 4. reduced dead 4 W 12 60.00% 14 66.67 skin 8 W 16
80.00% 17 80.95 5. ordered skin 4 W 12 60.00% 15 71.43 texture 8 W
16 80.00% 13 61.90 6. brightened 4 W 12 60.00% 12 57.14 skin 8 W 14
70.00% 16 76.19 7. healthy skin 4 W 15 75.00% 13 61.90 8 W 16
80.00% 17 80.95 Criteria for questionnaire.dagger.: 0, No change/1,
feels small change but do not know the change/2, feels a little
improvement/3, feels improved/4, very much improved. Examiners
answered positively (%): 2~4 Criteria for questionnaire 1. Not at
all/2. No/3. It seems no/4. It seems likely/5. Likely/6. Very
likely. Examiners answered positively (%): 4~6
[0101] 3-2. Questionnaire Evaluation Result on Product
Usability
[0102] In all items except for `chewing feeling`, 70% or more of
the subjects positively answered in the control product and the
test product. Particularly, in items of `taste of product` and
`overall product satisfaction`, 100% of the subjects answered that
the items were satisfied in the test group (Table 13).
TABLE-US-00013 TABLE 13 Questionnaire on product usability after
using product Control group Test group (n = 20) (n = 21) Positive
Satis- Positive Satis- responses faction responses faction Items
(N) (%) (N) (%) 1. How about the color 16 80.00 20 95.24% of the
product? 2. How about the flavor 18 90.00 19 90.48% of the product?
3. How about taste of 19 95.00 21 100.00% the product? 4. How about
the form 14 70.00 17 80.95% of the product? 5. How about feeling 14
70.00 11 52.38% on chewing the product? 6. Are you satisfied 18
90.00 21 100.00% with the product overall? 7. Do you have an 15
75.00 15 71.43% intention to purchase of the product, if the
product is released? Criteria for questionnaire 1. Not good at
all/2. Not good/3. Normal/4. It seems good/5. Very good. Examiners
answered positively (%): 4~5
[0103] 4. Safety Evaluation Result
[0104] For the present test period, no adverse reactions to skin
and whole body systemic symptoms were observed in all subjects.
[0105] As the above result, it is determined that the present test
product helps in improvement of skin moisture, dead skin quantity,
transepidermal water loss (TEWL) and skin color.
Example 9: Preparation of Cosmetics Including Lactobacillus
plantarum K8 Lysate (Lactobacillus plantarum K8-LTA) and Human Body
Application Test
[0106] Clinical Trial Result
[0107] Three types of cosmetics including Lactobacillus plantarum
K8-LTA were prepared and experiments for evaluating efficacy of
improving skin moisturizing, skin color, skin elasticity, eye
wrinkles and dermis density were performed.
[0108] Tests in wrinkles and pigmentation were conducted by
targeting 32 female subjects aged 35 to 50. While test products
(Table 14) were continuously used for 8 weeks of a test period, at
time points of before using the products, 4 weeks after using the
products, and 8 weeks after using the products, visual evaluation
(eye wrinkles, skin color) and device measurement (skin
moisturizing, skin color, skin elasticity, eye wrinkles, and dermal
density) were performed. Left/right measurement portions were
defined by Block randomization (A: moisturizing and dermal density
of the left cheek, measurement of skin color, elasticity,
measurement of eye wrinkles of the right cheek/B: moisturizing and
dermal density of the right cheek, measurement of skin color,
elasticity, and measurement of eye wrinkles of the left cheek), and
safety and questionnaire evaluation were performed in 4 weeks and 8
weeks after using the products.
TABLE-US-00014 TABLE 14 Information on Cosmetics Name of cosmetics
k-lac Bio Essential Intensive Toner k-lac Bio Essential Ample
Essence k-lac Bio Essential Intensive Lifting Cream Type of
cosmetics Toner: colorless and solubilized type Essence: white and
cream type Cream: white and cream type Duration of using For 8
weeks cosmetics Method of using apply the appropriate amount of
face with an cosmetics order of toner - essence - cream in the
morning and evening, after washing face Used part of product Whole
face
[0109] The subjects participated in device measurement after
stabilization for at least 20 minutes after cleansing in a space
where there are no movement of air and direct sunlight and constant
temperature and humidity conditions (22.+-.2.degree. C. and
50.+-.5%) are maintained.
[0110] 1. Visual Evaluation
[0111] 1-1. Visual Evaluation Analysis of Eye Wrinkles for Each
Time
[0112] As compared with before using the test products, in 4 weeks
and 8 weeks after using the products, the wrinkles were
significantly improved (P<0.05), and the maximum variation of
wrinkles improvement was a decrease (improvement) rate of 5.50%
(see Table 15).
TABLE-US-00015 TABLE 15 Changes in eye wrinkles before and after
using the test products depending on time (n = 32) decrease or Time
of improvement measurement Mean(Grade) S.D p-value.dagger. rate
.sup.1 (%) 0 W 4.73 0.85 -- -- 4 W 4.58 0.77 0.005** 3.17 8 W 4.47
0.78 0.000*** 5.50 .dagger.p-value: Paired t-test (*p < 0.05,
**p < 0.01, ***p < 0.001, Significant difference compared to
before using the product) .sup.1 decrease or improvement rate:
(W.sub.x - W.sub.0)/W.sub.0 .times. 100, calculated by mean
value
[0113] 1-2. Visual Evaluation Analysis of Skin Color for Each
Time
[0114] As compared with before using the test products, in 4 weeks
and 8 weeks after using the products, the skin color was
significantly improved (P<0.05), and the maximum variation of
skin color improvement was a decrease (improvement) rate of 9.04%
(see Table 16).
TABLE-US-00016 TABLE 16 Changes in skin color before and after
using the test products depending on time (n = 32) decrease or Time
of improvement measurement Mean(Grade) S.D p-value.dagger.
rate.sup.1 (%) 0 W 5.09 1.26 -- -- 4 W 4.77 1.39 0.000*** 6.29 8 W
4.63 1.36 0.000*** 9.04 .dagger.p-value: Paired t-test (*p <
0.05, **p < 0.01, ***p < 0.001, Significant difference
compared to before using the product) .sup.1decrease or improvement
rate: (W.sub.x - W.sub.0)/W.sub.0 .times. 100, calculated by mean
value
[0115] 2. Device Measurement
[0116] 2-1. Measurement of Moisture
[0117] As compared with before using the test products, in 4 weeks
and 8 weeks after using the products, the moisture was
statistically significantly increased (improved) (P<0.05), and
the maximum variation of moisture improvement was an increase
(improvement) rate of 42.97% (see Table 17).
TABLE-US-00017 TABLE 17 Changes in skin moisture before and after
using the test products depending on time (n = 32) decrease or Time
of improvement measurement Mean(Grade) S.D p-value.dagger.
rate.sup.1 (%) 0 W 27.51 6.80 -- -- 4 W 34.02 5.54 0.000***
.tangle-solidup. 23.66 8 W 39.33 6.15 0.000*** .tangle-solidup.
42.97 .dagger.p-value: Paired t-test (*p < 0.05, **p < 0.01,
***p < 0.001, Significant difference compared to before using
the product) .sup.1decrease or improvement rate: (W.sub.x -
W.sub.0)/W.sub.0 .times. 100, calculated by mean value
[0118] 2-2. Measurement of Skin Color Brightness (L*Value)
[0119] As compared with before using the test products, in 4 weeks
and 8 weeks after using the products, the skin color brightness
degree at the cheek portion was statistically significantly
increased (improved) (P<0.05), and in 8 weeks after using the
products, the brightness degree at the pigmented portion was
statistically significantly increased (improved) (P<0.05). The
maximum variation of skin color brightness improvement was an
increase (improvement) rate of 1.59% at the cheek portion and 1.25%
at the pigmented portion.
TABLE-US-00018 TABLE 18 Changes in skin color before and after
using the test products depending on time (n = 32) decrease or Time
of improvement Site measurement Mean(Grade) S.D p-value.dagger.
rate .sup.1 (%) Cheek 0 W 62.14 3.19 -- -- 4 W 62.78 3.07 0.005**
.tangle-solidup. 1.03 8 W 63.13 2.90 0.000*** .tangle-solidup. 1.59
Pigmen- 0 W 59.09 3.27 -- -- tation 4 W 59.34 3.23 0.155
.tangle-solidup. 0.42 8 W 59.83 3.06 0.000*** .tangle-solidup. 1.25
.dagger.p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p
< 0.001, Significant difference compared to before using the
product) .sup.1 decrease or improvement rate: (W.sub.x -
W.sub.0)/W.sub.0 .times. 100, calculated by mean value
[0120] 2-3. Measurement of Skin Elasticity (Moire Topography
System)
[0121] As compared with before using the test products, in 4 weeks
and 8 weeks after using the products, the total distance and an
average angle of the contour lines were statistically significantly
decreased (improved) (P<0.05).
[0122] The maximum decrease rate of the entire length of the
contour line was 9.64% and the maximum decrease rate of the angle
was 14.70% (Table 19).
TABLE-US-00019 TABLE 19 Analysis result of skin elasticity before
and after using the test products depending on time (n = 32)
decrease or Time of improvement Category measurement Mean S.D
p-value.dagger. rate .sup.1 (%) Distance 0 W 270.31 61.76 -- --
(pixel) Distance 4 W 253.81 63.78 0.000*** 6.10 (pixel) Distance 8
W 244.25 54.87 0.000*** 9.64 (pixel) Angle 0 W 26.74 5.78 -- --
(.degree.) Angle 4 W 24.68 5.81 0.000*** 7.70 (.degree.) Angle 8 W
22.81 5.18 0.000*** 14.70 (.degree.) .dagger.p-value: Paired t-test
(*p < 0.05, **p < 0.01, ***p < 0.001, Significant
difference compared to before using the product) .sup.1 decrease or
improvement rate: (W.sub.x - W.sub.0)/W.sub.0 .times. 100,
calculated by mean value
[0123] 2-4. Measurement of Eye Wrinkles
[0124] As compared with before using the test products, in 4 weeks
after using the products, a mean depth of wrinkles and a max
wrinkle depth were statistically significantly decreased (improved)
and in 8 weeks after using the products, the max wrinkle depth was
statistically significantly decreased (improved) (p<0.05).
[0125] The maximum decrease rate of the `mean depth of wrinkles`
was 8.36% and the maximum decrease rate of the `maximum depth of
the wrinkles` was 26.17% (Table 20).
TABLE-US-00020 TABLE 20 Analysis result of eye wrinkle before and
after using the test products depending on time (n = 32) decrease
or Time of improvement Category measurement Mean S.D
p-value.dagger. rate .sup.1 (%) Total wrinkle 0 W 34.82 6.81 -- --
area (mm.sup.2) 4 W 34.68 8.32 0.903 0.40 8 W 33.83 7.84 0.377 2.84
Wrinkle 0 W 65.43 35.01 -- -- number 4 W 65.25 34.97 0.976 0.28 (N)
8 W 65.34 31.97 0.986 0.14 Average 0 W 0.91 0.24 -- -- length of 4
W 0.91 0.32 0.883 .sup. 0.00 wrinkles (mm) 8 W 0.93 0.31 0.721
.tangle-solidup. 2.20 Average depth 0 W 80.86 14.67 -- -- of
wrinkles 4 W 74.10 15.49 0.009** 8.36 (.mu.m) 8 W 78.97 16.95 0.377
2.34 Maximum 0 W 407.69 151.83 -- -- wrinkle depth 4 W 301.01 96.64
0.000*** 26.17 (.mu.m) 8 W 330.20 104.30 0.005** 19.01
.dagger.p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p
< 0.001, Significant difference compared to before using the
product) .sup.1 decrease or improvement rate: (W.sub.x -
W.sub.0)/W.sub.0 .times. 100, calculated by mean value
[0126] 2-5. Measurement of Skin Dermal Density
[0127] As compared with before using the test products, in 8 weeks
after using the products, the thickness of dermis was statistically
significantly increased (improved) and in 4 weeks and 8 weeks after
using the products, the density of dermis was statistically
significantly increased (improved) (p<0.05).
[0128] The maximum increase rate of the dermis thickness was 10.16%
and the maximum increase rate of the dermis density was 13.55%
(Table 21).
TABLE-US-00021 TABLE 21 Analysis result of skin dermal density
before and after using the test products depending on time (n = 32)
decrease or Time of improvement Category measurement Mean S.D
p-value.dagger. rate .sup.1 (%) Distance 0 W 1.28 0.16 -- -- (mm)
Distance 4 W 1.29 0.15 0.569 .tangle-solidup. 0.78 (mm) Distance 8
W 1.41 0.16 0.000*** .tangle-solidup. 10.16 (mm) Intensity 0 W
15.28 1.92 -- -- (%) Intensity 4 W 16.88 1.66 0.000***
.tangle-solidup. 10.47 (%) Intensity 8 W 17.35 1.42 0.000***
.tangle-solidup. 13.55 (%) .dagger.p-value: Paired t-test (*p <
0.05, **p < 0.01, ***p < 0.001, Significant difference
compared to before using the product) .sup.1 decrease or
improvement rate: (W.sub.x - W.sub.0)/W.sub.0 .times. 100,
calculated by mean value
[0129] 3. Questionnaire Evaluation
[0130] 3-1. Questionnaire Evaluation of Efficacy
[0131] In 4 weeks and 8 weeks after using the products, the
evaluation result on the efficacy of the products answered by the
subjects through questionnaire was as follows (Table 22).
TABLE-US-00022 TABLE 22 Results of questionnaire evaluation in
effects Level of improving in skin color.dagger. 4 W 3 9.38 8 W 17
53.13 Level of improving in skin wrinkles.dagger. 4 W 3 9.38 8 W 9
28.13 1. moisturizing skin 4 W 22 68.75 8 W 29 90.63 2. softening 4
W 27 84.38 8 W 32 100.00 3. ordered skin texture 4 W 25 78.13 8 W
32 100.00 4. brightened skin 4 W 22 68.75 8 W 30 93.75 5. clear
skin 4 W 22 68.75 8 W 30 93.75 6. Reduced skin sagging 4 W 19 59.38
8 W 25 78.13 7. healthy skin 4 W 23 71.88 8 W 32 100.00 Criteria
for questionnaire.dagger.: 0, No change/1, feels small change but
do not know the change/2, feels a little improvement/3, feels
improved/4, very much improved. Examiners answered positively (%):
3~4 Criteria for questionnaire 1. Not at all/2. No/3. It seems
no/4. It seems likely/5. Likely/6. Very likely. Examiners answered
positively (%): 4~6
[0132] 3-2. Questionnaire Evaluation of Product Usability
[0133] In 8 weeks after using the products, the evaluation result
on the final usability of the products answered by the subjects
through questionnaire was as follows (Table 23).
TABLE-US-00023 TABLE 23 Questionnaire on product usability Items
Toner Essence cream Positive Positive Positive Response
Satisfaction Response Satisfaction Response Satisfaction Items (N)
(%) (N) (%) (N) (%) 1. How about 23 71.88 26 81.25 28 87.50 the
color of the product? 2. How about 19 59.38 18 56.25 17 53.13 the
flavor of the product? 3. How about the 18 56.25 23 71.88 20 62.50
viscosity of the product? 4. How about the 21 65.63 24 75.00 24
75.00 adsorption rate of the product? 5. How about the 18 56.25 30
93.75 26 81.25 level of skin moist after adsorption of the product?
6. Are you 28 87.50 29 90.63 27 84.38 satisfied with the product
overall? 7. Do you have 24 75.00 29 90.63 25 78.13 an intention to
purchase of the product, if have an opportunity?
[0134] 4. Evaluation of Safety
[0135] In 4 weeks and 8 weeks after using the products, the
evaluation of subjective and objective stimuli was performed by a
tester by observing and questioning the subject's skin
condition.
[0136] For the present test period, no adverse reactions to skin
were observed in all subjects (Table 24).
TABLE-US-00024 TABLE 24 Evaluation of safety: adverse reactions to
skin (n = 32) Subjective feeling of stimulation Objective feeling
of stimulation Time of Stinging dead Pimple/ measurement Symptoms
itch pain irritating burning tingling stiffness pulling erythema
edema skin rash 4 W Weak 0 0 0 0 0 0 0 0 0 0 0 4 W Moderate 0 0 0 0
0 0 0 0 0 0 0 4 W Severe 0 0 0 0 0 0 0 0 0 0 0 8 W Weak 0 0 0 0 0 0
0 0 0 0 0 8 W Moderate 0 0 0 0 0 0 0 0 0 0 0 8 W Severe 0 0 0 0 0 0
0 0 0 0 0 Number of adverse 0 0 0 0 0 0 0 0 0 0 0 reactions
(Total)
[0137] It is determined that the present test products (three
cosmetics) helps in the improvement of skin moisturizing, skin
color, skin elasticity, skin color, eye wrinkles, and dermis
density.
Preparation Examples
Preparation Example 1: Preparation of Candy Containing
Lactobacillus plantarum K8 Lysate (Lactobacillus plantarum
K8-LTA)
[0138] A candy containing vegetable Lactobacillus plantarum K8
lysates was prepared. Raw materials used in the preparation of the
candy included purified glucose, Lactobacillus plantarum K8 lysates
2.1%, xylitol 2%, vitamin C, blueberry extract powder, anhydrous
citric acid, DL-malic acid, synthetic flavoring agents (blueberry
flavor and raspberry flavor), enzyme-treated stevia, and magnesium
stearate and were listed in Table 5. Next, an experiment for
determining whether aglycone isoflavone was included in the
prepared candy was performed. Three test candies having different
lot numbers and three control candies (showing ingredients in Table
25) were selected, grinded by using a mortar, and then suspended in
sterile distilled water. The suspended candy samples were crushed
by using a sonicator and then extracted by using ethanol. An
ethanol layer was mixed with methanol and then the LC/MS-MS
analysis was performed. As a result, daidzein and genistein as
aglycone isoflavones were detected from the test candies, but were
not detected in the control candies (Table 26).
TABLE-US-00025 TABLE 25 Comparison of ingredients in candies Sample
Control Title Plant Probiotic-contained Plant Probiotic free candy
candy Label Sample Control Composition Glucose, Glucose, L.
plantarum K8-LTA cell Xylitol 2%, Vitamin C, lysates 2.1%, Xylitol
2%, Blueberry concentrate Vitamin C, Blueberry powder, Citric acid
concentrate powder, Citric anhydrous, DL-malic acid, acid
anhydrous, DL-malic combined congener acid, combined congener
(Blueberry flavor, (Blueberry flavor, raspberry flavor), raspberry
flavor), Enzymatically Modified Enzymatically Modified Stevia
Glucosyl Stevia, Stevia Glucosyl Stevia, Magnesium Stearate
Magnesium Stearate Property Mauve candy Mauve candy Package White
airtight container White airtight container Storage Room
temperature Room temperature
TABLE-US-00026 TABLE 26 Result of measuring aglycone isoflavone in
candies Daidzein.dagger. Genistein.dagger. Control Candy 0 0 Sample
Candy 0.103 .+-. 0.064 0.093 .+-. 0.021 .dagger.Aglycone isoflavone
contents in candies: Mean .+-. S.D. (ng/ml)
Preparation Example 2: Preparation of Intestinal Regulation Agent
of Alactic Acid Bacteria Containing Aglycone
[0139] Lactobacillus plantarum K8 was mass-incubated in an MRS
medium added with various glycosides such as saponin or isoflavone
glycoside according to a method known in the art to prepare lactic
acid bacteria raw powder. A small amount of calcium and vitamin D
was mixed with the prepared Lactobacillus plantarum K8 raw powder
to prepare an intestinal regulation product. The detailed
compositions were described in Table 27 below. In order to enhance
an intestinal regulation effect, a small amount of other lactic
acid bacteria raw powder having an intestinal regulation effect,
for example, bifidus bacteria raw powder known to exist in the
large intestine may be added.
TABLE-US-00027 TABLE 27 Components of intestinal regulation agent
including Lactobacillus plantarum K8 Components 1 pack (wt %)
Lactobacillus plantarum K8 70 Vitamin D 15 Calcium 15 Total 100
Preparation Example 3: Preparation of Raw Food Including Lactic
Acid Bacteria Containing Aglycone
[0140] Various kinds of cereal powder, seaweed powder, fruit and
vegetable powder, mushroom powder and the aglycone lactic acid
bacteria raw powder of the present disclosure were mixed with
soybean embryo fermentation powder to prepare raw food products.
The detailed compositions were described in Table 28 below.
TABLE-US-00028 TABLE 28 raw food including Lactobacillus plantarum
K8 Components 1 pack (wt %) Soybean germ fermentation powder 25
Brown rice powder 10 Barley powder 5 Corn powder 3 Soybean powder 3
Adlay powder 3 Sesame powder 3 Red bean powder 3 Weed powder 5
Seaweed powder 8 Kelp powder 12 Kale powder 5 Aloe powder 3 Carrot
powder 2 Shiitake mushroom powder 4 Ganoderma lucidum powder 4
Lactobacillus plantarum K8 2 Total 100
Preparation Example 4: Preparation of Fermented Milk Including
Lactic Acid Bacteria Containing Aglycone
[0141] Skim milk or crude milk was mixed with a culture solution 1%
in which Lactobacillus plantarum K8 of the present disclosure was
pre-incubated and fermented for 6 to 8 hrs at 40.degree. C. to
prepare fermented milk. The detailed compositions were described in
Table 29 below. When fermenting the skim milk or the crude milk, in
order to shorten a fermentation time and improve the flavor of the
fermented milk, Streptococcus thermophilus or Lactobacillus
acidophilus, which is commercially available, may be used
together.
TABLE-US-00029 TABLE 29 components of fermented milk including
aglycone lactobacillus Components 1 pack (wt %) Nonfat dry milk 95
Oligosaccharide 4 aglycone lactic acid bacteria 1 Total 100
Preparation Example 6: Preparation of Fermented Soybean Liquid
Using Lactobacillus plantarum K8
[0142] A soybean liquid was mixed with a culture solution 1% in
which Lactobacillus plantarum K8 of the present disclosure was
pre-incubated and fermented for 6 to 8 hrs at 40.degree. C. to
prepare a fermented soybean liquid. The detailed compositions were
described in Table 30 below. When fermenting the soybean liquid, in
order to shorten a fermentation time and improve the flavor of the
fermented milk, Streptococcus thermophilus or Lactobacillus
acidophilus, which is commercially available, may be used
together.
TABLE-US-00030 TABLE 30 components of fermented soybean liquid
containing Lactobacillus plantarum K8 Components 1 pack (wt %)
Soybean liquid 90 Oligosaccharide 9 Lactobacillus plantarum K8 1
Total 100
Preparation Example 7: Preparation of Red Ginseng Lactic Acid
Bacteria
[0143] Lactobacillus plantarum K8 of the present disclosure was
incubated in 100 L of a minimal medium including red ginseng
extracts 0.1% for 12 hrs and then the cells were collected. The
collected cells were washed with water and then lyophilized and
included in a microcapsule to prepare red ginseng lactic acid
bacteria. The microcapsule may improve adhesion and cohesion by
considering an addition condition of a plasticizer, a type of
plasticizer, and surface tension of the capsules when a matrix is
formed by a wall material and a coagulating liquid. Because the
outside and the inside of the capsule make hard gelation, the
survival rate of the lactic acid bacteria in stomach acid is
increased and lactic acid bacteria capsules having high activity in
the intestines are made. As a primary coating solution, Na alginate
1.8%, glycerol 10%, xanthan gum 0.32%, Tween 20 0.05%, and MRS 5%
were used, as a hardening solution, CaCl.sub.2 0.5 M and Tween 80
0.5% were used, and as a secondary coating solution, chitosan 1.5%
and lactic acid 1.5% were used. Through the experiment, it was
confirmed that the red ginseng lactic acid bacteria capsules
produced by the condition were dissolved in the artificial
intestinal fluid at pH 7.4. Further, for an anti-acid test, the
ginseng lactic acid bacteria capsules reacted in the artificial
stomach liquid at pH 1.4 up to 180 minutes at time intervals and
then released from the artificial intestinal liquid and the viable
cell count was measured, and as a result, the survival rate in the
artificial stomach liquid was about 95%.
[0144] According to the preparing method of the present disclosure,
through the microorganism having ability of converting the
glycoside into the aglycone or hydrolyzed glycoside and ability of
accumulating the converted aglycone or hydrolyzed glycoside in the
cells, a microorganism capable of preparing aglycones or hydrolyzed
glycoside in a high concentration may be provided. Further,
microorganism viable cells or lysates thereof containing the
aglycone or hydrolyzed glycoside produced by the preparing method
of the present disclosure are prepared and may be variously used
for preparation of antioxidant compositions, intestinal regulation
agents, probiotic compositions, feed additives, food additives, raw
materials for cosmetics and other fermented products.
[0145] Although the specific part of the present disclosure has
been described in detail, it is obvious to those skilled in the art
that such a specific description is just a preferred embodiment and
the scope of the present disclosure is not limited. Thus, the
substantial scope of the present disclosure will be defined by the
appended claims and equivalents thereof
* * * * *