U.S. patent application number 10/732340 was filed with the patent office on 2005-06-16 for novel microorganism pediococcus pentosaceus erom101, having immune enhancement, anticancer and antimicrobial activities.
This patent application is currently assigned to EROMLIFE CO., LTD.. Invention is credited to Choi, Chang Won, Hong, Sung Gil, Hwang, Sang Ho, Im, Jong Jun, Jang, Jung Soon, Kim, Hwa Young, Kim, Joong Hark, Park, Mi Hyoun, Song, Mi Kyung, Woo, Suk Gyu.
Application Number | 20050130288 10/732340 |
Document ID | / |
Family ID | 34809735 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130288 |
Kind Code |
A1 |
Choi, Chang Won ; et
al. |
June 16, 2005 |
Novel microorganism Pediococcus pentosaceus EROM101, having immune
enhancement, anticancer and antimicrobial activities
Abstract
The present invention relates to a novel Pediococcus genus
microorganism and more particularly, Pediococcus pentosaceus
EROM101 (KCCM-10517) originated from human intestines having immune
enhancement, anticancer and antiviral activities and a use thereof.
Due to its excellent immune enhancement, anticancer and
antimicrobial activities by activating macrophages/spleen cells and
inducing gut immunity, the Pediococcus pentosaceus EROM101 of the
present invention can be effectively used for the production of
various products such as immune enhancement agent, anticancer
agent, antimicrobial agent, food additive, intestinal
function-controlling agent, live bacterial agent, feed additive and
other fermented products.
Inventors: |
Choi, Chang Won;
(Kwacheon-shi, KR) ; Park, Mi Hyoun; (Seoul,
KR) ; Hwang, Sang Ho; (Seoul, KR) ; Woo, Suk
Gyu; (Suwon-shi, KR) ; Song, Mi Kyung; (Seoul,
KR) ; Im, Jong Jun; (Seoul, KR) ; Hong, Sung
Gil; (Seoul, KR) ; Kim, Joong Hark; (Seoul,
KR) ; Jang, Jung Soon; (Seoul, KR) ; Kim, Hwa
Young; (Seoul, KR) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
EROMLIFE CO., LTD.
|
Family ID: |
34809735 |
Appl. No.: |
10/732340 |
Filed: |
December 11, 2003 |
Current U.S.
Class: |
435/243 |
Current CPC
Class: |
A23V 2002/00 20130101;
A23K 40/25 20160501; A23K 30/18 20160501; C12R 2001/01 20210501;
C12N 1/205 20210501; A23K 40/20 20160501; A61K 2035/11 20130101;
C12N 1/20 20130101; A23L 33/135 20160801; A23K 10/18 20160501; Y10S
435/822 20130101; A23K 10/12 20160501; A23Y 2280/55 20130101; A23L
3/3571 20130101; A23V 2002/00 20130101; A23V 2200/308 20130101;
A23V 2200/3204 20130101; A23V 2200/324 20130101 |
Class at
Publication: |
435/243 |
International
Class: |
A61K 039/02; C12N
001/00 |
Claims
What is claimed is:
1. A biologically pure culture of a microorganism Pediococcus
pentosaceus EROM101 (Accession number: KCCM-10517), or a mutant or
variant thereof having the same immune enhancement, anticancer and
antimicrobial activities as the said microorganism.
2. A composition for immune enhancement agent containing the
biologically pure culture of Pediococcus pentosaceus EROM101, or
mutant or variant thereof of claim 1.
3. A composition for anticancer agent containing the biologically
pure culture of Pediococcus pentosaceus EROM101, or mutant or
variant thereof of claim 1.
4. A composition for antimicrobial agent containing the
biologically pure culture of Pediococcus pentosaceus EROM101, or
mutant or variant thereof of claim 1.
5. A composition for intestinal function-controlling agent
containing the biologically pure culture of Pediococcus pentosaceus
EROM101, or mutant or variant thereof of claim 1.
6. A composition for live bacterial agent containing the
biologically pure culture of Pediococcus pentosaceus EROM101, or
mutant or variant thereof of claim 1.
7. A composition for feed additive containing the biologically pure
culture of Pediococcus pentosaceus EROM101, or mutant or variant
thereof of claim 1.
8. A composition for food additive containing the biologically pure
culture of Pediococcus pentosoceus EROM101, or mutant or variant
thereof of claim 1.
9. A fermented product containing the biologically pure culture of
Pediococcus pentosaceus EROM101, or mutant or variant thereof of
claim 1.
10. A method for culturing the Pediococcus pentosaceus EROM101, or
mutant or variant thereof of claim 1, wherein the Pediococcus
pentosaceus EROM101, or mutant or variant thereof is cultured in a
medium consisting of carbon source, nitrogen source, vitamin and
mineral at 30-45.degree. C. for 10-40 hours.
11. A method for enhancing immunity in a subject comprising
administering an effective amount of a biologically pure culture of
the Pediococcus pentosaceus EROM 101, or mutant or variant thereof
of claim 1 to a subject.
12. A method for treating cancer in a subject comprising
administering an effective amount of a biologically pure culture of
the Pediococcus pentosaceus EROM101, or mutant or variant thereof
of claim 1 to a subject.
13. A method for treating microbial infection in a subject
comprising administering an effective amount of a biologically pure
culture of the Pediococcus pentosaceus EROM101, or mutant or
variant thereof of claim 1 to a subject.
14. A method for controlling the microbial growth comprising
contacting the Pediococcus pentosaceus EROM101, or mutant or
variant thereof of claim 1 with other microorganisms.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel Pediococcus genus
microorganism and more particularly, Pediococcus pentosaceus
EROM101 (KCCM-10517) originated from human intestines having immune
enhancement, anticancer and antimicrobial activities and a use
thereof.
BACKGROUND OF THE INVENTION
[0002] Lactic acid bacteria are the bacteria metabolizing
carbohydrate and producing lactic acid thereby. These bacteria
belong to facultative anaerobes or obligatory anaerobes which
proliferate well under anaerobic conditions. Lactic acid bacteria
are divided into 5 genuses, which are Streptococcus, Lactobacillus,
Leuconostoc, Bifidobacteria and Pediococcus. A Streptococcus genus
microorganism is a homofermentive bacterium that generates lactic
acid by fermenting milk to suppress putrefying bacteria or
pathogenic bacteria A Lactobacillus genus microorganism is a
bacilliform, and a homo- or heterofermentive bacterium, which is
widely seen in the fermentation of dairy products or vegetables. A
Leuconostoc genus microorganism, a diplococcus, is a
heterofermentive bacterium and mostly involved in fermenting
vegetables. A Bifidobacteria genus microorganism is an obligatory
anaerobe which generates L(+) lactic acid useful for children's
health, but cannot survive under aerobic conditions. Lastly, a
Pediococcus genus microorganism is a homofermentive bacterium
having the form of tetracoccus, which is mostly found in Kimchi or
brined foods and involved in the fermentation of meats including
sausages. Among them, Bifidobacteria are the major obligatory
anaerobic lactic acid bacteria found in human intestines, and they
are counted 100-1000 times as many as facultative anaerobic lactic
acid bacteria such as Lactobacillus and Streptococcus in human
intestine.
[0003] Lactic acid bacteria control pH of intestines to keep them
acidic condition, so that they inhibit the proliferation of harmful
bacteria like E. coli or Clostridium sp. And they moderate diarrhea
and constipation, and play an important role in vitamin synthesis,
anticancer activity, lowering serum cholesterol and the like.
Particularly, lactic acid bacteria have a specific protein that can
be adhered tightly to mucosa and epithelial cells of intestines, to
have an intestinal function-controlling effect inhibiting the
growth of harmful bacteria. In addition, lactic acid bacteria
stimulate the proliferation of macrophages, resulting in the
enhancement of the capability of macrophage to recognize and
sterilize harmful bacteria invaded in intestines. They, further,
stimulate the secretion of immune-related substances, resulting in
the enhancement of the immunity (Gabriela perdigon et al., J. of
food Protection 53:404-410, 1990; Katsumasa sato et al., Microbiol.
Immunol., 32(7):689-698, 1988). Especially, Lactobacilius genus
microorganism produces acidophillin to inhibit the growth of
harmful bacteria such as dysentery bacillus (Shigella), salmonella
and staphylococcus, and the proliferation of causal bacteria of
diarrhea, resulting in the normalization of intestinal flora and
the cease of diarrhea In addition, Pediococcus pentosaceus has been
confirmed to have an excellent ability to inhibit the growth of
harmful bacteria such as Helicobacter pylori, Listeria sp. and the
like by secreting a strain specific antibacterial peptide.
[0004] Recently, studies have actively been progressing about
separating lactic acid bacteria and developing a live bacterial
medicine, food additive and intestinal function-controlling agent
using the same, in order to keep minor lactic acid bacteria
inhabiting human intestines longer. Korean patent No.264295
discloses "Novel Bifidobacterium longum MK-G7 bifidus strain having
a physiological activity suitable to Koreans and a use thereof",
and Korean patent No.158049 discloses "Bifidobacterium longum HS90
producing mucopolysaccharide having an excellent humidness". Also,
Korean patent No.2000-3 16517 discloses "Novel Lactobacillus
acidophilus YD9904 strain having an excellent acid producing
ability and acid resistance, and a product containing the same",
and the Korean patent No.2001-70689 discloses "Lactobacillus
acidophilus 30SC having a great acid resistance and bile-tolerant
activity as well as an antibacterial activity of inhibiting the
growth of pathogenic bacteria and putrefying bacteria". Korean
patent No.2001-11797 discloses "Novel acid-tolerant Lactobacillus
luteri probio-16 having an antimicrobial activity against rotavirus
and harmful microorganism, and live bacterial activator containing
the same". That is, almost all the studies about lactic acid
bacteria have been focused on Lactobacillus genus microorganisms
and Bifidobacterium genus microorganisms having an acid-tolerant
and/or an anti-microbial activity.
SUMMARY OF THE INVENTION
[0005] Accordingly, it is an object of the invention to provide
novel lactic acid bacteria having an excellent physiological
activity and inhabiting human intestines stably and a use thereof.
In the course of conducting continuous studies to develop novel
lactic acid bacteria having an immune enhancement activity, the
present inventors separated a novel Pediococcus genus microorganism
from human excrements and identified that the said microorganism
has immune enhancement, anticancer and antimicrobial activities,
and thus they have completed the present invention. Pediococcus
pentosaceus EROM101, or mutant or variant thereof of the present
invention showed excellent immune enhancement, anticancer and
antimicrobial activities by stimulating the activities of
macrophages/spleen cells and gut immunity, so that they can be used
for the production of various products such as immune enhancement
agent, anticancer agent, antimicrobial agent, food additive,
intestinal function-controlling agent, live bacterial agent, feed
additive and other fermented products.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a microphotograph showing the Pediococcus
pentosaceus EROM101 of the present invention.
[0007] FIG. 2 is a graph showing the number of live bacteria
according to the culture time of the Pediococcus pentosaceus
EROM101 of the present invention.
[0008] FIG. 3 is a set of graphs showing the proliferation rate (%)
of macrophages induced by the Pediococcus pentosaceus EROM101 of
the present invention. The proliferation rate was represented by
the percentage to the proliferation rate of macrophages induced by
Lactobacillus acidophilus, a test strain.
[0009] A: Comparison with a test strain
[0010] B: Proliferation rate according to cell concentration
[0011] FIG. 4 is a set of graphs showing the macrophage activity
(%) induced by the Pediococcus pentosaceus EROM101 of the present
invention. The activity was represented by the percentage to a
positive control.
[0012] A: Comparison of macrophage activity between live bacteria
EROM101 (ER) and cell lysate thereof (EL)
[0013] B: Comparison of macrophage activity between EROM101 cell
lysate (EL) and Lactobacillus acidophilus (test strain) cell lysate
(LL)
[0014] FIG. 5 is a graph showing the proliferation rate(%) of
spleen cells induced by the Pediococcus pentosaceus EROM101 of the
present invention. The proliferation rate was represented by the
percentage to a positive control.
[0015] EL: Group treated with EROM110 cell lysate
[0016] LL: Group treated with Lactobacillus acidophilus cell
lysate
[0017] FIG. 6 is a set of graphs showing the weight changes (A) of
mice while the Pediococcus pentosaceus EROM101 of the present
invention was administered orally, and tissue weight (B) measured
after 4 weeks with oral administration.
[0018] Con: Control group
[0019] ER: Group administered orally with live bacteria of
EROM101
[0020] EL: Group administered orally with cell lysate of
EROM101
[0021] LR: Group administered orally with live bacteria of
Lactobacillus acidophilus
[0022] LL: Group administered orally with cell lysate of
lactobacillus acidophilus
[0023] FIG. 7 is a graph showing the macrophage activity of mice
administered orally with the Pediococcus pentosaceus EROM101 of the
present invention. The activity was represented by the ratio to a
positive control.
[0024] ER: Group administered orally with live bacteria of
EROM101
[0025] EL: Group administered orally with cell lysate of
EROM101
[0026] LR: Group administered orally with live bacteria of
Lactobacillus acidophilus
[0027] LL: Group administered orally with cell lysate of
lactobacillus acidophilus
[0028] FIG. 8 is a set of graphs showing the proliferation rate (%)
of spleen cells of mice administered with the Pediococcus
pentosaceus EROM101 of the present invention. The proliferation
rate was represented by the percentage to a positive control.
[0029] A: Positive control (LPS-treated group)
[0030] B: Positive control (ConA-treated group)
[0031] ER: Group administered orally with live bacteria of
EROM101
[0032] EL: Group administered orally with cell lysate of
EROM101
[0033] LR: Group administered orally with live bacteria of
Lactobacillus acidophilus
[0034] LL: Group administered orally with cell lysate of
lactobacillus acidophilus
[0035] FIG. 9 is a graph showing the amount of IgA secreted from
mucosal membrane epithelial cells of the mice administered orally
with the Pediococcus pentosaceus EROM101 of the present
invention.
[0036] EL: Group administered orally with cell lysate of
EROM101
[0037] LL: Group administered orally with cell lysate of
Lactobacillus acidophilus
[0038] FIG. 10 is a set of graphs showing the anticancer activity
of Pediococcus pentosaceus of the present invention to stomach
cancer cell line (AGS; A), lung cancer cell line (A549; B.) and
blood cancer cell line (HL60; C). The anticancer activity was
obtained by measuring the proliferation rate (%) of cancer cells.
The proliferation rate was represented by the percentage to a
negative control.
[0039] FIG. 11 is a graph showing the weight increase rates of
mice, in which ascites cancer was induced, after cell lysate of
Pediococcus pentosaceus EROM101 of the present invention was
administered. The weight increase rates were calculated in terms of
the weight increase rates of normal mice without being injected
with ascites cancer cells.
[0040] EC-group: Cell lysate of EROM101 of the present invention
was injected into the abdominal cavity after the injection of
cancer cells
[0041] LC-group: Cell lysate of Lactobacillus acidophilus was
injected into the abdominal cavity after the injection of cancer
cells
[0042] TC-group: Saline was administered after the injection of
cancer cells
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides a Pediococcus pentosaceus
EROM101 having immune enhancement, anticancer and antimicrobial
activities, which was originated from human intestines.
[0044] It is known in the art to obtain mutants of microorganisms
without altering the characteristic thereof. For instance, mutants
may be obtained by treatment with physical or chemical mutagens
such as UV light, X-rays, gamma-rays and
n-methyl-N'-nitro-N-nitrosoguanidine. It is also known in the art
to obtain natural variants by e.g. screening cultures of the parent
strain. Therefore, the invention also pertains to the mutants or
variants of Pediococcus pentosaceus EROM101 which retain the
characteristics of the strains e.g. the immune enhancement,
anticancer and antimicrobial activities.
[0045] The present invention also provides a composition containing
the above Pediococcus pentosaceus EROM101, or mutant or variant
thereof for immune enhancement agent, anticancer agent,
antimicrobial agent, intestinal function-controlling agent, live
bacterial agent, feed additive and food additive.
[0046] The present invention further provides fermented products
containing the above Pediococcus pentosaceus EROM101, or mutant or
variant thereof.
[0047] The present invention also provides a method for culturing
the above Pediococcus pentosaceus EROM101, or mutant or variant
thereof
[0048] The present invention also provides a method for enhancing
immunity in a subject, comprising administering an effective amount
of a biologically pure culture of the above Pediococcus pentosaceus
EROM101, or mutant or variant thereof to a subject.
[0049] The present invention further provides a method for treating
cancer in a subject, comprising administering an effective amount
of a biologically pure culture of the above Pediococcus pentosaceus
EROM101, or mutant or variant thereof to a subject.
[0050] The present invention also provides a method for treating
microbial infection in a subject, comprising administering an
effective amount of a biologically pure culture of the above
Pediococcus penrosaceus EROM101, or mutant or variant thereof to a
subject.
[0051] The present invention further provides a method for
controlling the microbial growth, comprising contacting the above
Pediococcus pentosaceus EROM101, or mutant or variant thereof with
other microorganisms.
[0052] The present invention will be described in detail.
[0053] The present invention provides a novel Pediococcus genus
microorganism having excellent immune enhancement, anticancer and
antimicrobial activities. In order for lactic acid bacteria to work
effectively in human, they ought to inhabit human intestines stably
and have anti-bile activity and acid resistance. Thus, in order to
produce products containing lactic acid bacteria for human, the
lactic acid bacteria are required to be originated from human. So,
the present inventors have separated a Pediococcus genus
microorganism from human excrements, which is originated from human
intestines and has an excellent immune enhancement activity. First,
a number of lactic acid bacteria were selectively separated from
human excrements by using LBS agar medium, a lactic acid bacteria
selection medium. Then, biomass obtained from the culture of each
strain was cultured along with macrophages. A microorganism that
showed the highest macrophage proliferation rate was separated and
identified. As macrophages for this invention, those separated from
the abdominal cavity of a mammal are preferable, and particularly
in this invention, macrophages separated from the abdominal cavity
of a mouse were used. The separated microorganisms can be
identified by using the classification standard of the Bergey's
manual and analysis program with microorganism identification kit,
based on the morphological, cultural and physiological
characteristics of those microorganisms. Also, the identification
can be accomplished by investigating the physiological phenomena
such as Gram staining, oxygen requirement, nutritional requirement,
magnetic susceptibility, metabolite, enzyme reaction, antibiotics
resistance and the like by the molecular genetic analysis method
using DNA base sequence and 16S RNA structure, by the chemical
analysis method using the contents of a cell wall, quinone type of
electron transport system and the MIDI composition, and by the
immunological method. Particularly, in this invention, the
microorganism of the invention was identified by analyzing the
result obtained by using API 50 CHL kit (BioMereux Co., France), a
microorganism identification kit, with API LAB plus database V 5.0.
As a result, the microorganism separated by the present inventors
was confirmed to have 99.9% identity with Pediococcus pentosaceus.
Accordingly, the present inventors named the microorganism
`Pediococcus pentosaceus EROM101`, and deposited it at Korean
Culture Center of Microorganism on Oct. 10, 2003 (Accession No:
KCCM-10517).
[0054] Pediococcus pentosaceus EROM101 of the present invention has
following physiological characteristics;
[0055] First, the microorganism stimulates the macrophage
proliferation depending on cell concentration, and enhances
cellular immunity by macrophages, humoral immunity by spleen cells
and gut immunity.
[0056] Second, the microorganism significantly inhibits the cancer
cell proliferation.
[0057] Third, the microorganism shows a wide antimicrobial spectrum
against various harmful microorganisms.
[0058] Thus, Pediococcus penrosaceus EROM101 of the present
invention, or mutant or variant thereof can be effectively used as
a use for the enhancement of human or animal health. i.e., a
composition for immune enhancement agent, anticancer agent,
antimicrobial agent, intestinal function-controlling agent, live
bacterial agent or feed additive. The composition can include
crushed cell wall fraction live bacteria killed bacteria, dried
bacteria or culture product of Pediococcus pentosaceus EROM101, or
mutant or variant thereof as an effective ingredient. In addition,
excipients or carriers can be added therein. The said `culture
product` comprises the culture solution itself cultured on a liquid
medium, the solution left after removing strain by filtering or
centrifuging the culture solution(supernatant after centrifuging),
and cell lysate resulted from the ultrasonification or lysozyme
treatment of the culture solution. The amount of Pediococcus
pentosaceus EROM101, or mutant or variant thereof in the
composition varies depending on the use and formulation form of the
composition.
[0059] The composition of the invention for immune enhancement,
anticancer, antimicrobial, intestinal function-controlling agent or
live bacterial agent can be prepared and administered in many
formulation forms and by various methods. For example, the
Pediococcus pentosaceus EROM101, or mutant or variant thereof of
the invention can be produced and administered in the form of
tablet, troche, capsule, elixir, syrup, powder, suspension or
granule, by mixing with carriers and flavoring agents which are
used conventionally in the art of pharmaceutics. As such a carrier,
binder, lubricator, disintegrating agent, excipient, solubilizer,
dispersing agent, stabilizer, suspending agent and the like can be
used. The administering route includes oral administration,
parenteral administration or plastering, and oral administration is
more preferred. The dosage can be determined based on the
absorptivity of active ingredients, inactivity rate, excretion
speed, age, sex and condition of a patient. And the feed
composition of the present invention can be produced in the form of
fermented feed, formula feed, pellet feed and silage. The fermented
feed can be prepared by adding lactic acid bacteria of the present
invention, other microorganisms and enzymes, and fermenting organic
substances thereby. Also, the formula feed can be prepared by
mixing the Pediococcus pentosaceus EROM101, or mutant or variant
thereof of the present invention with general feed. The pellet feed
can be prepared by formulating the above fermented feed or formula
feed with a pelletizing machine. Silage can be prepared by
fermenting greenchop feed with the Pediococcus pentosaceus EROM101,
or mutant or variant thereof of the invention.
[0060] Also, the Pediococcus pentosaceus EROM101, or mutant or
variant thereof of the invention can be used as a food additive for
baby food, Kimchi, beverage and the like. Besides, the Pediococcus
pentosaceus EROM101, or mutant or variant thereof can be used as
seed bacteria for preparing fermented products. The fermented
products include fermented meat products such as ham and sausage,
fermented raw products, Kimchi and the like. The fermented products
using the Pediococcus pentosaceus EROM101, or mutant or variant
thereof of the invention can be prepared by the conventional method
well known in the art. For example, in order to prepare fermented
raw products, powders of cereals such as brown rice and Job's tears
were treated with the lactic acid bacteria of the present invention
or 2-3 strains of lactic acid bacteria mixture including the same,
leading to the fermentation at a required temperature, and then,
various agricultural products such as glutinous rice and sorghum
can be added properly to make nutritional balance and good
taste.
[0061] Pediococcus pentosaceus EROM101, or mutant or variant
thereof can be mass-produced by the conventional method for
culturing a Pediococcus genus microorganism. As a culture medium, a
medium consisting of carbon source, nitrogen source, vitamin and
mineral is available, and for example, MRS (Man-Rogosa-Sharp)
medium or milk-added medium can be used. The culture conditions of
a microorganism follow the conventional lactic acid bacteria
culture conditions, i.e., the microorganism can be cultured at 30
to 45.degree. C. for 10 to 40 hours. Culturing at 37.degree. C. for
18 hours is preferred. Centrifugation or filtration can be
performed to recover only concentrated bacteria with removing
culture medium from the culture solution, and this step can be
performed if the skilled in the art deems it necessary. The
concentrated bacteria can be frozen or lyophilized not to lose
their activities by the conventional method.
[0062] The `effective amount` in this invention refers to enough
amount to enhance immunity, or treat cancer or microbial infection
in a subject. The term `subject` as used herein refers to all
mammals, and in particular humans. Such subjects are also referred
to herein as patients in need of treatment.
[0063] The Pediococcus pentosaceus EROM101 of the present invention
stimulates macrophage proliferation and enhances cellular immunity,
humoral immunity and gut immunity (see FIG. 3-FIG. 5 and FIG.
7-FIG. 9). Thus, the present invention provides a method for
enhancing immunity in a subject, comprising administering an
effective amount of a biologically pure culture of Pediococcus
pentosaceus EROM101, or mutant or variant thereof into a subject.
The preferable administration forms and methods of Pediococcus
pentosaceus EROM101, or mutant or variant thereof are as explained
above.
[0064] The Pediococcus pentosaceus EROM101 of the present invention
significantly inhibits the cancer cell proliferation (see FIG. 10
and FIG. 11). Thus, the present invention also provides a method
for treating cancer in a subject, comprising administering an
effective amount of a biologically pure culture of Pediococcus
pentosaceus EROM101, or mutant or variant thereof into a subject.
The above cancers are not limited to, but may include breast
cancer, large intestinal cancer, lung cancer, small cell lung
cancer, stomach cancer, liver cancer, blood cancer, bone cancer,
pancreatic cancer, skin cancer, head or neck cancer, cutaneous or
intraocular melanoma, uterine sarcoma, ovarian cancer, rectal
cancer, anal cancer, colon cancer, fallopian tube carcinoma,
endometrial carcinoma, cervical cancer, vulval cancer, vaginal
carcinoma, Hodgkin's disease, esophageal cancer, small intestine
cancer, endocrine cancer, thyroid cancer, parathyroid cancer,
adrenal cancer, soft tissue tumor, urethral cancer, penile cancer,
prostate cancer, chronic or acute leukemia, lymphocytic lymphoma,
bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma,
renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, bone
marrow tumor, brain stem nerve gliomas, pituitary adenoma, or a
combination thereof. Preferably, the cancer might be stomach
cancer, lung cancer, blood cancer or ascites cancer. The preferable
administration forms and methods of Pediococcus pentosaceus
EROM101, or mutant or variant thereof are as explained above.
[0065] The microorganism Pediococcus pentosaceus EROM101 of the
present invention has a broad spectrum of activities in vitro with
respect to Gram-positive and Gram negative microorganism (see Table
3-5). Therefore, the present invention provides a method for
treating microbial infection in a subject, comprising administering
an effective amount of a biologically pure culture of Pediococcus
pentosaceus EROM101, or mutant or variant thereof into a subject.
The preferable administration forms and methods of Pediococcus
pentosaceus EROM101, or mutant or variant thereof are as explained
above.
[0066] The present invention also provides a method for controlling
microbial growth, comprising contacting Pediococcus pentosaceus
EROM101, or mutant or variant thereof with other microorganisms.
The microorganism comprises both Gram-positive bacteria such as
Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and
Gram-negative bacteria such as E. coli, Salmonella typhimurium,
Vibrio parahaemolyticus, Yersinia enterocolitica, but is not
limited thereto.
EXAMPLES
[0067] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0068] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1
[0069] Separation and Identification of the Pediococcus Genus
Microorganism of the Present Invention
[0070] 1 g of human excrements was suspended in 100 ml of saline,
then a required amount was taken therefrom and distributed on LBS
agar medium (sodium acetate 25 g/L, glucose 20 g/L, pancreatic
digest casein 10 g/L, KH.sub.2PO.sub.4 6.0 g/L, yeast extract 5
g/L, ammonium citrate 2 g/L, polysorbic acid 80 1 g/L, MgSO.sub.4
0.575 g/L, FeSO.sub.4 0.034 g/L, MnSO.sub.4 0.12 g/L, acetic acid
1.32 mL/L, agar 15 g/L, pH 5.5) to separate a single colony. Each
colony was inoculated on MRS medium (peptone 10 g/L, yeast extract
5 g/L, beef extract 10 g/L, glucose 20 g/L, polysorbic acid 1 g/L,
ammonium citrate 2 g/L, sodium acetate 5 g/L, MgSO.sub.4 0.1 g/L,
MnSO.sub.4 0.05 g/L, Na.sub.2HPO.sub.4 2 g/L), and then cultured at
37.degree. C. for 24 hours. Bacteria were recovered from the
culture solution by centrifugation, which were then crushed by
ultrasonicator to prepare samples for this experiment. The samples
were cultured along with macrophages separated from the abdominal
cavity of a Balb/c mouse for 12 hours to select the strain showing
the highest macrophage proliferation rate. The macrophage
proliferation was investigated by measuring the amount of dye
accumulated in the survived cells by taking advantage of neutral
red assay. The selected strain was identified using API 50 CHL kit
(BioMereux Co., France). The strain was suspended in API CHL medium
and distributed on a strip for further culture. The result was
interpreted by investigating the change in color of fermented
carbohydrates by pH resulting from the generation of acid. All the
results were analyzed using API LAB plus database V 5.0, a
microorganism identification program. As a result, the selected
strain showed 99.9% identity with Pediococcus pentosaceus. Thus,
the present inventors named the strain `Pediococcus pentosaceus
EROM101` and deposited it at Korean Culture Center of Microorganism
on Oct. 10, 2003 (Accession No: KCCM-10517). FIG. 1 is a
microphotograph showing the Pediococcus pentosaceus EROM101 of the
present invention (referred `EROM101` hereinafter). The biochemical
characteristics of the strain are as follows and the sugar
utilization was listed in Table 1.
[0071] <Characteristics of the Strain>
[0072] 1. Growth temperature: Grow well at 30-45.degree. C., cannot
grow at 15.degree. C. (optimum growth temperature: 37.degree.
C.)
[0073] 2. Growth pH: 6-7 (optimum growth pH: 6.5)
[0074] 3. Mobility: None
[0075] 4. Gram staining: Positive
[0076] 5. .alpha.-glucosidase activity: Positive,
.beta.-galactosidase activity: Negative
[0077] 6. Oxygen requirement: Facultative anaerobic
1TABLE 1 Sugar utilization of Pediococcus pentosaceus EROM101 of
the invention Sugar Reaction Sugar Reaction Glycerol - Salicine +
Erythritol - Cellobiose + D-arabinose - Maltose + L-arabinose +
Lactose + Ribose + Melibiose + D-xylose + Saccharose + L-xylose -
Trehalose + Adonitol - Inuline - .beta.methyl-xyloside - Melezitose
- Galactose + D-Raffinose + D-glucose + Amidon - D-fructose +
Glycogene - D-mannose + Xylitol - L-Sorbose - .beta. Gentiobiose +
Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose
+ Mannitol - D-Fucose - Sorbitol - L-Fucose -
.alpha.-methyl-D-mannoside - D-Arabitol - .alpha.-methyl-D-glucosi-
de - L-Arabitol - N-acetylglucosamine + Gluconate - Amygdaline +
2-keto-gluconate - Arbutine + 5-keto-gluconate - Esculine +
Example 2
[0078] Investigation of the Immune Enhancement Activity of EROM101:
in vitro Test
[0079] <2-1> Investigation of Proliferation Rate and Activity
of Macrophage
[0080] a) Measuring Proliferation Rate of Macrophage
[0081] In order to investigate the immune enhancement activity of
EROM101 separated in the Example 1, the viable cells were measured
according to culturing time. The pre-cultured strain was inoculated
on MRS medium to 2% of the final density, which was further
cultured at 37.degree. C. During the culturing, viable cells were
measured according to culturing time. As a result, as shown in FIG.
2, the maximum viable cells were observed when they ha been
cultured for 18 hours.
[0082] In order to confirm the immune enhancement activity of
EROM101 of the present invention, EROM101 pre-cultured for 18 hours
was further cultured for 24 hours with macrophages originated from
the abdominal cavity of mice as seen in Example 1. At this time,
Lactobacillus acidophilus (ATCC 43121), a test strain was used as a
control. Macrophage proliferation rate was investigated by the same
method used in Example 1, and was represented by the percentage to
that of a control group. As a result, as shown in FIG. 3A,
macrophage proliferation by EROM101 of the invention was much
higher than that by Lactobacillus acidophilus. Also, in order to
investigate the macrophage proliferation rate by measuring viable
cells, EROM101 pre-cultured for 18 hours was diluted to each
concentration and macrophages originated from mice were added to
each concentration for co-culture. As a result, as shown in FIG.
3B, macrophage proliferation was increased depending on cell
concentration. From the result, it was confirmed that EROM101 of
the present invention stimulates macrophage proliferation in high
level.
[0083] b) Investigating Activity of Macrophage
[0084] 1 ml of sterilized thioglycoltate (Sigma-Aldrich, USA) was
injected into the abdominal cavity of a 5 week old Balb/c mouse
(Daehan Biolink, Korea), followed by the activation for 72 hours.
Then, its cervical vertebra was dislocated. 5 ml of RPMI 1640
medium (Gibco, New York, USA) was injected into the abdominal
cavity of the mouse for washing. After the washing, the medium was
recovered. The procedure was repeated 2-3 times. The recovered
solution was centrifuged at 4.degree. C. with 1,500 rpm for 10
minutes, which was suspended in a medium to the cell number of
1.times.10.sup.6 cells/ml. The solution was distributed into a
96-well plate by 100 .mu.l per each well and the final cell density
was adjusted to 1.times.10.sup.5 cells/ml. The plate was cultured
in a 5% CO.sub.2 incubator at 37.degree. C. for 2 hours. It was
confirmed whether macrophages were fixed on the plate to form a
monolayer. The culture solution was removed from the plate, then
live EROM101 and cell lysate thereof were added to RPMI 1640 medium
(+10% FBS) by concentrations (1 .mu.g, 10 .mu.g, 100 .mu.g and 1
mg). The medium was distributed by 200 .mu.l per each well. The
said live bacteria were prepared by filtrating the cell culture
solution (1.times.10.sup.9 cells therein) through 0.2 .mu.m filter.
The preparation of the said lysate was as follows; the cell culture
solution was centrifuged with 6,000 rpm for 15 minutes, and the
supernatant was removed and washed with PBS twice. Lysozyme from
hen egg white (Fluka Co. Switzerland) was treated thereto by the
concentration of 0.1 g/10 ml, which was left in a 37.degree. C.
shaking incubator for 1 hour and filtered by 0.2 .mu.m filter. In
the meantime, RPMI 1640 medium (+10% FBS) containing 20 .mu.l of
LPS (lipopolysaccharide; B-cell inducing substance extracted from
E. coli) was distributed for a positive control, and the medium was
distributed for a negative control. Macrophages were activated by
means of culturing in a 5% CO.sub.2 incubator for 24 hours.
Macrophage activity was investigated by measuring the activity of
enzyme (lysosomal enzyme) that was secreted during the macrophage
activation. After completing the culture, supernatant was removed
25 .mu.l of 0.1% triton-X-100 was added, followed by a reaction in
an incubator for 20 seconds. 150 .mu.l of 100 mM .rho.-nitrophenyl
phosphate and 50 .mu.l of 0.1 M citrate buffer solution(pH 6.0)
were added thereto, followed by a reaction in a 37.degree. C. 5%
Co.sub.2 incubator for 45 minutes. The reaction was stopped by
adding 50 .mu.l of 0.2 M borate buffer solution (pH 9.6).
OD.sub.405 was measured with ELSIA reader (TECAN GENios,
Austria).
[0085] As a result, as shown in FIG. 4A, the lysate of EROM101 of
the present invention showed higher macrophage activity than live
EROM101. The maximum activity of the lysate was 93.84%, which was
very high compared to that of a positive control (LPS-treated
group)(100%).
[0086] c) Comparison of the Macrophage Activity Between EROM101 and
Test Strain
[0087] The macrophage activity of EROM101 of the present invention
was compared with that of Lactobacillus acidophilus (ATCC 43121), a
test strain. The investigation procedure was the same as used in
the above b). As a result, as shown in FIG. 4B, EROM101 of the
present invention showed much higher macrophage activity than
Lactobacillus acidophilus.
[0088] <2-2> Investigation of the Spleen Cell Activity
[0089] The spleen of a 5-week-old Balb/c mouse was taken out and
spleen cells were separated therefrom using a mesh Centrifugation
was performed at 4.degree. C. with 1,700 rpm for 5 minutes and the
supernatant was removed. After washing with RPMI 1640 medium twice,
3 ml of lysis buffer (155 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM
EDTA) was added in order to eliminate red blood cells included in
the spleen cell solution. After it had been put in a 37.degree. C.
water bath for 5 minutes, centrifugation was performed again at
4.degree. C. with 1,700 rpm for 5 minutes. Supernatant was removed,
followed by washing with RPMI 1640 medium twice. The final
concentration of the cell solution was adjusted to 5.times.10.sup.6
cells/ml and then the solution was distributed to each well of a
96-well plate by 200 .mu.l. The lysate of EROM101 of the present
invention and the lysate of Lactobacillus acidophilus (ATCC 43121)
were grouped by concentrations for further treatment. The said
lysates were prepared by the same method as used in Example
<2-1> b). 10 .mu.g/.mu.l of LPS was treated to a positive
control and the sterilized water was treated to a negative control.
The spleen cell activity was investigated by measuring the
proliferation rate of the spleen cell using MTS assay. After it had
been cultured in 5% CO.sub.2 incubator at 37.degree. C. for 72
hours, 20 .mu.g/ml of MTS (CellTiter 96 Aqueous One Solution Cell
Proliferation Assay. Promega, Cat No: G3580) was treated thereto,
followed by a reaction for 1-3 hours. OD.sub.492 was measured with
ELISA reader (TECAN GENios, Austria).
[0090] As a result, as shown in FIG. 5, EROM101 of the present
invention showed much higher spleen cell activity than
Lacrobacillus acidophilus, a test strain. The maximum activity was
123.13%, which was very high compared to that of a positive control
(100%).
Example 3
[0091] Investigation of Immune Enhancement Activity of EROM101: in
vivo Test
[0092] <3-1> Sample Ingestion
[0093] 3 week old Balb/c female mice(Daehan Biolink, Korea) were
classified. by 6 mice per each group, into 5 groups; 1) control
group (sterilized water administered group), 2) live EROM101
orally-administered group (ER), 3) EROM101 lysate
orally-administered group (EL), 4) live Lactobacillus
orally-administered group (LR), and 5) Lactobacillus lysate
orally-administered group (LL), to which 200 .mu.l of each sample
were orally administered once a day for 4 weeks. Except filtering
process, the said live bacteria and lysate were prepared by the
same methods as used in Example <2-1> b). In the experiments
for investigating the activities of macrophages, spleen cells and
gut immunity, LPS, a B-cell inducing material, and ConA
(concanvali), a T-cell inducing material, were used by 10 .mu.g/ml,
respectively, for a positive control. The mice of each group were
raised under the fixed condition in which temperature was set at
32.+-.2.degree. C., humidity was set to .+-.20%, and the light
exposure for 12 hours (from AM 07:00 to PM 07:00). Solid feed for a
mouse and tap water were given to the mice freely.
[0094] <3-2> Measuring the Change in Total Weight and the
Weight of the Internal Organs
[0095] Weight changes were observed to investigate the growth of
the mice of each group. As a result, as shown in FIG. 6A, there
were no significant changes in both experimental groups and control
groups. In order to investigate if the internal organs of each
mouse were normal, the abdominal cavities of mice were opened 4
weeks after the oral administration and the internal organs (spleen
and liver) were separated to measure their weights. As a result, as
shown in FIG. 6B, all the groups showed similar aspects.
[0096] <3-3> Measuring Biochemical and Hematological Indexes
in Blood
[0097] 4 weeks after the oral administration, blood was taken from
orbit, and serum was separated from each mouse. Biochemical indexes
in blood--liver functional indexes (GOT and GPT) and kidney
functional indexes (BUN and creatine)--were measured by a
biochemical analyzer (Cobas Mira plus), and hematological indexes
(red blood cell, platelet and blood figment) were also measured by
a blood corpuscle analyzer (Medonic Ca 620, Boule, Sweden). As a
result, as shown in below Table 2, the biochemical and
hematological indexes of the mice from every group were all
normal.
2TABLE 2 Biochemical and hematological indexes Measured Index
Normal value measured range Group (Mean .+-. SD) Biochemical GOT
Under Control 85.9 .+-. 8.99 Index 247 U/L .sup.aER 104.46 .+-.
11.22 .sup.bEL 117.72 .+-. 24.47 .sup.cLR 110.27 .+-. 7.87 .sup.dLL
130.18 .+-. 7.87 GPT Under Control 28.67 .+-. 1.96 132 U/L ER 33.98
.+-. 2.11 EL 36.86 .+-. 5.85 LR 30.72 .+-. 4.17 LL 37.56 .+-. 5.02
BUN Under Control 28.59 .+-. 3.97 29 U/L ER 26.12 .+-. 4.61 EL
26.26 .+-. 4.47 LR 27.63 .+-. 5.49 LL 26.61 .+-. 3.41 Creatine
Under Control 0.65 .+-. 0.14 0.9 U/L ER 0.84 .+-. 0.14 EL 0.73 .+-.
0.16 LR 0.66 .+-. 0.09 LL 0.77 .+-. 0.08 Hematological RBC 6-12
Control 9.42 .+-. 0.54 Index ER 8.69 .+-. 0.77 EL 10.66 .+-. 0.27
LR 9.39 .+-. 0.49 LL 8.93 .+-. 0.54 PLT 190-1000 Control 431.75
.+-. 192.12 ER 423.83 .+-. 182.84 EL 577.5 .+-. 66.11 LR 673.75
.+-. 86.44 LL 437.75 .+-. 75.61 HGB 12-17 Control 14.25 .+-. 0.66
ER 13.8 .+-. 2.9 EL 18.4 .+-. 0.81 LR 16.9 .+-. 1.62 LL 13.82 .+-.
0.65 .sup.aER: Group administered orally with live ERM101 of the
present invention .sup.bEL: Group administered orally with cell
lysate of EROM101 of the present invention .sup.cLR: Group
administered orally with live Lactobacillus acidophilus .sup.dLL:
Group administered orally with cell lysate of Lactobacillus
acidophilus
[0098] <3-4> Investigation of Macrophage Activity
[0099] Macrophages were separated from each mouse of every group.
The macrophage activity was investigated by measuring the activity
of an enzyme secreted during the macrophage activation using the
same method as used in the above Example <2-1> b). As a
result, as shown in FIG. 7, the highest activity was seen in
EROM101 lysate orally-administered group (EL) and Lacrobacillus
lysate orally-administered group (LL), live EROM101
orally-administered group (ER) and live Lactobacillus
orally-administered group (LR) follows in that order. In addition,
the EROM101 lysate orally-administered group showed higher activity
than the positive control group.
[0100] <3-5> Investigation of Spleen Cell Activity
[0101] Spleen cells were separated from each mouse of every group
by using same method as Example <2-2> to investigate the
spleen cell activity by using MTS assay. As a result, as shown in
FIG. 8, the highest activity was seen in EROM101 lysate
orally-administered group (EL) and live EROM101 orally-administered
group (ER), Lactobacillus lysate orally-administered group (LL) and
live Lactobacillus orally-administered group (LR) follows in that
order. Particularly, EROM101 lysate orally-administered group (EL)
showed higher activity than the positive controls, LPS treated
group and ConA treated group.
[0102] <3-6> Investigation of the Activity of Gut
Immunity
[0103] In order to investigate the activity of gut immunity, the
amount of IgA secreted in peyer's patch was measured. First, all
mice were sacrificed by dislocation of cervical vertebrae. Then,
the abdominal cavities of the mice were opened to take out small
intestines. Peyer's patch was separated to extract target cells
using a mesh The obtained cells were suspended in RPMI 1640 medium
(+10% FS) by the concentration of 1.times.10.sup.6 cells/ml. The
suspension was distributed by 1 ml to each well of a 24-well plate.
10 .mu.g/ml of LPS was additionally treated to some wells (positive
control). Peyer's patch was activated by culturing the plate in 5%
CO.sub.2 incubator at 37.degree. C. for 5 days. After finishing the
culture, supernatant was taken to measure the total antibody
content of IgA. For that purpose, anti-mouse IgA antibody
(Phamingen, USA) was fixed on a 96-well plate, to which Block ace
(PBS containing 10% FBS) was treated, resulting in the inhibition
of unspecific binding of antibody. Then, the sample containing IgA
was mixed The reaction with peroxidase-conjugated anti-mouse IgA
antibody (Phamingen, USA) was followed. OD.sub.450 was measured
with ELISA reader (TECAN GENios, Austria).
[0104] As a result, as shown in FIG. 9, EROM101 lysate administered
group showed the highest IgA generation, which was 2.8 times higher
than that of the positive control.
[0105] From all the above results, EROM101 of the present invention
has been confirmed to have much more excellent immune enhancement
activity than Lactobacillus acidophilus, a test strain.
Example 4
[0106] Investigation of the Anticancer Activity of EROM101
[0107] <4-1> In vitro Test
[0108] In order to investigate the anticancer activity of EROM101
of the present invention, the capability of EROM101 to inhibit the
cancer cell proliferation in human cancer cell lines was measured.
The stomach cancer cell line AGS (adenocarcinoma, stomach, human,
KCLB No.21739), the lung cancer cell line A549 (carcinoma, lung,
human, KCLB N. 10185), and the blood cancer cell line HL60
(leukemia, blood, human, KCLB No. 10240) were distributed in a
96-well plate by 2.0-2.5.times.10.sup.4 cells/ml per each well.
EROM101 lysate and Lactobacillus acidophilus (test strain, ATCC
43121) lysate were treated by concentrations, which were, then,
cultured for 24 or 72 hours. RPMI 1640 medium containing
heat-inactivated FBS, 100 .mu.g/ml penicillin and 100 .mu.g/ml
streptomycin was used for the culture in 5% CO.sub.2 (95% air)
incubator at 37.degree. C. The said lysate was prepared by the same
method as used in the above Example <2-1> b). Serum-free
medium was treated to a negative control. After finishing the
culture, 20 .mu.l of MTS (Cat. No. G3581, Promega Co., USA) was
treated to induce color development. In one hour, OD.sub.492 was
measured with ELISA reader (TECAN GENios, Austria).
[0109] As a result, as shown in FIG. 10, EROM101 of the present
invention was confirmed to significantly inhibit cancer cell
proliferation in stomach, lung, blood cancer cell lines, comparing
to Lactobacillus acidophilus, a test strain. Particularly, the
inhibition effect was at maximum 42.5% in a stomach cancer cell
line, 35.5% in a lung cancer cell line, and was high depending on
concentration in a stomach cancer cell line. All the results were
statistically significant.
[0110] <4-2> In vivo Test
[0111] 8 week old 40 ICR mice (Daehan Biolink, Korea) were
classified into 4 groups and 1.times.10.sup.6 sarcoma 180 cell line
(KCLB 40066) were injected into the abdominal cavities of the mice
in three groups to induce ascites cancer. Two of the above three
groups were treated with EROM101 lysate and Lactobacillus
acidophilus (ATCC 4356) lysate every other day after the sarcoma
180 cell line injection, respectively. The lysates were injected
into the abdominal cavities of mice once two day for 14 days. The
said lysates were prepared by the same method as used in Example
<2-1> b) except filtering. One of the above three groups was
injected with saline using the same method as said two groups. The
above four groups were as follows; 1) N-group in which cancer cells
were not injected, 2) TC-group in which saline was injected in
their abdominal cavity after the injection of cancer cells, 3)
EC-group in which EROM101 lysate was injected in their abdominal
cavity after the injection of cancer cells, and 4) LC-group in
which Lactobacillus acidophilus lysate was injected in their
abdominal cavity after the injection of cancer cells. AU the mice
of each group were raised under the pathogen-free, standard
conditions. For the next two weeks, the weight changes caused by
the increase of ascites cancer cells were observed. The increased
weight was converted into the increase rate to the weight of
N-group mice in which cancer cells were not injected, leading to
the determination of anticancer effect by immune enhancement.
[0112] As a result, as shown in FIG. 11, the weight increase rate
was 44% in TC-group compared to N-group, which seemed to be caused
by the growth of ascites cancer cells. And the weight increase rate
of LC-group injected with Lactobacillus acidophilus was 15%,
compared to the rate of TC-group. On the other hand, EC-group
injected with EROM101 of the invention showed only 4% weight
increase. Therefore, EROM101 of the present invention was confirmed
to have an anticancer activity.
Example 5
[0113] Investigation of the Antimicrobial Activity of EROM101
[0114] <5-1> Investigation of the Antimicrobial Activity to
Gram-Positive and Gram-Negative Bacteria
[0115] EROM101 of the present invention was cultured in 250 ml
Erlenmeyer flask at 35.degree. C. for 48 hours. At that time,
volume was adjusted to 100 ml and MRS medium (Difco Lab., USA) was
used. As test strains for the investigation of the antimicrobial
activity, the strains listed in Table 3 were used. TSA medium
(Difco Lab., USA) was used for the culture. The antimicrobial
activity of EROM101 of the present invention was investigated by
using paper disk method. 5.5 ml of soft agar (0.75%), on which the
test strain was inoculated, was piled up on the TSA medium to
prepare a plate for the investigation of the antimicrobial
activity. On that plate, paper disk (8 mm in diameter) (Adventec
Toyo RoshiKaisha. Ltd., Japan) was placed, onto which the culture
broth, supernatant and cell lysate of EROM101 were distributed by
80 .mu.l respectively, followed by a further culture at 37.degree.
C. for 18 hours. The supernatant was obtained by centrifuging the
culture broth at 4.degree. C. with 8,000 rpm. In order to obtain
the lysate. PBS was added to the pellet generated from the above
centrifugation to prepare suspension. And the suspension was
treated with sonic Dismenbrator (Model 500 Fisher scientific USA),
resulting in lysate. The generation of a growth inhibition ring was
identified.
[0116] As a result, as shown in Table 3, EROM101 of the present
invention was confirmed to inhibit the growth of all test strains.
The antimicrobial activity was detected in the culture broth and
the supernatant, but not in the lysate.
3TABLE 3 Antimicrobial activity of EROM101 Inhibition Culture Type
Test Strain Broth Supernatat Lysate Gram- Bacillus cereus, + + -
positive ATCC 14579 Staphylococcus aureus, + + - KCTC 1621 Listeria
monocytogenes, + + - KFRI 799 Gram- Escherichia coli, + + -
negative ATCC 11775 Escherichia coli O 157:H7 + + - 933 Salmonella
typhimurium, + + - ATCC 12023 Vibrio parahaemolyticus, + + - KCCM
41664 Yersinia enterocolitica, + + - KTCC 9610
[0117] <5-2> Comparison of the Antimicrobial Activity of EROM
With Test Strains
[0118] The antimicrobial activity of EROM101 of the present
invention was compared with those of Pediococcus pentosaceus (KCCM
11902) and Lactobacillus acidophilus (ATCC 43121). At that time,
Bacillus cereus (ATCC 14579) and Listeria monocytogenes (KFRI 799)
were used as indicators. For the investigation of the antimicrobial
activity, the above two indicator bacteria were smeared on TSA agar
plate by 1.times.10.sup.6 CFU/ml using a cotton rod. The growth
inhibition ring was confirmed by using the same method as used in
the above Example <5-1>. The antimicrobial activity unit was
calculated by assuming that 1 AU (Arbitrary Unit) is determined to
be 1 mm which is the size of the remaining dimensions after
subtracting the dimensions of paper disk from the dimensions of the
generated growth inhibition ring.
[0119] As a result, as shown in Table 4 and Table 5, EROM101 of the
present invention showed much higher antimicrobial activity against
the two indicators than other test strains.
4TABLE 4 Antimicrobial activity of EROM101 to Bacillus cereus
Strain Antimicrobial activity (AU) EROM101 72 Pediococcus
pentosaceus KCCM 11902 44 Lactobacillus acidophilus ATCC 43121
28
[0120]
5TABLE 5 Antimicrobial activity of EROM101 to Listeria
monocytogenes Strain Antimicrobial activity (AU) EROM101 209
Pediococcus pentosaceus KCCM 11902 126 Lactobacillus acidophilus
ATCC 43121 87
[0121] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *