Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Microorganism Pediococcus pentosaceus EROM101, having immune enhancement, anticancer and antimicrobial activities
United States Patent: 
7,241,441
Issued: 
July 10, 2007
Inventors:   Choi; Chang Won (Gyeonggi-do, KR), Park; Mi Hyoun (Seoul, KR), Hwang; Sang Ho (Seoul, KR), Woo; Suk Gyu (Gyeonggi-do, 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)
Assignee: 
Eromlife Co., Ltd. (Seoul, KR)
Appl. No.: 
11/450,452
Filed: 
June 12, 2006


 

Patheon


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.

Description of the Invention

FIELD OF THE INVENTION

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

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.

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, Lactobacillus 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.

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.2000-316517 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

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.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a Pediococcus pentosaceus EROM101 having immune enhancement, anticancer and antimicrobial activities, which was originated from human intestines.

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 parent strains e.g. the immune enhancement, anticancer and antimicrobial activities.

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.

The present invention further provides fermented products containing the above Pediococcus pentosaceus EROM101, or mutant or variant thereof.

The present invention also provides a method for culturing the above Pediococcus pentosaceus EROM101, or mutant or variant thereof.

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.

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.

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 pentosaceus EROM101, or mutant or variant thereof to a subject.

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.

The present invention will be described in detail.

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 the Korean Culture Center of Microorganisms, 361 221, Yurim B/D, Hongje-1-dong, Sodaemun gu, Seoul 120-091, Republic of Korea, on Jul. 19, 2002. The original deposit was requested on Oct. 10, 2003 to be converted under the Budapest Treaty and was assigned Accession No. KCCM-10517.

Pediococcus pentosaceus EROM101 of the present invention has following physiological characteristics;

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.

Second, the microorganism significantly inhibits the cancer cell proliferation.

Third, the microorganism shows a wide antimicrobial spectrum against various harmful microorganisms.

Thus, Pediococcus pentosaceus 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.

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.

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.

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. for 10 to 40 hours. Culturing at 37.degree. 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.

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.

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.

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.

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.

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.
 


Claim 1 of 1 Claim

1. A biologically pure culture of a microorganism Pediococcus pentosaceus EROM101 having the Accession number KCCM-10517.

 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]