The present invention provides an isolated microorganism strain, Lactobacillus fermentum GM-090, which is found to be effective in stimulating IFN-.gamma. secretion and/or treating allergy. The use of the Lactobacillus fermentum GM-090 in stimulating IFN-.gamma. secretion and/or treating allergy is also provided.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention mainly relates to a novel microorganism strain, Lactobacillus fermentum GM-090 and its use for stimulating IFN-.gamma. secretion and/or treating allergy.

2. Description of the Related Art

Allergy refers to an acquired potential to develop immunologically mediated adverse reaction to normally innocuous substances. Allergic reaction provokes symptoms such as itching, coughing, wheezing, sneezing, watery eyes, inflammation and fatigue. It is believed that allergic reaction includes an early specific immune response and a late inflammatory reaction. It was reported that allergens (e.g. pollens and mite dust) mediated the early phase of allergy by stimulating high affinity immunoglobulin (IgE) receptors. For instance, mast cells and basophils, when stimulated by allergens, would release histamine and cytokines. The cytokines released from mast cells and basophils then mediated the late phase of allergy by recruiting inflammatory cells (see Serafin, W E, In Goodman and Gillmans, "The Pharmacological Basis of Therapeutics", Hardmen, Ja; Limbird, L, E eds, McGraw-Hill, N.Y., 659-682, 1996). It was also reported that the influx of eosinophils, macrophages, lymphocytes, neutrophils and platelets started the vicious inflammatory cycle. The late phase of allergy amplified the initial immune response, which in turn triggered the release of more inflammatory cells (Townley, R G and Okada, C, Annals of Allergy, 68: 190-196, 1991).

Various therapies have been pursued in order to treat the symptoms of allergies. Among them, anti-allergics and histamine H-receptor antagonists (anti-histamines) have been used. Histamine antagonists were administered to antagonize the action of histamine released from mast cells in response to the presence of allergens. They reduced the redness, itching and swelling caused by the action of histamine on the target tissues, and served to prevent or alleviate many of the symptoms resulting from degranulation of mast cells. However, anti-histamines were also reported to be associated with adverse reactions such as diminished alertness, slowed reaction times and somnolence (Goodman and Gillman, The Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon Press, New York, pages 575-588, 1990).

There are also some reports on the treatment of allergies by regulating cytokines. Among them, interferon-.gamma. (IFN-.gamma.) was found to inhibit the over-expression of cytokines in Th2 lymphocytes, especially the secretion of IL-4 to lower the proliferation of B cells. Besides, IFN-.gamma. could stimulate the immune response of Th1 and repress the synthesis of IgE (Sareneva T et al., Influenza A virus-induced IFN-.alpha./.beta. and IL-18 synergistically enhance IFN-.gamma. gene expression in human T cells. J Immunol 160:6032-6038, 1998; Shida K et al., Lactobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte culture. Int Arch Allergy Immunol 115:278-287, 1998). Since IFN-.gamma. can repress B cell proliferation and IgE secretion, it is believed that IFN-.gamma. is effective in treating allergy.

Lactic acid bacteria, which are gram-positive bacteria, are commonly used in industrial food fermentations. In recent studies, lactic acid bacteria were shown to stimulate IFN-.gamma. secretion of cells (Blum S et al., Intensinal microflora and the interaction with immunocompetent cells. Antonie Van Leeuwenhoek 67:199-205, 1999; Contractor NV et al., Lymphoid hyperplasia, autoimmunity and compromised intestinal intraepithelial lymphocyte development in colits-free gnotobiotic IL-2-deficient mice. J Immunol 160:385-394, 1998; Delneste Y et al., Functional foods: Mechanism of action on immuncompetent cells. Nutr Rev 56:593-98, 1998; Haller D et al., Non-pathogenic bacteria elicit a different cytokine response by intestinal epithelial cell/leucocyte co-culture. Gut 47:79-87, 2000). Some specific lactic acid bacteria, such as Bifidobacterium lactis and Lactobacillus brevis subsp., were found to stimulate IFN-.gamma. secretion of lymphocytes in blood derived from mice and humans (Arunachalam K et al., Enhancement of natural immune function by dietary consumption of Bifidobacterium lactis (HN 019). Eur J Clin Nutr 54:1-5, 2000; Kishi A et al., Effect of the oral administration of Lactobacillus brevis subsp. Coagulans on interferon-.alpha. producing capacity in humans. J Am Coll Nutr 15:408-412, 1996). It was also reported that lactic acid bacteria could stimulate lymphocytes derived from humans or mice to secret Interleukin-12 (IL-12), which was a T cell stimulatory cytokine activating T cells and NK cells to secrete IFN-.gamma. (Pouwels P H et al., The potential of Lactobacillus as a carrier for oral immunization: development and preliminary characterization of vector systems for targeted delivery of antigens. J Biotechnol 44:183-92, 1996).

A topic dermatitis is one of the most common allergic diseases in children. The conventional method for treating a topic dermatitis is topic steroid therapy. However, the symptoms persist and recur frequently after stopping administrating steroids. Probiotic therapy is regarded as a safer and more effective way to treat a topic dermatitis. Some lactic acid bacteria were suggested to be effective in prevention and/or treatment of a topic dermatitis, such as L. rhamnosus strain GG (Kalliomaki, M., S. Salminen, et al., Probiotics and prevention of a topic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet 361(9372): 1869-71, 2003; Kirjavainen, P. V., S. J. Salminen, et al., Probiotic bacteria in the management of a topic disease: underscoring the importance of viability. J Pediatr Gastroenterol Nutr 36(2): 223-7, 2003; Dreborg, S., The implications of nomenclature. Ann Allergy Asthma Immunol 89(6 Suppl 1): 83-5, 2002; Pessi, T., Y. Sutas, et al., Interleukin-10 generation in a topic children following oral Lactobacillus rhamnosus GG. Clin Exp Allergy 30(12): 1804-8, 2000; Isolauri, E., T. Arvola, et al., Probiotics in the management of a topic eczema. Clin Exp Allergy 30(11): 1604-10, 2000), the combination of L. rhamnosus 19070-2 and L. reuteri DSM 122460 (Rosenfeldt, V., E. Benfeldt, et al., Effect of probiotic Lactobacillus strains in children with a topic dermatitis. J Allergy Clin Immunol 111(2): 389-95, 2003), and Bifidobacterium lactitis Bb-12 (Isolauri, E., T. Arvola, et al., 2000).

SUMMARY OF THE INVENTION

The invention provides a novel microorganism strain, Lactobacillus fermentum GM-090.

In another aspect, the invention provides a method for stimulating IFN-.gamma. secretion in a subject comprising administrating said subject with a composition comprising the microorganism strain, Lactobacillus fermentum GM-090.

In still another aspect, the invention provides a method for treating allergy in a subject comprising administrating said subject with a composition comprising the microorganism strain, Lactobacillus fermentum GM-090.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel microorganism strain, Lactobacillus fermentum GM-090, which is capable of stimulating IFN-.gamma. secretion and/or treating allergy. The strain GM-090 was deposited on Jul. 19, 2004 with the China Center for Type Culture Collection (CCTCC) under the accession number of CCTCC M 204055.

The Lactobacillus fermentum GM-090 was isolated from human gastrointestinal tract.

The mycological characteristics of the Lactobacillus fermentum GM-090 are shown below:

(a) Morphological Characteristics: (1) Shape and size of cell: bacillus, which has a rod-like shape with round edge when the cells after cultured at 37.degree. C. overnight in MRS broth were observed with a microscope. (2) Motility: non-motile (3) Flagella: none (4) Sporulation: no spore-forming (5) Gram-stain: positive

(b) Cultural Characteristics: (1) Medium: MRS broth (DIFCO.RTM. 0881) (as shown in Table 1 (see Original Patent)), final pH 6.5.+-.0.2

(2) Cultural condition: 37.degree. C. anaerobic

(c) Physiological Characteristics: (1) API 50 CHL test: API 50 CHL system is used for identification of lactic acid bacteria. By assaying the responses of a serious of enzymes, the characters of the lactic acid are established. Identification of the strain GM-090 was carried out by a carbohydrate fermentation test using API 50 CH strips and an API 50 CHL medium (API.RTM. Systems SA, Montalieu Vercieu, France)The result of API 50 CHL test of GM-090 is listed in Table 2 (see Original Patent).

(d) Genetic Characteristics: The Lactobacillus species specific primers were used to identify the species of the strain GM-090 according to Yeung et al (Yeung, P. S. M., Sanders, M. E., Kitts, C. L., Cano, R., Tong, P. S., Species-specific Identification of Commercial Probiotic Strains. J. Diary. Sci. 85:1039-1051, 2002). The 16s rDNA sequence is as shown in SEQ ID NO: 1. Also, randomly amplified polymorphic DNA (RAPD analysis) was performed. It shows that GM-090 belongs to Lactobacillus fermentum, but has a specific RAPD patterns. Given the above, GM-090 is a novel Lactobacillus fermentum strain.

(e) Cell wall proteins of the strain GM-090: The cell wall proteins of the strain GM-090 show specific pattern when compared with other conventional Lactobacillus fermentum strains. The SDS-PAGE patterns of the cell wall proteins of the strain GM-090 are shown in FIG. 4 (see Original Patent).

The present invention provides a method for stimulating IFN-.gamma. secretion and/or treating allergy in a subject comprising administrating said subject with a composition comprising the strain GM-090.

L. fermentum is usually found during malt whisky fermentation (van Beek, S. and Pries, F. G. Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study. Appl Environ Microbiol 68(1): 297-305, 2002; and Simpson, K. L. et al., Characterization of lactobacilli from Scotch malt whisky distilleries and description of Lactobacillus ferintoshensis sp. nov., a new species isolated from malt whisky fermentations. Microbiology 147: 1007-1016, 2001). Intravenous injection of saline washed L. fermentum was shown to elicit hypersensitivity reactions in rabbits (Jackson, D. E., C. R. Howlett, et al., Induction of hypersensitivity reactions to Lactobacillus fermentum and lipoteichoic acid in rabbits. Part II. Int Arch Allergy Appl Immunol 65(3): 304-12, 1981; Jackson, D. E., G. D. Jackson, et al., Induction of IgM immunological memory to lipoteichoic acids in rabbits. Part I. Int Arch Allergy Appl Immunol 65(2): 198-202, 1981; Jackson, D. E., A. J. Wicken, et al., Immune responses to lipoteichoic acid: comparison of antibody responses in rabbits and mice. Part II. Int Arch Allergy Appl Immunol 65(2): 203-11, 1981). On the other hand, one strain, L. fermentum CP34 was found to have the significant effect of decreasing the serum antigen-specific IgE levels compared to a control group (Ishida, Y. et al., Decrease in ovalbumin specific IgE of mice serum after oral uptake of lactic acid bacteria. Biosci Biotechnol Biochem 67(5): 951-957, 2003).

It is surprisingly found in the invention that the strain GM-090 has ability to stimulate IFN-.gamma. secretion. In the animal model of the invention, the splenocytes cultured with the strain GM-090 were stimulated to increase IFN-.gamma. secretion. The effect of GM-090 in stimulating IFN-.gamma. secretion is even better than the positive control, PHA.

According to the invention, L. fermentum is for treating allergy in a subject. Preferably, the allergy is preferably dermatitis. More preferably, the dermatitis is a topic dermatitis. The term "allergy" used herein refers to IFN-.gamma. mediated allergy. The allergic disorders include rhinitis, sinusitis, asthma, hypersensitive pneumonia, extrinsic allergic alveolitis, conjunctivitis, urticaria, eczema, dermatitis, anaphylaxis, angioedema, allergic and migraine headache, and certain gastrointestinal disorders.

According to the examples of the invention, the conditions of dermatitis were dramatically improved by treating with GM-090. Furthermore, the symptom is nearly dispelled.

According to the invention, the lactic acid bacterial strain can be live or inactive. For instance, the live bacterial strains can be treated by a heating step, or other treatments commonly used in the art for killing the lactic acid bacterial strains to obtain inactive strains. Preferably, the lactic acid bacterial is lyophilized.

In another aspect, the invention provides a composition comprising the strain L. fermentum GM-090.

According to the invention, the lactic acid bacterial strain can be included in a pharmaceutical composition, dietary supplement, food, healthy food, medical food, or the components thereof, which are normally administrated by people. In a preferred embodiment of the invention, the lactic acid bacterial strain can be delivered in food form, such as in a coagulated milk product that prepared through the fermentation of a lactic acid bacterium in milk. The food products prepared thus can be conveniently administrated to infants or children.

In still another aspect, the invention provides a method for stimulating IFN-.gamma. secretion in a subject comprising administrating said subject with a composition comprising the isolated strain L. fermentum GM-090.

In still another aspect, the invention provides a method for treating allergy in a subject comprising administrating said subject with a composition comprising the isolated strain GM-090.
 

Claim 1 of 7 Claims

1. An isolated microorganism of strain, Lactobacillus fermentum GM-090, deposited at the China Center for Type Culture Collection under the accession number of CCTCC M 204055.

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