Composition comprising a lactic acid bacteria for preventing and treating vaginosis and the use thereof

Abstract

A composition comprising combination of salt, sugar and lactic acid bacteria as an active ingredient to treat vaginosis. The composition showed potent antibacterial activity through various experiments, for example, (1) the indirect inhibitory activity test from the growth of vaginosis causing bacteria by determining the change of pH and lactic acid level; (2) the direct inhibitory activity test from the growth of vaginosis causing bacteria by determining the susceptibility of test sample; (3) brief clinical tests, and finally confirmed that the combination showed potent antibacterial activity in the test. Accordingly, the combination may be useful to alleviate and treat vaginosis in the form of a pharmaceutical composition, health functional food, food additive, topical composition, and detergent composition.

Claims

1.-17. (canceled)

18. A composition comprising salt, sugar and lactic acid bacteria.

19. The composition of claim 18, wherein the salt is selected from the group consisting of a refined salt, a processed salt and an un-refined salt.

20. The composition of claim 18, wherein the salt is sodium chloride.

21. The composition of claim 18, wherein the sugar is selected from the group consisting of a mono-saccharide, a disaccharide, an oligosaccharide and a polysaccharide.

22. The composition of-claim 18, wherein the sugar is selected from the group consisting of xylose, arabinose, glucose, mannose, fructose, galactose, lactulose, lactitol, sucrose, lactose, maltose, trehalose, fructo-oligosaccharide, raffinose, stachyose, maltodextrin, amylose, cellulose and pectin.

23. The composition of claim 18, wherein the salt, sugar and lactic acid bacteria are in a mixed ratio of 1:100-0.01:100-0.01 (w/w).

24. The composition of claim 18, wherein the lactic acid bacteria is selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., Bacillus spp., Streptococcus spp., Enterococcus spp., Saccharomyces spp., and Leuconostoc spp.

25. The composition of claim 24, wherein the Lactobacillus spp. bacteria is selected from the group consisting of lactobacillus plantarum, lactobacillus pentosus, lactobacillus casei, lactobacillus casei ssp. paracasei, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus delbrueckii, lactobacillus delbrueckii ssp. bulgaricus, lactobacillus delbrueckii ssp. delbrueckii, lactobacillus fermentum, lactobacillus gasseri, lactobacillus reuteri, lactobacillus brevis, lactobacillus cellobiosus, lactobacillus crispatusi, lactobacillus GG, lactobacillus johnsonii, lactobacillus lactis, and lactobacillus salivarius.

26. The composition of claim 25, wherein the Lactobacillus spp. bacteria is selected from the group consisting of lactobacillus plantarum, lactobacillus pentosus, lactobacillus casei, lactobacillus casei ssp. paracasei, lactobacillus rhamnosus, and lactobacillus acidophilus.

27. The composition of claim 24, wherein the Bifidobacterium spp. bacteria is selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium bereve, Bifidobacterium animalis ssp. lactis, Bifidobacterium adoescentis, Bifidobacterium pseuocatenulatum, Bifidobacterium catenulatum, Bifidobacterium infantis, and Bifidobacterium thermophilum.

28. The composition of claim 18, wherein the composition is a pharmaceutical composition.

29. The composition of claim 28, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

30. The composition of claim 18, wherein the composition is a health functional food.

31. The composition of claim 30, wherein the health functional food is selected from the group consisting of a food, a health beverage and a dietary supplement.

32. The composition of claim 30, wherein the health functional food further comprises a sitologically acceptable additive.

33. The composition of claim 18, wherein the composition is incorporated into a form selected from the group consisting of topical composition, detergent composition, liquid solution and insert preparation.

34. The composition of claim 33, wherein the topical composition is selected from the group consisting of a aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized preparation, suppository preparation, cleansing liquid, gel, jelly, foam, cream, ointment, lotion, balm, patch, paste, spray solution and aerosol.

35. The composition of claim 33, wherein the insert preparation is selected from the group consisting of vaginal tablet, vaginal cream, vaginal ointment, dressing solution, spraying preparation, vaginal capsule, vaginal film, vaginal sponge, spreading cream, tampon, sanitary pad, diaper and panties.

36. A method of treatment comprising administering to a mammal in need of treatment for vaginosis an effective amount of the composition of claim 18, together with a pharmaceutically acceptable carrier thereof.

37. The method of claim 36, wherein the vaginosis is selected from the group consisting of bacterial vaginosis, fungal vaginosis and Tricomonas vaginitis.

Description

BEST MODE

Best Mode for Carrying Out the Invention

[0075] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0076] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES

[0077] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Preparation of an Inventive Combination

1-1. Preparation of Salt

1-1-1. Preparation of Melted Salt

[0078] 900 mg of un-refined salt (Shinan salt coop, 88-6, Dongniseonchakjang-gil, Sangtaedong-ri, Sinui-myeon, Sinan-gun, Jeollanam-do, Republic of Korea, 58857) was melted for 24 hours at 850-1000 C. using by heater (MS-E104, TOPS Co. Ltd.) to obtain 400 mg of the melted salt (designated as MS hereinafter).

1-1-2. Preparation of Refined Salt

[0079] 400 mg of refined salt (NaCl, F.W. 58.44) was procured the company (SPPO-91701, Duksan Pure Chemicals, Co., Ltd, 53, Siwon-ro, 133beon-gil, Danwon-gu, Ansan-si, Gyeonggi-do, Korea) (designated as RS hereinafter).

1-1-3. Preparation of Unrefined Salt

[0080] 400 mg of un-refined salt (natural salt) was procured the company (Shinan salt coop, 88-6, Dongniseonchakjang-gil, Sangtaedong-ri, Sinui-myeon, Sinan-gun, Jeollanam-do, Republic of Korea, 58857) (designated as US hereinafter).

1-2. Preparation of Sugar

1-2-1. Preparation of Glucose

[0081] 800 mg of glucose (D(+)-glucose) was procured the company (Cat. No. jb01, J. T. Baker Chemical Company). (designated as GL hereinafter).

1-2-2. Preparation of Fructo-Oligosaccharide

[0082] 800 mg of fructo-oligosaccharide (derived from chicory) was procured the company (Cat. No. F8052050 g, Sigma-Aldrich Co. LLC). (designated as FO hereinafter).

1-2-3. Preparation of Lactulose

[0083] 800 mg of lactulose was procured the company (Cat. No., 126-03732, Wako Pure Chemical Industries). (designated as LS hereinafter).

1-2-4. Preparation of Fructose

[0084] 800 mg of fructose was procured the company (Cat. No., 64505-0410, Junsei Chemical Co. Ltd). (designated as FS hereinafter).

1-2-5. Preparation of Lactitol

[0085] 800 mg of lactitol was procured the company (Cat. No., sc488686, Santa Cruz Biotechnology, Inc). (designated as LT hereinafter).

1-3. Preparation & Culture of Lactic Acid Bacteria

1-3-1. Preparation of Lactic Acid Bacteria

[0086] Various lactic acid bacteria listed in Table 1 were apportioned from KCTC (Korean Collection for Type Cultures, Korea) and used in the following experiments.

TABLE-US-00001 TABLE 1 Lactic Acid Bacteria Name Scientific name Deposit No. BLA Lactobacillus acidophilus KCTC No. 3164 BLB Lactobacillus brevis KCTC No. 13094 BLC Lactobacillus casei KCTC No. 13086 BLD Lactobacillus delbrueckii subsp. bulgaricus KCTC No. 3635 BLLC Lactobacillus casei subsp. casei KCTC No. 3260 BLDD Lactobacillus delbrueckii subsp. delbrueckii KCTC No. 13721 BBL Bifidobacterium longum sub sp. longum KCTC No. 3128 BBA Bifidobacterium adolescentis KCTC No. 3267 BBB1 Bifidobacterium bifidum KCTC No. 3357 BBB2 Bifidobacterium breve KCTC No. 3441 BBC2 Bifidobacterium catenulatum KCTC No. 3221 BBC Bacillus cereus KCTC No. 13123 BSS Streptococcus sp. KCTC No. 5644

1-3-2. Culture of Bacteria

[0087] (1) Facultative anaerobic and microaerophilic bacteria, i.e., Lactobacillus acidophilus (KCTC No. 3164), Streptococcus sp. (KCTC No. 5644), Bifidobacterium longum sub sp. longum (KCTC No. 3128) etc and aerobic bacteria, i.e., Bacillus cereus (KCTC No. 13123) etc were used in the experiment.

[0088] (2) In addition to the above lactic acid bacteria, vaginosis causing bacteria, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097) were used in the experiment.

[0089] (3) The vaginosis causing bacteria, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097) were inoculated into Casmans medium supplemented with 5% sheep blood (12 g of Tryptone, 5 g of meat peptone, 5 g of sodium chloride, 3 g of yeast extract, 3 g of beef extract, 1 g of starch casein, 0.5 g of D-glucose, 0.05 g of nicotinamide, 0.005 g of p-aminobenzoic acid, 1 L of distilled water, pH 7.3) and performed to shaking incubation at 37 C. at the speed of 150 rpm using by GasPak TMEZ Pouch system (BD, Cat. No. 26083, USA).

[0090] (4) Facultative anaerobic and microaerophilic bacteria, i.e., Lactobacillus acidophilus (KCTC No. 3164), Streptococcus sp. (KCTC No. 5644), Bifidobacterium longum sub sp. longum (KCTC No. 3128) etc and aerobic bacteria, i.e., Bacillus cereus (KCTC No. 13123) etc were inoculated into MRS medium (10 g of Protease peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of D-glucose, 1 ml of Tween 80, 2 g of K.sub.2HPO.sub.4, 5 g of sodium acetate, 2 g of diammonium hydrogencitrate, 0.2 g of MgSO.sub.47H.sub.20, 0.2 g of MnSO.sub.4H.sub.20, 1 L of distilled water, pH 6.2-6.5) and performed to shaking incubation at 37 C. at the speed of 150 rpm.

1-4. Preparation of Inventive Combination

[0091] 50 mg of the melted salt prepared in the above step was thoroughly mixed with 5 mg of dried glucose and 1 mg of dried Lactobacillus acidophilus prepared in the above steps, together to obtain the inventive combination (designated as CL1 hereinafter).

[0092] The combinations were kept at 75 C. in refrigerator and used in following experiments by dissolving in distilled water before use.

[0093] The other combinations of the present invention listed in Table 2 were prepared according to the similar method to the above.

TABLE-US-00002 TABLE 2 Combinations Group A. B. C. Name salt* sugar** lactic acid bacteria*** CL1 RS (5 mg) GL (5 mg) BLA (1 mg) CL2 MS (5 mg) FO (10 mg) BLB (1 mg) CL3 US (5 mg) LS (25 mg) BLC (1 mg) CL4 RS (5 mg) FS (2.5 mg) BLD (1 mg) CL5 MS (5 mg) LT (1 mg) BLLC (1 mg) CB1 RS (5 mg) GL (5 mg) BBC (1 mg) CB2 MS (5 mg) FO (10 mg) BBC (1 mg) CB3 US (5 mg) LS (25 mg) BBC (1 mg) CB4 RS (5 mg) FS (2.5 mg) BBC (1 mg) CB5 MS (5 mg) LT (1 mg) BBC (1 mg) CF1 RS (5 mg) GL (5 mg) BBL (1 mg) CF2 MS (5 mg) FO (10 mg) BBA (1 mg) CF3 US (5 mg) LS (25 mg) BBB1 (1 mg) CF4 RS (5 mg) FS (2.5 mg) BBB2 (1 mg) CF5 MS (5 mg) LT (1 mg) BBC2 (1 mg) CS1 RS (5 mg) GL (5 mg) BSS (1 mg) CS2 MS (5 mg) FO (10 mg) BSS (1 mg) CS3 US (5 mg) LS (25 mg) BSS (1 mg) CS4 RS (5 mg) FS (2.5 mg) BSS (1 mg) CS5 MS (5 mg) LT (1 mg) BSS (1 mg) CP1 BLA (1 mg) CP2 BBC (1 mg) CP3 BBL (1 mg) CP4 BSS (1 mg) *: RS (Refined salt), MS (melted salt), US (Unrefined salt) **: GL (glucose), FO (Fructo-oligosaccharide), LS (lactulose), FS (Fructose), LT (lactitol) ***: BLA (Lactobacillus acidophilus), BLB (Lactobacillus brevis), BLC (Lactobacillus casei), BLD (Lactobacillus delbrueckii subsp. bulgaricus), BLLC (Lactobacillus casei subsp. Casei), BLDD (Lactobacillus delbrueckii subsp. Delbrueckii), BBL (Bifidobacterium longum subsp. Longum), BBA (Bifidobacterium adolescentis), BBB1 (Bifidobacterium bifidum), BBB2 (Bifidobacterium breve), BBC2 (Bifidobacterium catenulatum), BBC (Bacillus cereus), BSS (Streptococcus sp)

Example 2. Preparation of Inventive Vaginal Tablet Composition

[0094] The combination prepared in Example 1 comprising 400 mg of melted salt, 800 mg of glucose and 40 mg of dried Lactobacillus acidophilus was mixed with 2 mg of magnesium stearate in order to formulating into inventive vaginal tablet composition combination (designated as SGL2 hereinafter) using by entableting apparatus (KT2000, Kumsungkigong).

Example 3. Preparation of Inventive Vaginal Cleansing Solution Composition

[0095] The vaginal cleansing solution composition comprising the combination prepared in Example 1 comprising 400 mg of refined salt, 800 mg of glucose and 40 mg of dried Bifidobacterium longum sub sp. longum was prepared by mixing with following ingredients as shown in Tablet 3 (designated as SGL3 hereinafter) for 48 hours with stirring.

TABLE-US-00003 TABLET 3 SGL4 solution (100 ml) Ingredient Amount SGL3 0.5 g Lactic acid 1 g adjuvant Whey 180 mg Ethanol 1 g Preservatives Trace amount (benzalkonimum HCl and menthol) Distilled water Appropriate amount to adjusted to 100 ml

Experimental Example 1. Inhibition Effect on the Growth of Vaginosis Causing Strains

[0096] To determine the inhibitory effect of inventive combinations prepared in Example 1 on the growth of vaginosis-causing strains indirectly, following test was performed according to the procedure disclosed in the literature (Choi, J. G. et al, Antibacterial activity of Ecklonia cava against methicillin-resistant Staphylococcus aureus and Salmonella spp., Foodborne Pathog. Dis., 2010 (April): 7(4), pp 435-441).

1-1. Testing Strains

[0097] The vaginosis causing bacteria, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097) were inoculated into Casmans medium supplemented with 5% FBS (Fetal bovine Serum) (12 g of Tryptone, 5 g of meat peptone, 5 g of sodium chloride, 3 g of yeast extract, 3 g of beef extract, 1 g of starch casein, 0.5 g of D-glucose, 0.05 g of nicotinamide, 0.005 g of p-aminobenzoic acid, 1 L of distilled water, pH 7.3, 5% FBS) and performed to shaking incubation at 37 C. at the speed of 150 rpm using by GasPakEZ Pouch system (Anaerobic Gas Generation Pouch System with indicator, BD, Cat. No. 26083, USA) according to the cited literature (D. D. Charles, et al., Identification of haemophilus vaginalis, 1960, Journal of bacteriology p277, R. P. Morgan et al., Amniotic fluid and maternalrace influence responsiveness of fetal membranes to bacteria, Journal of reproductive immunology, 96 (2012) 68-78).

1-2. Experimental Materials

[0098] The inventive combinations shown in Table 2 were used as test samples and lactic acid was purchased from the company (Cat. No. 252476, ACS reagent, 85-90%, L-lactic acid in water, Sigma-Aldrich Co. LLC).

1-3. Purpose of Experiment

[0099] The purpose of experiment is to determine the change of OD (optical density), pH and level of lactic acid in the test group treated with test samples prepared in Example 1 comparing with control group treated with only vaginosis-causing strains.

1-4. Experimental Procedure

1-4-1. Control Group

[0100] The test samples prepared in Example 1 were added to the culturing medium of vaginosis-causing strains, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097). The pre-cultured vaginosis-causing strains (Absorbance: OD.sub.660=1.0 A, 1/100 v/v) were inoculated into Casman medium containing FBS again and performed to shaking incubation at 37 C. at the speed of 150 rpm for 12 hours. The control group was treated with test samples prepared in Example 1

1-4-2. Comparative Group

[0101] Various lactic acid bacteria as shown in Table 1, were only added to the culturing medium of vaginosis-causing strains, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097). The pre-cultured vaginosis-causing strains (Absorbance: OD.sub.660=1.0 A, 1/100 v/v) were inoculated into Casman medium containing FBS again and performed to shaking incubation at 37 C. at the speed of 150 rpm for 12 hours. The comparison group treated with only lactic acid bacteria.

1-4-3. Test Sample Group

[0102] The combination of 1 mg of lactic acid bacteria and the test samples prepared in Example 1 were added to the culturing tube of vaginosis-causing strains, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097) and performed to shaking incubation at 37 C. at the speed of 150 rpm for 18 hours. The test sample group treated with 1 mg of lactic acid bacteria and the test samples prepared in Example 1.

1-4-4. Determination of pH Value

[0103] The change of pH value in the control group and test sample groups was determined as follows.

[0104] The pH of cultured cell solution in the control group and test sample groups was determined using by apparatus (pH meter, SevenEasy, Mettler Toledo, Swiss).

1-4-5. Determination of the Level of Lactic Acid

[0105] The level of lactic acid in the control group and test sample groups was determined using by assay kit (D-/L-Lactic acid (Rapid) Assay kit, megazyme, Cat. No. K-DLATE) as follows.

[0106] 1.5 ml of distilled water, 0.1 ml of test sample and 0.02 ml of buffer solution (Buffer; plus D-glutamate and sodium azide (0.02% w/v), 0.1 ml of NAD, DGP (D-Glutamate-pyruvate transaminase suspension) were poured to cuvettes and mixed together for 3 mins. The absorbance of A1 value (wavelength: 340 nm) is determined using by spectrophotometer (Spectronic Genesys 2, Thermo, USA) and 0.02 ml of D-LDL (D-Lactate dehydrogenase suspension, D-/L-lactic acid kit, Megazyme, Ireland) was added to the solution to mix together for 5 mins.

[0107] The absorbance of A2 value (wavelength: 340 nm) is determined using by spectrophotometer (Spectronic Genesys 2, Thermo, USA).

[0108] Finally, 0.02 ml of D-LDL (D-Lactate dehydrogenase suspension, D-/L-lactic acid kit, Megazyme, Ireland) was added to the solution to mix together for 10 mins and the absorbance of A3 value (wavelength: 340 nm) is determined using by spectrophotometer (Spectronic Genesys 2, Thermo, USA).

[0109] The level of lactic acid in the control group and test sample groups was determined using by software (Mega-Calc, Megazyme, IDA Business Park, Bray, Co. Wicklow, A98 YV29, Ireland) and the resulting absorbance values of A1-A3.

1-5. Test Result

[0110] The test results on the change of OD (optical density), pH and level of lactic acid in the test group treated with test samples prepared in Example 1 comparing with control group treated with only vaginosis-causing strains were shown in Tables 4 & 5 (Gadnerella vaginalis strain) and Tables 6 & 7 (Bacterioides fragilis strain).

1-5-1. Gardnerella vaginalis Strain (See Tables 4 & 5)

[0111] In case of the experiment treated to Gardnerella vaginalis strain, the test group treated with inventive combinations showed acidic environment, i.e., pH range of 4.0 to 4.9, providing with the inhibiting environment from the growth of Gardnerella vaginalis strain in vagina whereas the control group without inventive combinations showed pH range of 5.19 to 5.52.

[0112] As to level of lactic acid having inhibiting activity from the growth of detrimental bacteria in vagina, the level of lactic acid in the test groups treated with inventive combinations ranged 0.970 mg/ml to 1.712 mg/ml, providing with more potent inhibiting activity from the growth of Gardnerella vaginalis strain in vagina compared with the comparative group treated with only lactic acid bacteria whereas the control group without inventive combinations showed the level of lactic acid ranging from 0.711 mg/ml to 0.765 mg/ml.

1-5-2. Bacterioides fragilis Strain (See Tables 6 & 7)

[0113] In case of the experiment treated to Bacterioides fragilis strain, the test group treated with inventive combinations showed acidic environment, i.e., pH range of 4.4 to 4.9, providing with the inhibiting environment from the growth of Bacterioides fragilis strain in vagina whereas the control group without inventive combinations showed pH range of 5.21 to 5.62.

[0114] As to level of lactic acid having inhibiting activity from the growth of detrimental bacteria in vagina, the level of lactic acid in the test groups treated with inventive combinations ranged 0.4420 mg/ml to 1.049 mg/ml, providing with more potent inhibiting activity from the growth of Bacterioides fragilis strain in vagina compared with the comparative group treated with only lactic acid bacteria whereas the control group without inventive combinations showed the level of lactic acid of 0.000 mg/ml.

[0115] Accordingly, it has been confirmed that the inventive combinations promoted the growth of lactic acid bacteria by way of providing with necessary nutrients, resulting in stimulating the reproduction of lactic acid maintaining a vagina with an acidic environment.

TABLE-US-00004 TABLE 4 Change of pH and level of lactic acid (Gardnerella vaginalis strain) Gardnerella vaginalis Lactic acid bacteria* Gardnerella vaginalis BLA BBC BBL BSS Control group (media pH = 7.3) 61 Ctrl. 62 Ctrl. 63 Ctrl. 64 Ctrl. Control group pH = 5.45 pH = 5.52 pH = 5.45 pH = 5.19 (media pH = 7.3) Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.762 (mg/ml) = 0.711 (mg/ml) = 0.751 (mg/ml) = 0.711 (D:0.016/L:0.746) (D:0.015/L:0.696) (D:0.014/L:0.737) (D:0.014/L:0.697) Test group I CL1 CB1 CF1 CS1 Test group I pH = 4.32 pH = 4.65 pH = 4.22 pH = 4.26 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.052 (mg/ml) = 1.022 (mg/ml) = 1.061 (mg/ml) = 1.025 (D:1.052/L:0.000) (D:0.014/L:1.008) (D:0.017/L:1.044) (D:0.014/L:1.011) Test group II CL2 CB2 CF2 CS2 Test group II pH = 5.01 pH = 5.18 pH = 5.22 pH = 5.07 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.005 (mg/ml) = 0.970 (mg/ml) = 0.758 (mg/ml) = 1.712 (D:0.218/L:0.787) (D:0.017/L:0.953) (D:0.016/L:0.742) (D:0.014/L:0.673) Comparative group (media pH = 7.3) CP1 CP2 CP3 CP4 Comparative group pH = 5.43 pH = 5.55 pH = 5.47 pH = 5.22 (media pH = 7.3) Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.753 (mg/ml) = 0.709 (mg/ml) = 0.754 (mg/ml) = 0.715 (D:0.015/L:0.738) (D:0.011/L:0.698) (D:0.015/L:0.739) (D:0.013/L:0.702) *BLA (Lactobacillus acidophilus), BBC (Bacillus cereus), BBL (Bifidobacterium longum subsp. Longum), BSS (Streptococcus sp); **D; D-LDL/L; L-LDL

TABLE-US-00005 TABLE 5 Change of pH and level of lactic acid (Gardnerella vaginalis strain) Gardnerella vaginalis Lactic acid bacteria* Gardnerella vaginalis BLA BBC BBL BSS Control group (media pH = 7.3) 61 Ctrl. 62 Ctrl. 63 Ctrl. 64 Ctrl. Control group pH = 5.45 pH = 5.52 pH = 5.45 pH = 5.19 (media pH = 7.3) Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.762 (mg/ml) = 0.711 (mg/ml) = 0.751 (mg/ml) = 0.711 (D:0.016/L:0.746) (D:0.015/L:0.696) (D:0.014/L:0.737) (D:0.014/L:0.697) Test group III CL3 CB3 CF3 CS3 Test group III pH = 4.27 pH = 4.99 pH = 4.20 pH = 5.09 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.046 (mg/ml) = 0.940 (mg/ml) = 1.064 (mg/ml) = 0.665 (D:0.991/L:0.055) (D:0.017/L:0.923) (D:0.021/L:1.043) (D:0.013/L:0.652) Test group IV CL4 CB4 CF4 CS4 Test group IV pH = 4.38 pH = 4.89 pH = 4.55 pH = 4.87 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.045 (mg/ml) = 1.017 (mg/ml) = 1.033 (mg/ml) = 0.806 (D:0.764/L:0.281) (D:0.021/L:0.996) (D:0.015/L:1.018) (D:0.013/L:0.793) Test group V CL5 CB5 CF5 CS5 Test group V pH = 5.01 pH = 5.34 pH = 5.17 pH = 5.12 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.006 (mg/ml) = 0.942 (mg/ml) = 0.737 (mg/ml) = 0.693 (D:0.237/L:0.767) (D:0.019/L:0.923) (D:0.014/L:0.723) (D:0.013/L:0.680) Comparative group (media pH = 7.3) CP1 CP2 CP3 CP4 Comparative group pH = 5.43 pH = 5.55 pH = 5.47 pH = 5.22 (media pH = 7.3) Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.753 (mg/ml) = 0.709 (mg/ml) = 0.754 (mg/ml) = 0.715 (D:0.015/L:0.738) (D:0.011/L:0.698) (D:0.015/L:0.739) (D:0.013/L:0.702) *BLA (Lactobacillus acidophilus), BBC (Bacillus cereus), BBL (Bifidobacterium longum subsp. Longum), BSS (Streptococcus sp); **D; D-LDL/L; L-LDL

TABLE-US-00006 TABLE 6 Change of pH and level of lactic acid (Bacterioides fragilis strain) Bacterioides fragilis Lactic acid bacteria* Bacterioides fragilis BLA BBC BBL BSS Control group (media pH = 7.3) 51 Ctrl. 52 Ctrl. 53 Ctrl. 54 Ctrl. Control group pH = 5.58 pH = 5.36 pH = 5.62 pH = 5.21 (media pH = 7.3) Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.000 (mg/ml) = 0.000 (mg/ml) = 0.000 (mg/ml) = 0.000 (D:0.000/L:0.000) (D:0.000/L:0.000) (D:0.000/L:0.000) (D:0.000/L:0.000) Test group I CL1 CB1 CF1 CS1 Test group I pH = 4.47 pH = 4.70 pH = 4.48 pH = 4.55 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.049 (mg/ml) = 0.278 (mg/ml) = 1.007 (mg/ml) = 0.324 (D:1.007/L:0.042) (D:0.278/L:0.000) (D:0.327/L:0.680) (D:0.3244/L:0.000) Test group II CL2 CB2 CF2 CS2 Test group II pH = 5.22 pH = 4.99 pH = 5.38 pH = 5.04 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.451 (mg/ml) = 0.015 (mg/ml) = 0.006 (mg/ml) = 0.004 (D:0.190/L:0.261) (D:0.010/L:0.005) (D:0.005/L:0.001) (D:0.000/L:0.004) Comparative group (media pH = 7.3) CP1 CP2 CP3 CP4 Comparative pH = 5.42 pH = 5.35 pH = 5.60 pH = 5.22 group Lactate** Lactate** Lactate** Lactate** (media pH = 7.3) (mg/ml) = 0.002 (mg/ml) = 0.003 (mg/ml) = 0.002 (mg/ml) = 0.004 (D:0.002/L:0.000) (D:0.001/L:0.002) (D:0.001/L:0.001) (D:0.003/L:0.001) *BLA (Lactobacillus acidophilus), BBC (Bacillus cereus), BBL (Bifidobacterium longum subsp. Longum), BSS (Streptococcus sp); **D; D-LDL/L; L-LDL

TABLE-US-00007 TABLE 7 Change of pH and level of lactic acid (Bacterioides fragilis strain) Bacterioides fragilis Lactic acid bacteria* Bacterioides fragilis BLA BBC BBL BSS Control group (media pH = 7.3) 51 Ctrl. 52 Ctrl. 53 Ctrl. 54 Ctrl. Control group pH = 5.58 pH = 5.36 pH = 5.62 pH = 5.21 (media pH = 7.3) Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.000 (mg/ml) = 0.000 (mg/ml) = 0.000 (mg/ml) = 0.000 (D:0.000/L:0.000) (D:0.000/L:0.000) (D:0.000/L:0.000) (D:0.000/L:0.000) Test group III CL3 CB3 CF3 CS3 Test group III pH = 4.45 pH = 4.80 pH = 4.85 pH = 4.73 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 1.029 (mg/ml) = 0.026 (mg/ml) = 0.116 (mg/ml) = 0.050 (D:1.016/L:0.013) (D:0.023/L:0.003) (D:0.096/L:0.020) (D:0.049/L:0.001) Test group IV CL4 CB4 CF4 CS4 Test group IV pH = 4.74 pH = 4.60 pH = 4.85 pH = 4.52 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.939 (mg/ml) = 0.043 (mg/ml) = 0.442 (mg/ml) = 0.514 (D:0.710/L:0.229) (D:0.043/L:0.000) (D:0.432/L:0.010) (D:0.514/L:0.000) Test group V CL5 CB5 CF5 CS5 Test group V pH = 5.30 pH = 5.03 pH = 5.37 pH = 5.03 Lactate** Lactate** Lactate** Lactate** (mg/ml) = 0.431 (mg/ml) = 0.015 (mg/ml) = 0.011 (mg/ml) = 0.000 (D:0.183/L:0.248) (D:0.012/L:0.003) (D:0.005/L:0.006) (D:0.000/L:0.000) Comparative group (media pH = 7.3) CP1 CP2 CP3 CP4 Comparative pH = 5.42 pH = 5.35 pH = 5.60 pH = 5.22 group Lactate** Lactate** Lactate** Lactate** (media pH = 7.3) (mg/ml) = 0.002 (mg/ml) = 0.003 (mg/ml) = 0.002 (mg/ml) = 0.004 (D:0.002/L:0.000) (D:0.001/L:0.002) (D:0.001/L:0.001) (D:0.003/L:0.001) *BLA (Lactobacillus acidophilus), BBC (Bacillus cereus), BBL (Bifidobacterium longum subsp. Longum), BSS (Streptococcus sp); **D; D-LDL/L; L-LDL

Experimental Example 2. Direct Inhibition Effect on the Growth of Vaginosis Causing Strains

[0116] To reconfirm the direct inhibitory effect of inventive combinations prepared in Example 1 on the growth of vaginosis-causing strains, following test was performed as follow.

2-1. Purpose of the Preliminary Test

[0117] The susceptibility of the vaginosis-causing strains and lactic acid bacteria used in the experiment was determined by using various antibiotic-resistance of the vaginosis-causing strains and lactic acid bacteria and the inhibitory activity or promoting effect of the inventive combinations on vaginosis-causing strains can be quantitatively and directly determined.

[0118] Various concentrations of various antibiotics, i.e., 50 microgram/ml of ampicillin (Cat. No. AM0510, Georgia Chem. USA), 50 microgram/ml of kanamycin (Cat. No. KA2003, Georgia Chem. USA), 34 microgram/ml of chloramphenicol (Art. 3886.2, Germany), 15 microgram/ml of tetracycline (Art. Nr. HP63.1, Germany), 50 microgram/ml of Streptomycin (Cat. No. S1027, Biosesang, Korea), 20 microgram/ml of gentamycin (Cat. No. GS3007, Georgia Chem. USA) and 25 microgram/ml of rifampicin (Cat. No. R3501, Sigma-Alrich, USA) were used in the experiment.

2-2. Preliminary Test on the Susceptibility of the Vaginosis-Causing Strains

2-2-1. Experiment Procedure

[0119] The vaginosis-causing strains, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097) were inoculated into Casmans medium supplemented with 5% sheep blood and 1.5% agar and performed to shaking incubation for 36 hrs under anaerobic condition using by GasPak TMEZ Gas Generation Pouch system (BD, Cat. No. 26083, USA).

[0120] After the incubation, the colony was collected by platinum loop and inoculated to Casmans medium supplemented with 5% FBS (Fetal bovine Serum) (12 g of Tryptone, 5 g of meat peptone, 5 g of sodium chloride, 3 g of yeast extract, 3 g of beef extract, 1 g of starch casein, 0.5 g of D-glucose, 0.05 g of nicotinamide, 0.005 g of p-aminobenzoic acid, 1 L of distilled water, pH 7.3, 5% FBS). The media was performed to shaking incubation at 37 C. for 24 hrs at the speed of 150 rpm using by GasPakEZ Pouch system (Anaerobic Gas Generation Pouch System with indicator, BD, Cat. No. 26083, USA) according to the cited literatures (Treatment of Gardnella vaginalis infection., J I, Adinma et al., J. Obstetrics and Gynecology, 17(6), pp. 573-575, 1997; Treatment of urinary tract infection by Gardnella vaginalis; a composition of oral metronidazole versus ampicillin, AG. PA., et al., Rev Latioam Microbiol. 2001, 43(2):pp 65-69; Antimicrobial susceptibilities of Gardnella vaginalis., Kharsany A B., et al., antimicrobial agents and chemotherapy, 1993, pp 2733-2735; Susceptibilities of Bacteroides fragilis to six antibiotics determined by standardized antimicrobial disc susceptibility testing., Sutter V L., et al., antimicrobial agents and chemotherapy, 1973, pp 188-193).

2-2-2. Test Result

[0121] At the result, the antibiotic susceptibility of various antibiotics against vaginosis-causing bacteria was shown in Table 8.

TABLE-US-00008 TABLET 8 Antibiotic susceptibility of various antibiotics against vaginosis-causing bacteria vaginosis-causing bacteria antibiotics Bacteroides fragilis Gardnella vaginalis Ampicillin (50 microgram/ml) +++ Kanamycin(50 microgram/ml) +++ +++ Chloramphenicol (34 microgram/ml) Tetracyclin (15 microgram/ml) Streptomycin (50 microgram/ml) +++ Gentamycin (20 microgram/ml) +++ +++ Ripampicin (25 microgram/ml) *; +++: being normally grown without the effect of antibiotic, : being not grown caused by the effect of antibiotic

2-3. Preliminary Test on the Susceptibility of the Lactic Acid Bacteria Strains

2-2-1. Experiment Procedure

[0122] In order to determine the antibiotic resistance of the lactic acid bacteria strains reproducing abundant lactic acids, i.e., Lactobacillus acidophilus (KCTC No. 3164), Bifidobacterium longum sub sp. longum (KCTC No. 3128) etc, the bacteria was inoculated into MRS medium (10 g of Protease peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of D-glucose, 1 ml of Tween 80, 2 g of K.sub.2HPO.sub.4, 5 g of sodium acetate, 2 g of diammonium hydrogencitrate, 0.2 g of MgSO.sub.47H.sub.20, 0.2 g of MnSO.sub.4H.sub.20, 1 L of distilled water, pH 6.2-6.5) and performed to shaking incubation at 37 C. at the speed of 150 rpm.

[0123] For anaerobic incubation, the media was performed to shaking incubation at 37 C. for 24 hrs at the speed of 150 rpm using by GasPak TMEZ Pouch system (Anaerobic Gas Generation Pouch System with indicator, BD, Cat. No. 26083, USA) according to the cited literatures (Antibiotic susceptibility of members of the lactobacillus acidophilus group using broth microdilution and molecular identification of their resistance determinants, M. Sigrids et al., International Journal of Food Microbiology 144 (2010), pp 81-87; Antibiotic sensitivity of acid stressed probiotic Lactobacillus acidophilus ncdc 291, NNK, GS., The internet Journal of microbiology Vol. 9 (2010); Antimicrobial susceptibility of bifidobactria., F. CM., et al., Journal of antimicrobial Chemotherapy (2005) 55, pp. 38-44).

2-2-2. Test Result

[0124] At the result, the antibiotic susceptibility of various antibiotics against lactic acid bacteria strains was shown in Table 7.

TABLE-US-00009 TABLET 9 Antibiotic susceptibility of various antibiotics against lactic acid bacteria strains AB* LB** A50 K50 C34 T15 S50 G20 R25 BLA *** +++ BLB +++ BLC +++ BLD +++ BBL +++ +++ +++ BBA ++ ++ +++ BBB1 +++ ++ ++ BBB2 +++ +++ ++ BBC2 ++ ++ +++ BBC ++ ++ +++ BSS +++ ++ ++ *AB (Antibiotics), A50 (Ampicillin, 50 microgram/ml), K50 (Kanamycin, 50 microgram/ml), C34 (Chloramphenicol, 34 microgram/ml), T15 (Tetracyclin, 15 microgram/ml), S50 (Streptomycin, 50 microgram/ml), G20 (Gentamycin, 20 microgram/ml), R25 (Ripampicin, 25 microgram/ml) **LB (Lactic acid bacteria), BLA (Lactobacillus acidophilus), BLB (Lactobacillus brevis), BLC (Lactobacillus casei), BLD (Lactobacillus delbrueckii subsp. bulgaricus), BBL (Bifidobacterium longum subsp. Longum), BBA (Bifidobacterium adolescentis), BBB1 (Bifidobacterium bifidum), BBB2 (Bifidobacterium breve), BBC2 (Bifidobacterium catenulatum), BBC (Bacillus cereus), BSS (Streptococcus sp), ***+++: being normally grown without the effect of antibiotic, : being not grown caused by the effect of antibiotic

2-3. Principal Experiment on Antibiotic Susceptibility

2-3-1. Experiment Procedure

[0125] In order to determine the antibiotic resistance of the vaginosis-causing strains, i.e., Bacterioides fragilis (KCTC No. 5013) and Gardnerella vaginalis (KCTC No. 5097) as well as various lactic acid bacteria strains reproducing abundant lactic acids, i.e., Lactobacillus acidophilus (KCTC No. 3164), Bifidobacterium longum sub sp. longum (KCTC No. 3128) etc, following experiment was performed.

[0126] The growth rate of Bacterioides fragilis was determined by treating 50 microgram/ml of ampicillin to the groups treated with both Bacterioides fragilis and various Lactobacillus strains, resulting in inhibition of growth of various Lactobacillus strains.

[0127] The growth rate of Bacterioides fragilis was determined by treating 50 microgram/ml of ampicillin to the groups treated with both Bacterioides fragilis and various Bifidobacterium strains, resulting in inhibition of growth of various Bifidobacterium strains.

[0128] The growth rate of Gardnerella vaginalis was determined by treating 20 microgram/ml of ampicillin to the groups treated with both Gardnerella vaginalis and various Lactobacillus strains, resulting in inhibition of growth of various Lactobacillus strains.

[0129] The growth rate of Gardnerella vaginalis was determined by treating 20 microgram/ml of ampicillin to the groups treated with both Bacterioides fragilis and various Bifidobacterium strains, resulting in inhibition of growth of various Bifidobacterium strains.

[0130] Based on the test result from preliminary test, the test groups were divided into five groups, i.e., (a) Group I treated with the combination with refined salt and glucose (1:1, w/w), such as CL1, CF1, CB1, CS1, (b) Group II treated with the combination with melted salt and fructo-oligosaccharide (1:2, w/w), such as CL2, CF2, CB2, CS2, (c) Group III treated with the combination with unrefined salt and lactulose (1:5, w/w), such as CL3, CF3, CB3, CS3, (d) Group IV treated with the combination with refined salt and fructose (2:1, w/w), such as CL4, CF4, CB4, CS4, and (e) Group VI treated with the combination with melted salt and lactitol (5:1, w/w) such as CL5, CF5, CB5, CS5. The diluted test samples were inoculated into the selected media comprising 50 microgram/ml of ampicillin or 20 microgram of gentamycin again. 20 microliter of pre-incubated media in liquid media was diluted to 1,000 microliter of Casmans medium supplemented with 5% FBS (Fetal bovine Serum) (12 g of Tryptone, 5 g of meat peptone, 5 g of sodium chloride, 3 g of yeast extract, 3 g of beef extract, 1 g of starch casein, 0.5 g of D-glucose, 0.05 g of nicotinamide, 0.005 g of p-aminobenzoic acid, 1 L of distilled water, pH 7.3, 5% FBS) and 20 microliter of media was collected to inoculated into 3 ml of Casmans medium supplemented with 5% FBS comprising selected antibiotics again. Both of agar containing solid medium and liquid medium were performed to shaking incubation at 37 C. for 24 hrs at the speed of 150 rpm using by GasPakEZ Pouch system (Anaerobic Gas Generation Pouch System with indicator, BD, Cat. No. 26083, USA).

[0131] The growth rate of the bacteria cultured in liquid media was determined by using spectrophotometer (Spectronic genesis 2, Thermo, USA, O. D. value at wavelength of 600 nm) and that in solid media was determined by counting the number of colonies.

2-3-2. Test Result

[0132] As can be seen in Table 10 showing the test result of Bacterioides fragilis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Bacterioides fragilis in the Groups I, III and V cultured with various Lactobacillus strains was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 10.

TABLE-US-00010 TABLE 10 Test result on the growth of Bacterioides fragilis cultured with various Lactobacillus strains B.F* O.D.sub.600** Colony No. Group Combination*** 0.815 2862 Group I CL1 0.007 0 Group II CL2 0.680 520 Group III CL3 0.011 0 Group IV CL4 0.627 512 Group V CL5 0.007 0 *B.F: Bacterioides fragilis, **O.D.sub.600: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0133] As can be seen in Table 11 showing the test result of Bacterioides fragilis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Bacterioides fragilis in the Groups I, II, III, IV and V cultured with Bifidobacterium strains was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 11.

TABLE-US-00011 TABLE 11 Test result on the growth of Bacterioides fragilis cultured with Bifidobacterium strains B.F* O.D.sub.600** Colony No. Group Combination*** 0.858 3125 Group I CF1 0.050 0 Group II CF2 0.039 0 Group III CF3 0.046 0 Group IV CF4 0.045 0 Group V CF5 0.037 0 *B.F: Bacterioides fragilis, **O.D.sub.600: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0134] As can be seen in Table 12 showing the test result of Bacterioides fragilis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Bacterioides fragilis in the Groups I, II, III, IV and V cultured with Bacillus cereus strain was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 12.

TABLE-US-00012 TABLE 12 Test result on the growth of Bacterioides fragilis cultured with Bacillus cereus strain B.F* O.D600** Colony No. Group Combination*** 0.841 3004 Group I CB1 0.035 0 Group II CB2 0.028 0 Group III CB3 0.041 0 Group IV CB4 0.038 0 Group V CB5 0.031 0 *B.F: Bacterioides fragilis, O.D600**: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0135] As can be seen in Table 13 showing the test result of Bacterioides fragilis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Bacterioides fragilis in the Groups I, II, III, IV and V cultured with Streptococcus sp strain was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 13.

TABLE-US-00013 TABLE 13 Test result on the growth of Bacterioides fragilis cultured with Streptococcus sp strain B.F* O.D.sub.600** Colony No. Group Combination*** 0.818 3724 Group I CS1 0.031 0 Group II CS2 0.038 0 Group III CS3 0.037 0 Group IV CS4 0.027 0 Group V CS5 0.016 0 *B.F: Bacterioides fragilis, **O.D.sub.600: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0136] As can be seen in Table 14 showing the test result of Gardnerella vaginalis in selected medium (Casmans medium: MRS medium (v/v), containing 20 microgram/ml of gentamycin), it has been confirmed that the growth of Gardnerella vaginalis in the Group III cultured with various Lactobacillus strain was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 14.

TABLE-US-00014 TABLE 14 Test result on the growth of Gardnerella vaginalis cultured with various Lactobacillus strains GV* O.D.sub.600** Colony No. Group Combination*** 0.533 3851 Group I CL1 0.044 42 Group II CL2 0.031 33 Group III CL3 0.030 35 Group IV CL4 0.063 59 Group V CL5 0.061 55 *GV: Gardnerella vaginalis, O.D.sub.600**O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0137] As can be seen in Table 15 showing the test result of Gardnerella vaginalis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Gardnerella vaginalis in the Groups I, II, III, IV and V cultured with various Bifidobacterium strains was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 15.

TABLE-US-00015 TABLE 15 Test result on the growth of Gardnerella vaginalis cultured with Bifidobacterium strains *G.V: O.D.sub.600** Colony No. Group Combination*** 0.831 3125 Group I CF1 0.036 0 Group II CF2 0.031 0 Group III CF3 0.035 0 Group IV CF4 0.028 0 Group V CF5 0.025 0 *GV: Gardnerella vaginalis, **O.D.sub.600: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0138] As can be seen in Table 16 showing the test result of Gardnerella vaginalis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Gardnerella vaginalis in the Groups I, II, III, IV and V cultured with Bacillus cereus strain was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 16.

TABLE-US-00016 TABLE 16 Test result on the growth of Gardnerella vaginalis cultured with Bacillus cereus strain G.V* O.D.sub.600** Colony No. Group Combination*** 0.856 3447 Group I CB1 0.035 0 Group II CB2 0.021 0 Group III CB3 0.026 0 Group IV CB4 0.015 0 Group V CB5 0.026 0 *GV: Gardnerella vaginalis, **O.D.sub.600: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0139] As can be seen in Table 17 showing the test result of Gardnerella vaginalis in selected medium (Casmans medium: MRS medium (v/v), containing 50 microgram/ml of ampicillin), it has been confirmed that the growth of Gardnerella vaginalis in the Groups I, II, III, IV and V cultured with Streptococcus sp strain was inhibited and the test result on O.D (optical density) value and the counting number of colonies, was shown in Table 17.

TABLE-US-00017 TABLE 17 Test result on the growth of Gardnerella vaginalis cultured with Streptococcus sp strain GV* O.D.sub.600** Colony No. Group Combination*** 0.832 3486 Group I CS1 0.025 0 Group II CS2 0.027 0 Group III CS3 0.031 0 Group IV CS4 0.019 0 Group V CS5 0.010 0 *GV: Gardnerella vaginalis, **O.D.sub.600: O.D. value at wavelength of 600 nm ***combinations listed in Table 2

[0140] Accordingly, it has been confirmed that the inventive combination of the present invention showed more potent inhibiting effect on vaginosis-causing bacteria comparing with the sole treatment of lactic acid bacteria and the combination of salt and sugar through the above experiments.

Experimental Example 3. Brief Clinical Test (1)

[0141] 1,200 mg of the vaginal tablet composition (SGL2) prepared in Example 2 was administrated externally once a day for 5 days to 100 volunteers consisting of 35 patients suffering from vaginosis, and 65 normal women ranging from 20 to 50 years who live in Korea to conduct a questionnaire survey and the difference of various contents, (a) preventive effect from unpleasant scent, (b) the level of freshness, (c) and the alleviating activity of skin psoriasis was surveyed.

[0142] The investigated result was classified into four groups, (1) very satisfied, (2) satisfied, (3) normal and (4) unsatisfied and the surveyed result shown in Table 18.

TABLE-US-00018 TABLE 18 Result of survey Surveyed content Content very satisfied satisfied normal unsatisfied sum (a) 79 16 5 0 100 (b) 74 16 8 2 100 (c) 71 19 10 0 100

[0143] At the result, as can be seen in Table 18, as to the (a) preventive effect from unpleasant scent, 94% volunteers after the treatment with inventive composition was satisfied and in particular, 79% volunteers was very satisfied.

[0144] As to the (b) the level of freshness, 86% volunteers after the treatment with inventive composition were satisfied and in particular, 72% volunteers were very satisfied. As to the alleviating activity of skin psoriasis, 85% volunteers after the treatment with inventive composition were satisfied and in particular, 67% volunteers were very satisfied.

[0145] Accordingly, it has been proved that the inventive vaginal tablet composition has potent favorable effect, for example, (a) preventive effect from unpleasant scent, (b) the level of freshness, (c) and the alleviating activity of skin psoriasis etc and it can be useful as a vaginal tablet composition for treating or preventing the patients from vaginal vaginosis.

[0146] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Experimental Example 4. Brief Clinical Test (2)

[0147] 200 ml of the vaginal cleansing composition (SGL4) prepared in Example 3 was administrated externally once a day for 5 days to 100 volunteers consisting of 42 patients suffering from vaginosis, and 58 normal women ranging from 20 to 50 years who live in Korea and the difference of vaginal pH between the pH of (A) before and (B) after the treatment with inventive composition was determined using by pH meter (MP-103, www.yuyuinst.co.kr).

[0148] At the result, as can be seen in Table 19, the vaginal pH of 85% test group before the treatment with inventive composition had reached to more than 5.5 however that of 90% test group after the treatment with inventive composition reached to normal pH range.

TABLE-US-00019 TABLE 19 pH difference pH <3.5 4 4.5 5 5.5 6 >6.5 Sum A 0 1 7 8 17 49 19 100 B 1 22 45 23 8 1 0 100

[0149] Accordingly, it has been proved that the inventive cleansing composition can be SGL4 can be useful in decreasing the vaginal pH of the patients suffering with vaginal akalisation.

[0150] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Experimental Example 5. Acute Toxicity Test of Oral Administration in Rat

[0151] The acute toxicity test was performed by administrating inventive combinations (SGL 2 and SGL4) to 6-weeks aged SPF Sprague-Dawley rats.

[0152] 250 mg/kg, 500 mg/kg, 1000 mg/kg, 5000 mg/kg of inventive combination was orally administrated to each group consisting of 2 rats and the symptoms of rats were observed for 14 days. After administrating the inventive combinations, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as haematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

[0153] There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there showed any toxicity in test group treated with 5000 mg/kg of inventive combinations.

[0154] Accordingly, it has been confirmed that the inventive combinations prepared in the present invention was potent and safe substance showing LD.sub.50 (more than 5000 mg/kg) in oral administration.

MODE FOR INVENTION

[0155] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of Injection

[0156] CB5 combination (100 mg) [0157] Sodium metabisulfite (3.0 mg) [0158] Methyl paraben (0.8 mg) [0159] Propyl paraben (0.1 mg) [0160] Distilled water for injection optimum amount

[0161] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.

Preparation of Powder

[0162] CL1 combination (500 mg) [0163] Corn Starch (100 mg) [0164] Lactose (100 mg) [0165] Talc (10 mg)

[0166] Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of Tablet

[0167] CL3 combination (200 mg) [0168] Corn Starch (100 mg) [0169] Lactose (100 mg) [0170] Magnesium stearate optimum amount

[0171] Tablet preparation was prepared by mixing above components and entabletting.

Preparation of Capsule

[0172] CB3 combination (100 mg) [0173] Lactose (50 mg) [0174] Corn starch (50 mg) [0175] Talc (2 mg) [0176] Magnesium stearate optimum amount

[0177] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of Liquid

[0178] CS3 combination (1000 mg) [0179] Sugar (20 g) [0180] Polysaccharide (20 g) [0181] Lemon flavor (20 g)

[0182] Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000 ml ample and sterilizing by conventional liquid preparation method.

Preparation of Health Food

[0183] CS5 combination (1,000 mg) [0184] Vitamin mixture (optimum amount) [0185] Vitamin A acetate (70 g) [0186] Vitamin E (1.0 mg) [0187] Vitamin B.sub.10 (13 mg) [0188] Vitamin B.sub.2 (0.15 mg) [0189] Vitamin B6 (0.5 mg) [0190] Vitamin B1 (20.2 mg) [0191] Vitamin C (10 mg) [0192] Biotin (10 mg) [0193] Amide nicotinic acid (1.7 mg) [0194] Folic acid (50 mg) [0195] Calcium pantothenic acid (0.5 mg) [0196] Mineral mixture (optimum amount) [0197] Ferrous sulfate (1.75 mg) [0198] Zinc oxide (0.82 mg) [0199] Magnesium carbonate (25.3 mg) [0200] Monopotassium phosphate (15 mg) [0201] Dicalcium phosphate (55 mg) [0202] Potassium citrate (90 mg) [0203] Calcium carbonate (100 mg) [0204] Magnesium chloride (24.8 mg)

[0205] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

Preparation of Health Beverage

[0206] CL4 combination (1000 mg) [0207] Citric acid (1000 mg) [0208] Oligosaccharide (100 g) [0209] Apricot concentration (2 g) [0210] Taurine (1 g) [0211] Distilled water (900 ml)

[0212] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 C. for 1 hour, filtered and then filling all the components in 1000 ml ample and sterilizing by conventional health beverage preparation method.

[0213] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

[0214] As described in the present invention, the present invention provides a composition comprising an inventive combination of salt, sugar and lactic acid bacteria as an active ingredient to treat or prevent vaginosis. The inventive composition showed potent antibacterial activity through various experiments, for example, (1) the indirect inhibitory activity test from the growth of vaginosis causing bacteria by determining the change of pH and lactic acid level (Experimental example 1); (2) the direct inhibitory activity test from the growth of vaginosis causing bacteria by determining the susceptibility of test sample (Experimental example 2); (3) brief clinical tests, and finally confirmed that the combination showed potent antibacterial activity in the test. Accordingly, the inventive combination may be useful to alleviate, treat or prevent vaginosis in the form of a pharmaceutical composition, health functional food, food additive, topical composition, and detergent composition.