WO2013093941A2

WO2013093941A2 - Compositions comprising probiotic lactobacillus strains for improved vaginal health

Info

Publication number: WO2013093941A2
Application number: PCT/IN2012/000828
Authority: WO
Inventors: Gursaran Parshad TALWAR; Kavita GARG
Original assignee: Talwar Research Foundation
Priority date: 2011-12-21
Filing date: 2012-12-18
Publication date: 2013-06-27
Also published as: WO2013093941A3; WO2013093941A4

Abstract

The present invention relates to a composition comprising: one or more isolated probiotic bacterial strain selected from the group consisting of: a) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617; b) Lactobacillus gasseri TRF #8 deposited under number MTCC 5615; and c) Lactobacillus salivarius TRF #30 deposited under number MTCC 5616 or variants thereof; at least one or more pharmaceutically acceptable excipients; optionally one or more herbal extract and/or prebiotics.

Description

COMPOSITIONS COMPRISING PROBIOTIC LACTOBACILLUS STRAINS FOR IMPROVED VAGINAL HEALTH

TECHNICAL FIELD

The present invention relates to probiotic lactobacillus strains, compositions comprising said strains and uses thereof.

BACKGROUND

In course of evolution, some bacteria have evolved to co-exist with human tissues, particularly in cavities such as the gastrointestinal tract and vagina. These non pathogenic friendly bacteria contribute to maintain the health of the tissue on which they are colonized. Doderlein (Zentralbl Gynakol 18: 10-14, 1894), a German scientist observed over a century back the presence of such micro-organisms in the vagina of women. Over time much has been learnt on the characteristics and function of these bacteria classified as "Probiotics".

Probiotics comprise live microorganisms beneficial to the host organism. According to definition adopted by FAO/WHO, probiotics are: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host". As far as the reproductive cavity of females is concerned, probiotic bacteria include the genus of Lactobacillus mostly and in postnatal years, bifidobacteria. The pH of normal healthy vagina is maintained around 4 (instead of the pH 7.4 of normal blood) by virtue of lactic acid that these bacteria make and secrete. Also many, but not all, strains of Lactobacilli secrete H₂0₂, around them which could be construed to act as a local antiseptic. The acidic pH created by Lactobacilli discourages the growth of several pathogens. Besides that, various lactobacilli also make and secrete peptides, termed as Bacteriocins, with anti-microbial properties. Many, but not all, lactobacilli have the enzyme arginine deiminase, which prevents the making of foul odour derivatives of this amino acid by other microbiota.

Lactobacilli Important for Healthy Vagina

Absence/Depletion of Lactobacilli in vagina disposes them to reproductive tract infections (RTIs). A number of women suffer from recurring episodes of RTIs, vaginosis/vaginitis l caused by a range of aerobic, anaerobic micro-organisms. They have abnormal vaginal discharge with foul odour. Invariably their vaginal pH is above 5, indicating that the lactobacilli normally resident in healthy vagina have disappeared from their vagina. Hence, there is a need for a composition comprising probiotic bacterial strains of the genera Lactobacillus, which have; an ability to colonize in human Vagina and produce high levels of lactic acid and have preferably the capability of producing and secreting H₂0₂ and have arginine deiminase to prevent the occurrence of foul odour. The composition is used for treatment and /or prophlaxis of vaginosis/ vaginitis, infections in the vagina and sexually transmitted infections.

SUMMARY

An object of the present subject matter is to provide a composition comprising: isolated probiotic bacterial strains of the genera lactobacillus which have the ability to colonize in human vagina and produce high levels of lactic acid to restore the acidic pH range of the vagina akin to that in normal vagina; at least one or more pharmaceutically acceptable excipient and/or prebiotics and/or polyherbal microbicide for treatment of vaginosis/ vaginitis, infections in the vagina, sexually transmitted diseases, infections endangering the foetus in pregnant women, preterm labour, urinary tract infection, and a microbial infection of the reproductive tract.

Another object of the present subject matter is to provide novel probiotic bacterial strains of the genera lactobacillius which have the ability to colonize in human Vagina and produce high levels of lactic acid to restore the acidic pH range of the vagina akin to that in normal vagina.

Another object of the present subject matter is to provide novel probiotic bacterial strains of the genera lactobacillus which in addition to producing high levels of lactic acid have the capability of producing and secreting H₂0₂ and/or have arginine deiminase to prevent the occurrence of foul odour.

Another objective of the present invention is to provide novel probiotic bacterial strains of the genera lactobacillus which can be administered intra-vaginally or orally, and are capable of colonizing in human vagina. Another objective of the present invention is to provide novel probiotic bacterial strains of the genera lactobacillus which can be administered intra rectally or orally to benefit the urogenital tract health. Arlother object of the present invention is to provide novel probiotic bacterial strains of the genera lactobacillus which prevent, alleviate the effects of, and/or treat vaginosis/ vaginitis, infections in the vagina and sexually transmitted infections.

Accordingly, the subject matter described herein is directed to a composition comprising: one or more isolated probiotic bacterial strains selected from the group consisting of:

a) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617;

b) Lactobacillus gasseri TRF #8 deposited under number MTCC 5615; and c) Lactobacillus salivarius TRF #30 deposited under number MTCC 5616

or variants thereof; at least one pharmaceutically acceptable excipients selected from the group comprising of diluent, binder, disintegrant, lubricant, glidant, stabilizer, surfactant, organic solvent, water, film forming polymer, opacifier, plasticizer, modified release polymer.

The composition of the present invention can optionally comprise one or more herbal extract selected from the group comprising of curcumin, amla (Emblica officinalis) extract , Neem {Azadirachta indica) leaves extract , Aloe Vera extract, Sapindus mukerosii , rose water and/or prebiotics.

Said bacterial strains have been deposited according to the Budapest Treaty at the MTCC in Chandigarh, India on June 1, 2011, and comprise the strain of:

(1) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617

(2) Lactobacillus gasseriJRF #8 deposited under number MTCC 5615

(3) Lactobacillus sa/ivariusTRF #30 deposited under number MTCC 5616 BRIEF DESCRIPTION OF DRAWINGS:

These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Fig. 1 illustrates a Genus specific PCR product (200bp amplicon) of 6 of the isolates obtained from the vagina of 80 women having healthy vagina.

Fig. 2 illustrates results of a Species specific PCR for 10 species of Lactobacilli obtained from the vagina of 80 women with healthy vagina. The representative figure shows the profiles of 2 isolates of 6 species and 1 isolate of the rest 4 species viewed on 2% agarose gel.

Lane SI and S2 is L. salivarius strain's 400bp product. Lane Rl and R2 is L reuteri strains's 300bp product. Lane PI and P2 is L plantarum strains' 250 bp amplicon. Lane Fl and F2 is L. fermentum strain's, 200bp amplicon. Lane Gl and G2 is L gasseri strain's 350bp product. Lane CI and C2 is L crispatus strain's 500bp PCR product. Lane Jl is L Jensen/Is 700bp amplicon. Lane Al is L. acidophilus strain's 200bp PCR product. Lane PC is L paracasei strain's 300bp product. Lane Rhl is L rhamnosus strain's lOObp product.

Fig. 3 illustrates a 350 bp polymorphic sequence from 16S r DNA of one of the isolate L fermentumTRF # 36 obtained from the vagina of 80 women with healthy vagina

Fig. 4 illustrates a RAPD (Random Amplified Polymorphic DIMA) profile of 7 strains of L salivarius, 7 strains of L reuteri and 6 strains of L. fermentum obtained from the vagina of 80 women with healthy vagina illustrating the heterogeneity of the strains in each species.

DESCRIPTION:

The invention relates to a composition comprising probiotic bacterial strains of the genera lactobacillus which in addition to producing high levels of lactic acid have the capability of producing and/or secreting H₂0₂ and have arginine deiminase to prevent the occurrence of foul odour along with at least one pharmaceutically acceptable excipients and/or prebiotics and/or vaginal use polyherbal microbicide for treatment and /or prophlaxis of vaginosis/ vaginitis, infections in the vagina, sexually transmitted diseases, infections endangering the foetus in pregnant women, preterm labour, urinary tract infection, and microbial infections of the reproductive tract. The said bacterial strains are chosen from the group comprising of;

(1) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617

(2) Lactobacillus gasseril ? #8 deposited under number MTCC 5615

(3) Lactobacillus sa/ivariusTRF #30 deposited under number MTCC 5616

or a variant thereof

The phrase " variant thereof as used herein in reference to the bacterial strains of the invention, especially those which have been deposited, is defined as a bacteria belonging to the cluster having a RAPD similarity of at least 80 % to said probiotic bacterial strains; and/or having the ability to colonize human vagina; and/or producing high levels of lactic acid and have preferably the capability of producing and secreting H₂0₂ and / or have arginine deiminase. The phrase "probiotic bacterial strain(s)" 'as used refers to bacterial strains which when administered in adequate amounts alone or in combination confer a health benefit on the host. In addition, the bacterial strains must colonize the vagina and produce lactic acid in high amounts. In one embodiment of the invention the probiotic bacterial strain is Lactobacillus fermentum ssp mucosae TRF#36, which was deposited, under the provisions of the Budapest Treaty, at MTCC Chandigarh on June 1, 2011 and has been assigned accession number MTCC 5617, or a variant thereof. In another embodiment of the invention the probiotic bacterial strain is Lactobacillus gasseri TRF #8, which was deposited under the provisions of the Budapest Treaty, at MTCC Chandigarh on June 1, 2011 and has been assigned accession number MTCC 5615, or a variant thereof.

In still another embodiment of the invention the probiotic bacterial strain is Lactobacillus salivariusJ ? #30, which was deposited, under the provisions of the Budapest Treaty, at MTCC Chandigarh on June 1, 2011 and has been assigned accession number MTCC 5616, or a variant thereof. In yet another embodiment the bacterial strains described above are used as a medicament.

In another aspect the present invention relates to the use of a bacterial strain as described above or a variant thereof along with an effective microbicide for treatment and /or prophlaxis of vaginosis/ vaginitis, infections in the vagina, sexually transmitted diseases, infections endangering the foetus in pregnant women, preterm labour, urinary tract infection, and a microbial infection of the reproductive tract.

The invention further relates to a composition comprising: one or more isolated probiotic bacterial strain selected from the group consisting of: a) Lactobacillus fermentum ssp mucosae T F#36 deposited under number MTCC 5617;

In a preferred embodiment of the present Invention the composition comprises one or more herbal extract selected from the group comprising curcumin , amla (Emblica officinalis) extract , eem {Azadirachta indica) leaves extract , Aloe Vera extract , Sapindus mukerosii , rose water.

In a preferred embodiment of the present Invention the composition comprises herbal extract as under: Ingrdient Preferable Amount curcumin 0.18 to 1.5 %

amla {Emblica officinalis) extract 1.5 to 15 %

Neem Azadirachta indica) leaves 5 to 15 %

extract

Aloe Vera extract 1.5 to 15 %

Sapindus mukerosii 0.5 to 2 %

Rose water 1.5 to 5 %

In another preferred embodiment the composition is administered along with BASANT, a polyherbai anti microbial composition comprising; Aloe vera, Emblica officinalis extract, Sapindus mukerosii, Curcumin and Rose water.

In another preferred embodiment the composition further comprises prebiotics.

The preferred embodiment of the invention further relates to a composition comprising: at least two lyophilized probiotic bacterial strains selected from the group consisting of: a) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617;

b) Lactobacillus gasseri TRF #8 deposited under number MTGC 5615; and c) Lactobacillus salivarius TRF #30 deposited under number MTCC 5616

or variants thereof; at least one pharmaceutically acceptable excipients selected from the group comprising of diluent, binder, disintegrant, lubricant, glidant, stabilizer, surfactant, organic solvent, water, film forming polymer, opacifier, plasticizer, modified release polymer; and one or more herbal extract selected from the group comprising of curcumin , amla Emblica officinalis) extract , Neem (Azadirachta indica) leaves extract , Aloe Vera extract Sapindus mukerosii , rose water and/or prebiotics. The most preferred embodiment of the invention relates to a composition comprises: at least 3 lyophilized probiotic bacterial strains selected from the group consisting of: a) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617;

b) Lactobacillus gasseri TRF #8 deposited under number MTCC 5615;and c) Lactobacillus salivarius TRF #30 deposited under number MTCC 5616

or variants thereof; at least one pharmaceutically acceptable excipients selected from the group comprising of diluent, binder, disintegrant, lubricant, glidant, stabilizer, surfactant, organic solvent, water, film forming polymer, opacifier, plasticizer, modified release polymer; and one or more herbal extract selected from the group comprising of curcumin , amla {Emblica officinalis) extract , Neem {Azadirachta indica) leaves extract , Aloe Vera extract, Sapindus mukerosii , rose water and/or prebiotics. In another preferred embodiment the composition of the present invention further comprises at least an herbal extract selected from the group comprising purified curcumin ,purified amla {Emblica officinalis) extract , Neem {Azadirachta indica) leaves extract , Aloe Vera extract, rose water or combination of these. In another aspect of the present invention the diluents are selected from microcrystalline cellulose, directly compressible grade microcrystalline cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol and the like, preferably starch.

In another aspect of the present invention the surfactant are selected from anionic surfactant, cationic surfactants and nonionic surfactant. Examples of the above- mentioned nonionic surfactant are, for instance, polyoxyethylamine oxides, alkylamine oxides, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerine esters, polyoxyethylene alkylamine, the derivatives thereof, and the like. Anionic surfactants are selected from higher fatty acid and its salt, alkyl sulfate, alkyl sulfonate, alkyl aryl sulfonate, alkyl phosphoric acid ester and the like, preferably Sodium lauryl sulphate.

In another aspect of the present invention the lubricants are selected from Magnesium stearate, Sodium stearyl fumarate, Stearic acid, Talc, Colloidal silicon dioxide and the like, preferably Magnesium stearate. In another aspect of the present invention the disintegrants are selected from alginic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate and starch, preferably sodium starch glycolate or crospovidone . In another aspect of the present invention the solvents are selected from purified water, alcohols, ketones, esters, ethers, halogenated solvents, hydrocarbons, nitrites, or mixtures thereof.

In yet another aspect of the invention, at least one probiotic bacterial strain is encapsulated or coated, and preferably present in an amount giving, an effective daily dose of 10³ - 10¹⁰ CFU. In yet another aspect of the invention, pharmaceutically acceptable excipients are selected from the group consisting of; Citric Acid; Sorbitol; Microcrystaline Cellulose; Sodium Starchglycolate; Starlac; Crospovidones; Sodium alginate and Magnesium Stearate.

In still another preferred embodiment of the present invention, the composition comprises at least one of the lactobacilli selected from the group consisting of: a) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617

b) Lactobacillus gasseriTRF #8 deposited under number MTCC 5615

c) Lactobacillus sal/variusTRF #30 deposited under number MTCC 5616 in the range of from lxlO⁶ to lOxlO⁹ , preferably a total of 3xl0⁹ along with the following ingredients; Ingredients Range

curcumin 0.18 to 1.5 %

amla {Emblica officinalis) extract 1.5 to 15 %

Neem {Azadirachta indica) leaves extract 5 to 15 %

Aloe Vera extract 1.5 to 15 %

Sapindus mukerosii 0.5 to 2 %

Rose water 1.5 to 5 %

Citric Acid 2-6%

Sorbitol 2-10%

Microcrystaline Cellulose 15-30%

Sodium Starchglycolate 5-15%

Starlac 15-30%

Crospovidones 0.5-5%

Sodium alginate 1-10%

Magnesium Stearate 0.25-2%

In one embodiment said pharmaceutical composition is administered vaginally along with pharmaceutically acceptable excipients and one or more herbal extracts.

In a further embodiment said pharmaceutical composition is administered orally.

In another embodiment said pharmaceutical composition is administered in the form of vaginal tablets, vaginal cellulose capsules, vaginal cream, gel, ointment, lotion, pessaries.

In another embodiment of the invention the composition is administered via a rectal suppository for treatment and relief from the symptoms of urogenital infections. Another embodiment of the present invention relates to a hygiene product comprising composition as described above. In one embodiment said hygiene product is chosen from the group comprising tampons, sanitary napkins, sanitary pads, diapers. In one embodiment said probiotic bacterial strain is viable. In still another embodiment it is genetically modified.

DESCRIPTION OF EMBODIMENT

With the consent of women and approval of the Institutional Ethics Committee, swabs were taken from the vagina of 80 women with healthy vagina in Delhi attending the Antenatal Clinics. These were cultured in MRS Broth.

Morphologically the isolates were rods. All of them were Gram positive, catalase negative. They were identified at molecular level by Polymerase Chain Reaction (PCR) employing genus specific primers. Figure 1 illustrates the result of a Genus specific PCR product of 6 of the isolates. All 80 isolates gave ~ 200 bp amplicon. The isolates were further identified by a Polymerase Chain Reaction (PCR) employing species specific primers. Figure 2 shows the profiles of 2 isolates of each of 6 species and 1 isolate of 4 species viewed on 2% agarose gel.

The identification of the species as revealed by the above investigation was confirmed by gene sequencing of 16s rDNA and Fig 3 gives as an illustration of such confirmation at genetic level of L fermentumTRF#36.

These investigations revealed that the most predominant Lactobacilli in Indian women are Lfermentum, Lreuteri, L.salivarius and Lplantarum belonging to Group IV (Garg et al, Ind. J. Med. Res. 2009, 129(6), 652-657), whereas Group II consisting of L crispatus, L. jensenii, and Group III consisting of L. rhamnosus are the most predominant Lactobacilli reported from vagina of women in USA, Canada, and Sweden {Vasquez et al, J Clin Microbiol (2002) 40(8): 2746-2749; Vallor et al, J Infect Diseas (2001) 184: 1431- 1436; Burton et al, Appl Environ Microbiol (2003) 69: 97-101}.

Thus, the probiotics for replenishment of vagina in India may be preferably chosen from the species prevalent in the country, although there is no reason to believe that once exposed, women elsewhere cannot benefit from colonization of Lactobacilli strains of the present invention.

After conducting extensive studies on 80 isolates form ecohealthy vagina of women, the following 3 strains were found highly beneficial: (1) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617

(2) Lactobacillus gasser/TRF #8 deposited under number MTCC 5615

(3) Lactobacillus salivariuslR? #30 deposited under number MTCC 5616

They have good growth rates. They are high producers of D lactic acid. Lactobacillus fermentum and L. gasseri make and secrete H₂0₂. They are highly hydrophobic as measured by Microbial adhesion to hydrocarbon (MATH) assay. They have arginine deiminase to prevent the occurrence of foul odour.

L. fermentum TRF #36 and L. gasseri TRF #8 are H₂0₂ producers. H₂0₂ has antiseptic and anti-viral properties. Peroxidase, H₂0₂ and halides constitute a strong anti-microbial system in phagocytes and tissue fluids (Klebanoff, 3 Bacteriol 1968; 95:2131-2138). Peroxidase and halide ions are already present in the vaginal fluid. In vitro, H₂0₂ producing Lactobacillus strains have strong inhibitory action against Gardnerella vaginalis, Prevotella bivia, Neisseria gonorrhoae and HIV-1 (Klebanoff et al, J Infect Diseas 1991; 164: 94-100). The strains L fermentum TRF#36, L. salivariuslR? #30 and L gasseri RF #8 produce arginine deiminase which prevents the production of foul smelling derivatives of arginine. The enzyme has anti-viral activity against HIV-l (Kubo et al, J General Virol 2006; 87:1589-1593). Furthermore, it prevents the formation of polyamines. Polyamines like spermine, spermidine, putrescine and cadaverine are found in elevated concentration in vaginal discharge of women with bacterial vaginosis contributing to elevated vaginal pH. These polyamines account for transudation and exfoliation of the epithelial cells thereby causing copious discharge.

All the three strains namely, L. fermentum TRF # 36, L salivarius TRF # 30 and L. gasseri TRF # 8 produce high amount of lactic acid sufficient to acidify the vagina. Healthy vagina has a pH of 3.8-4.5. Vaginal pH of women with bacterial vaginosis is elevated >5 because of depletion of resident lactobacilli of good properties.

All the three strains namely, L. fermentum TRF # 36, L. salivarius TRF # 30 and L. gasseri TRF # 8 are resistant to antibiotics metronidazole, clotrimazole, vancomycin, ciprofloxacin up to concentrations usually employed for therapy against banal infections. They are also tolerant to Polyherbal microbicide Praneem, Basant and Nauroz. Hence, these polyherbal microbicides can be employed for prevention of STIs without affecting the presence of these lactobacilli in the vagina. The selected strains are however sensitive to antibiotics, clindamycin, amoxicillin, erythromycin.

Clinical Evaluation of Colonization Potential and Efficacy of bacterial strains to Protect against RTIs. Clinical trials were conducted with the permission of the Drugs Controller General of India and Institutional Ethics committees in 2 premiere Medical Institutions viz The All India Institute of Medical Sciences and Sir Gangaram Hospital, New Delhi. These trials were conducted in women with recurring episodes of vaginosis/vaginitis with vaginal pH > 5. The objective of the trials was to determine the colonization potential of the selected probiotics and their ability to maintain the pH of vagina and protect women from getting recurrence of reproductive tract infections such as vaginosis /vaginitis.

To restore the Reproductive Health of women, a composition comprising the isolated probiotic bacterial strain was administered intra-vaginally in women. The composition comprised 3 billion Probiotics of either one or more than one selected strains of Lactobacillus TRF # 36, TRF # 30 and TRF # 8 along with purified diferuloyl methane {(E,E)-l,7-bis(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione} commonly known as curcumin, purified extract of Embilca officinalis (Amla), purified extracts of Neem leaves and Aloe vera. These ingredients are formulated in pharmaceutically approved excipients selected from the group comprising of: Citric Acid, Sorbitol, Microcrystaline Cellulose, Sodium Starchglycolate, Starlac, Crospovidones and Sodium alginate. This formulation is easily insertable in the vagina, with washed fingers, when dispensed in a cellulose capsule, the preferred mode of delivery. The composition comprises 3 l0⁹ lactobacilli alone or combination of 2 or 3 of selected strains of Lactobacillus TRF # 36, TRF # 30 and TRF # 8 along with;

Amla 2.5-15% 5%

Neem leaves extract 5-15% 6%

Aloe vera 2.5-15% 5%

Citric Acid 2-6% 4%

Sorbitol 2-10% 5%

Microcrystaline Cellulose 15-30% 28%

Sodium Starchglycolate 5-15% 10%

Starlac 15-30% 24%

Crospovidones 0.5-5% 3%

Sodium alginate 1-10% 6%

Magnesium Stearate 0.25-2% 1%

Typical observations on efficacy and utility of the Formulation are indicated by the observations given below in women suffering from recurring episodes of vaginosis/vaginitis, whose vaginal pH was above 5 at the time of enrolment.

A few illustrative examples of efficacy of administration of these bacilli given alone or in combination are given below.

Table 1: Follow up chart of subject (APB) who after treatment with Poly herbal microbicide BASANT was given a single Probiotic selected strain.

Day 26 - 4.5 1 +

Day 29 - 4.5 0 +

Day 36 - 4.5 1 +

Day 53 - 4.5 2 +

Day 54 - 4.5 2 +

Table 2: Follow up chart of subject (Asukh)

Table 3: Follow up chart of subject (G)

Day 22 5.0 4 L salivarius

TRF#30 (3xl0⁹ lyophilized

bacilli)

Day 40 - 4.5 2 +

Day 53 - 4.3 2 +

Day 82 - 4.5 3 -

Day 83 - 4.5 0 +(L.fermentum

TRF#36&

Lgasseri TRF#8)

Table 5: Follow up chart of subject (UD):

Table 6: Follow up chart of Subject with Code: BV-138/IIB

After 20 days After 30 days

Symptom Abnormal + +

Vaginal Discharge

Discharge-Amount + + • -

Colour

Odour -

Quality Watery -

Itching/Burning + - - pH of vagina 6.0 4.5 4.5

Subjective Relief

from Itching, 90% Relief highly >90% Relief fully Burning and satisfied satisfied

Discharge

Side Effects None None

These trials have amply shown the merit of 3 of these selected Lactobacillus strains to colonizie for periods of 1-3 months in the vagina lacking lactobacillus after a single intra- vaginal administration of 3xl0⁹ lyophilized bacilli. The strains are Lactobacillus fermentum strain TRF#36, Lactobacillus gasseri strain TRF #8 and Lactobacillus salivarius strain TRF #30. The combination of TRF#36 and TRF#8 maintained the vagina healthy at pH 4 free of recurrence of vaginosis in women getting recurring episodes of infections for longer than 1 month and in some recipients up to 3 months of observation period. Although each one of the three selected Probiotics strain have ability to colonize vagina of women suffering from recurring episodes of vaginosis/vaginitis, combination of two is more effective. The most effective results were obtained with a triple combination of the three selected strains. The Table given below provides a summary statement: Table 7: Table shows properties of Probiotic bacterial strains of the present invention when administered alone or in combination for their ability to colonize and restore pH to acidic range in women suffering recurrently from vaginoisis.

In table 1 and 7

"-"indicates- "Negative result"

"+" indicates- "Positive result"

"++" indicates- "a positive result that is better than "+""

"+++" indicates- "a positive result that is better than "++""

The best results were obtained when the 3 probiotic strains of the invention were used in combination.

Although the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present invention as defined.

Claims

We claim:

1. A composition comprising: one or more isolated probiotic bacterial strain selected from the group consisting of:

a) Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617;

or variants thereof; at least one or more pharmaceutically acceptable excipients selected from the group comprising of diluent, binder, disintegrant, lubricant, glidant, stabilizer, surfactant, organic solvent, water, film forming polymer, opacifier, plasticizer, modified release polymer; and optionally one or more herbal extract or combination selected from the group comprising of curcumin, amla {Emblica officinalis) extract , Neem {Azadirachta indica) leaves extract , Aloe Vera extract , Sapindus mukerosii , rose water and/or prebiotics.

2. The composition as claimed in claim 1, wherein the probiotic bacterial strains are present in the range of from l l0⁶to lOxlO⁹.

3. The composition as claimed in claim 1, wherein any two of said lyophilized probiotic bacterial strains are in consortium.

4. The composition as claimed in claim 1, wherein all 3 of said lyophilized probiotic bacterial strains are in consortium.

5. The composition as claimed in any of the claims 1 to 4, wherein curcumin is present in the in the range of from 0.18 to 1.5 %, amla {Emblica officinalis) extract is present in the range of from 1.5 to 15 %, Neem {Azadirachta indica) leaves extract is present in the of from 5 to 15 %, Aloe Vera extract is present in the range of from 1.5 to 15 %, Sapindus mukerosii is present in the range of from 0.5 to 2 %, rose water is present in the range of from 1.5 to 5 %.

6. The composition as claimed in any of the claims 1 to 4, wherein the diluents are selected from the group comprising of; microcrystalline cellulose, directly compressible grade microcrystalline cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol.

7. The composition as claimed in any of the claims 1 to 4, wherein the binders are selected from the group comprising of; acacia, alginic acid, carbomer, carboxymethylcellulose sodium, dextrin, ethyl cellulose, guar gum, hydrogenated vegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone, pregelatinized starch, sodium alginate, sorbitol, and starch.

8. The composition as claimed in any of in any of the claims 1 to 4, wherein the surfactant may be selected from the group comprising of anionic surfactant, cationic surfactants and non ionic surfactant.

9. The composition as claimed in any of the claims 1 to 4, wherein the lubricants are selected from magnesium stearate, sodium stearyl fumarate, stearic acid, talc, colloidal silicon dioxide.

10. The composition as claimed in any of the claims 1 to 4, wherein the disintegrants and stablizer are selected from alginic acid, croscarmellose sodium, sodium starch glycdlate, crospovidone, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, starlac and starch.

11. The composition as claimed in any of the claims 1 to 4, wherein the solvents are selected from purified water, alcohols, ketones, esters, ethers, halogenated solvents, hydrocarbons, nitrites, or mixtures thereof.

12. The composition as claimed in claim 1, wherein said one or more of the lactobacilli are in the range of from lxl0⁶to lOxlO⁹ ; Said pharmaceutically acceptable excipients are, citric acid preferably in the range of from 2 to 6%; sorbitol preferably in the range of from 2 to 10%; microcrystaline cellulose preferably in the range of from 15 to 30% ; sodium starchglycolate preferably in the range of from 5 to 15%; starlac preferably in the range of from 15 to 30%; crospovidones preferably in the range of from 0.5 to 5%; sodium alginate preferably in the range of from 1 to 10% and magnesium stearate preferably in the range of from 0.25 to 2%;

Said herbal extracts are, curcumin preferably in the range of from 0.18 to 1.5 %, amla (Emblica officinalis) extract preferably in the range of from 1.5 to 15 %, neem {Azadirachta indica) leaves extract preferably in the of from 5 to 15 %, Aloe

Vera extract preferably in the range of from 1.5 to 15 % optionally also Sapindus mukerosii preferably in the range of from 0.5 to 2 %, rose water preferably in the range of from 1.5 to 5 %.

13. The composition as claimed in claim 12, comprising

3xl0⁹ of said lactobacilli strains alone or in combination;

4 % citric Acid; 5% sorbitol; 28% microcrystaline cellulose; 10 % sodium starchglycolate; 24 % starlac; 3% crospovidones; 6% sodium alginate; 1% magnesium stearate;

1% curcumin; 5 % amla extract; 6 % Neem leaves extract and 5 % Aloe Vera extract.

14. The composition as claimed in claim 12, comprising 3xl0⁹ lactobacilli alone or combination of said three selected strains of Lactobacillus.

15. The composition as claimed in claim 1 to 14, wherein the probiotic bacterial strains are encapsulated or coated.

16. The composition as claimed in any of the preceding claims, wherein the probiotic bacterial strains are present in an amount giving an effective dose of from 10³ to 10¹⁰ CFU.

17. The composition as claimed in any of the preceding claims, wherein said composition is capable of vaginal or oral administration.

18. The composition as claimed in any of the preceding claims, wherein said composition is in a form selected from the group consisting of vaginal tablets, vaginal vegetarian (cellulose) capsules, cream, gel, ointment, lotion, pessaries.

19. The composition as claimed in claims 1 to 17, wherein said composition is in the form of a rectal suppository.

20. A hygiene product comprising the composition claimed in any one of the preceding claims.

21. The hygiene product as claimed in claim 20, wherein said hygiene product is selected from the group comprising of tampons, sanitary napkins, sanitary pads, diapers.

22. An isolated Lactobacillus fermentum ssp mucosae TRF#36 deposited under number MTCC 5617 or variants thereof.

23. An isolated Lactobacillus gasserilR? #8 deposited under number MTCC 5615 or variants thereof.

24. An isolated Lactobacillus salivariuslK? #30 deposited under number MTCC 5616 or variants thereof.

25. A consortium of one or more of the isolated bacteria claimed in claim 22 to 24.