Composition of lactobacillus crispatus for nutraceutical and therapeutic uses and method thereof

Abstract

The present invention comprises a consortium of Lactobacillus crispatus with pathogen resistance and anti-steatosis functions for nutraceutical and therapeutic uses and method thereof. The present invention comprises an indigenous microbiota that inhibit the growth of pathogenic bacteria, including Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus, Gardnerella vaginalis and reduce the accumulation of visceral and hepatic fat.

Description

[0001] COMPOSITION OF LACTOBACILLUS CRISPATUS FOR NUTRACEUTICAL AND THERAPEUTIC USES AND METHOD THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The invention, in general, relates to the field of Nutraceuticals and Therapeutics. More particularly, the invention relates to a consortium of Lactobacillus crispatus with pathogen resistance and anti-steatosis functions for nutraceutical and therapeutic uses and method thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006] Presently, the healthy vaginal microbiome is mostly dominated by the different members of Lactobacillus species, which are lactic acid-producing bacteria. They maintain a lower pH through the fermentation of glycogen into lactic acid, creating an acidic environment that helps prevent the growth of acid-sensitive pathogens. Despite the global use of antibiotics like metronidazole and clindamycin to treat microbial imbalances associated with bacterial vaginosis (BV) and restore normal vaginal flora, recurrence rates as high as 69% have been observed post-treatment.

[0007] Research into the human microbiome demonstrated significant genomic and functional variations among autochthonous microbes residing in different body sites such as the gastrointestinal tract, vagina, oral cavity, and skin. These variations are driven by the flexible gene pool acquired horizontally from related or unrelated microbes, leading to population- and niche-specific adaptations. Lactobacillus species exhibit high genomic, ecological, and phenotypic heterogeneity, particularly evident in sites like the reproductive tract, oral cavity, and gastrointestinal tract. The greatest diversity of lactobacilli is typically found in the vaginal environment of reproductive-aged women.

[0008] Lactobacilli contribute to host immunity by producing antimicrobial peptides and maintaining a low vaginal pH through glycogen fermentation. Disruptions to the endogenous vaginal microbiome by pathogenic bacteria can result in adverse pregnancy outcomes. There is a list of few already available in the market like LACTINV, INLIFE Prebiotics and Probiotic and Rephresh Pro-B Probiotic Supplement. LACTIN-V, also known as CTV-05 or as Lactobacillus crispatus CTV-05 and Rephresh Pro-B Probiotic Supplement were developed which focused on the treatment of bacterial vaginosis (BV) only. INLIFE Prebiotic and Probiotic is a consortium of five different bacteria which is mainly involved for gut health.

[0009] For example, US10842834B2 discloses a method for reducing the likelihood of developing liver cancer in an individual diagnosed with non-alcoholic fatty liver disease involves providing in the gut of an individual a population of beneficial bacteria selected from the group consisting of Lactobacillus species and administering fiber to the individual to maintain a therapeutically effective amount of the beneficial bacteria in the gut of the individual. In certain embodiments, monoacylglycerolacyltransferase-3 (MGAT3) synthesis is inhibited to lower triacylglycerol (TAG) production, while in others, expression of diacylglycerolacyltransferase-2 (DGAT-2) is inhibited. The beneficial bacteria are preferably modified to produce increased amounts of butyrate and may also be encapsulated in a frangible enclosure. Levels of Roseburia are preferably increased while the levels of Akkermansia spp. in the individual's gut microbiome are reduced. In other embodiments, a therapeutically effective amount of a bacterial formulation comprising Faecalibacterium prausnitzii is administered, or a composition comprising modified Lactobacillus reuteri bacteria having the ability to survive conditions in the duodenum or jejunum of the individual's small intestine. Other embodiments include the administration of a bacterial formulation comprising at least one of Coprococcus, Veillonella, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii and Prevotella.

[0010] Therefore, there is a need for developing probiotics composed of Lactobacillus species aimed at restoring a balanced vaginal and gut microbiomes and sustaining an acidic environment. The present invention offer cost-effective strategies to enhance human microbiome health. OBJECTIVE OF THE INVENTION:

[0011] The primary object of the present invention is to overcome the drawbacks associated with prior art.

[0012] Another object of the present invention is to provide a unique indigenous microbial consortium containing Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9-4-C11 for nutraceutical and therapeutic uses.

[0013] Another object of the present invention is to provide a unique indigenous microbial consortium with reproductive tract, respiratory tract and gastrointestinal tract associated bacterial pathogens resistance and anti-steatosis functions.

[0014] Another object of the present invention is to prevent bacterial infections through targeted probiotic interventions, thereby improving overall human microbiome health.

[0015] Another object of the present invention is to provide an indigenous microbiota that can inhibit the growth of both Gram negative and Gram positive pathogenic bacteria, including Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus and Gardnerella vaginalis and reduce the accumulation of visceral and hepatic fat.

[0016] SUMMARY OF THE INVENTION:

[0017] The present invention is described in the following sections by various embodiments. However, it should be understood that the invention can be implemented in various forms and is not limited to the specific embodiment provided herein. In the context of the present disclosure, it should be understood that the described embodiments in this section are put forth for illustrative purposes only. Those skilled in the art will appreciate that various modifications, adaptations, and alternative designs may be employed without departing from the scope and spirit of the invention. Accordingly, the present invention should not be limited to the specific embodiments illustrated herein, but rather should be construed according to the claims and description that follow.

[0018] Embodiments of the present invention provide a composition comprising a microbial consortium that includes a plurality of Lactobacillus crispatus strains fS10-5-C2-2 and S9-4-C11) isolated from high vaginal swab samples, wherein each strain is genetically characterized and found to harbour genes encoding bacteriocins, anti-inflammatory peptides, acid tolerance proteins, and glycogen degrading enzymes. The composition comprises a pharmaceutically acceptable carrier, and the consortium produces lactic acid from glycogen metabolism to establish a local pH ranging from about 3.8 to about 4.5. This targeted acidification not only creates an environment unfavourable to pathogenic bacteria but also supports a balanced microbiome, which together contribute to enhanced pathogen inhibition and modulation of metabolic functions, thereby overcoming limitations of conventional broad-spectrum treatments.

[0019] In accordance with an embodiment of the present invention, the microbial consortium further comprises Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9- 4-C11 strains that genetically express a plurality of CRISPR-Cas type II A systems. Additionally, these genetic elements provide adaptive immunity against foreign genetic materials and ensure the genomic stability and safety of the strains when used as probiotics. This further enhances the overall robustness and reliability of the composition, as the CRISPR-Cas systems play a critical role in maintaining the functional integrity of the active strains.

[0020] In accordance with an embodiment of the present invention, the composition is configured for administration in encapsulated dosage forms, sachets, or suppositories. In addition, the components included in the formulation are selected to preserve the viability and metabolic activity of the probiotic strains during processing, storage, and delivery. The formulation therefore contributes not only to local antimicrobial activity but also to effective systemic delivery, ensuring that the bioactive components are released at the target site to exert their intended therapeutic and nutraceutical effects.

[0021] Embodiments of the present invention provide a method for preparing a composition comprising collecting high vaginal swab samples from reproductive-age women under sterile conditions and isolating a plurality of Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9-4-C11 strains from the collected samples under anaerobic conditions. The isolated strains are cultured in a growth media formulated to simulate an acidic environment and subjected to genome sequencing along with biochemical assays to identify genes encoding bacteriocins, anti-inflammatory peptides, acid tolerance proteins, and glycogen degrading enzymes. The selected strains are then assembled into a consortium in ratios sufficient to maintain metabolic activity that results in lactic acid production lowering the pH to a range of about 3.8 to 4.5, and the consortium is subsequently incorporated into a formulation comprising a pharmaceutically acceptable carrier and buffering agents. This integrated process ensures that the composition harnesses both the antimicrobial and metabolic modulation functionalities of the selected strains.

[0022] In accordance with an embodiment of the present invention, the genome sequencing step includes identifying a plurality of gene clusters encoding for ribosomally and non- ribosomally synthesized bacteriocins. Additionally, this identification further verifies the presence of multiple genes that confer enhanced antibacterial properties, substantiating the potency and specificity of the antimicrobial response attributed to the probiotic strains.

[0023] In accordance with an embodiment of the present invention, the assembly of the Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9-4-C11 strains is conducted in a ratio that preserves the inherent microbial balance characteristic of healthy vaginal microbiota. Additionally, this carefully controlled ratio ensures that the consortium synergistically promotes lactic acid production and the secretion of antimicrobial substances, reinforcing its dual function of pathogen inhibition while modulating metabolic pathways to reduce visceral and hepatic fat accumulation.

[0024] In accordance with an embodiment of the present invention, the method further comprises administering the formulated composition to a subject. Upon administration, the indigenous Lactobacillus crispatus consortium produces lactic acid by metabolizing glycogen present in the host, thereby establishing a local pH between about 3.8 and about 4.5. This acidification not only provides a direct antimicrobial effect against pathogens such as Gardnerella vaginalis, Escherichia coli, Enterobacter hormaechei, Staphylococcus aureus and Klebsiella pneumoniae but also contributes to broader systemic benefits by influencing gut permeability, bile acid metabolism, and fatty acid oxidation which are key factors in reducing metabolic disorders.

[0025] These embodiments collectively illustrate a novel and integrated approach wherein the indigenous microbial consortium is optimized for both targeted pathogen inhibition and metabolic regulation. The synergistic interaction among the isolated Lactobacillus crispatus strains, their genomic and biochemical modules, the corresponding production of antimicrobial and lactic acid metabolites, and the supporting formulation and delivery system, comprises a robust composition that addresses both infectious and metabolic health challenges in a unified manner, offering significant therapeutic and nutraceutical advantages over existing solutions.

[0026] DETAILED DESCRIPTION OF THE DRAWINGS:

[0027] To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting in their scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:

[0028] Fig. 1 : Illustrate the Graphical representation of the development of a consortium of Lactobacillus crispatus with pathogen resistance and anti -steatosis functions for nutraceutical and therapeutic uses of the present invention.

[0029] DETAILED DESCRIPTION:

[0030] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0031] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0032] In an aspect of the present invention, there is provided an indigenous microbiota that inhibit the growth of pathogenic bacteria, including Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus, Gardnerella vaginalis and reduce the accumulation of visceral and hepatic fat.

[0033] In an embodiment, the present invention provides different species of Lactobacillus found in higher abundance in the vaginal microbiota of reproductive-age women across the globe, including in India. There is evidence that the Lactobacillus dominant vaginal microbiome reduces the risk of preterm birth (PTB).

[0034] In an embodiment, the present invention isolated, identified, and characterised Lactobacillus crispatus from the reproductive tract of pregnant Indian women enrolled in the GARBH-Ini cohort. The genome of Lactobacillus crispatus is enriched with pathogen resistance and anti-inflammatory functions. These functions are constitutively expressed, can inhibit the growth of Gram-negative bacterial pathogens, and reduce the risk of infections. The pathogen resistance function of the Lactobacillus crispatus cell free supernatants were evaluated by both well diffusion assay and time kill assay. Briefly, the cell free supernatant of the Lactobacillus crispatus strains cultured for 48 hours were collected and added into 8mm wells cut into Mueller Hinton agar plates swabbed with specific bacterial pathogens. The plates were incubated at 37°C and zones of inhibitions were measured at designated time intervals. Additionally, the bactericidal activity of the cell free supernatants was assessed through time kill assay by directly incubating the pathogens with the cell free supernatants and evaluating the bacterial viability at specified time points. Furthermore, this Lactobacillus crispatus consortium could further prove to be potential candidates for nutraceutical and biotherapeutics. The two Lactobacillus crispatus strains used in the consortium have been deposited at the Microbial Type Culture Collection and Gene Bank (MTCC), an International Depository Authority (IDA) under the Institute of Microbial Technology, as Lactobacillus crispatus MTCC 25902 and Lactobacillus crispatus MTCC 25903.

[0035] In an embodiment, the present invention comprises a distinctive indigenous microbial consortium based on Lactobacillus crispatus isolated from the vaginal milieu. This specialized blend exhibits resistance to bacterial pathogens associated with the reproductive, respiratory, and gastrointestinal tracts, and offers anti-steatosis benefits. Oral supplementation of the Lactobacillus crispatus effectively mitigated the systemic metabolic, hepatic, and cardiac pathologies in a diet induced cardiometabolic disease mice model. Lactobacillus crispatus supplementation significantly attenuated hepatic lipid accumulation, cellular ballooning, and collagen deposition, as confirmed by histology and non-invasive ultrasound echogenicity. It effectively inhibits the growth of harmful bacteria such as Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus Gardnerella vaginalis, while also reducing the accumulation of visceral and hepatic fat. Lactobacillus crispatus (consortium) produces lactic acid as a metabolic byproduct, which lowers the vaginal pH. The acidic environment (typically pH 3.8-4.5) created by Lactobacillus crispatus is inhospitable to many pathogenic bacteria and helps maintain the natural balance of the vaginal microbiota.

[0036] In an embodiment of the present invention, the indigenous consortia of Lactobacillus crispatus produces various antimicrobial substances, including bacteriocins and lactic acid. Bacteriocins are proteins that specifically target and inhibit the growth of other bacteria, while lactic acid reduces the environments pH which kills or inhibit many pathogenic bacteria. These antimicrobial effects can also help prevent infections that might otherwise contribute to respiratory conditions.

[0037] In an embodiment of the present invention, the developed consortium can also alter the composition of the gut microbiota. By increasing the abundance of beneficial bacteria and reducing the number of harmful bacteria, Lactobacillus crispatus can impact the metabolism of dietary fats and carbohydrates, leading to reduced fat accumulation. It reduces the absorption of dietary fats in the gut. This is achieved through the modulation of bile acid metabolism or changes in gut permeability, leading to decreased fat accumulation in visceral and hepatic tissues. It also enhances fatty acid oxidation in the liver and other tissues, reducing the storage of fats.

[0038] Thus, by improving gut barrier integrity and reducing intestinal permeability, Lactobacillus crispatus can prevent endotoxins from entering the bloodstream, which can otherwise contribute to inflammation and fat accumulation.

[0039] In an embodiment of the present invention, the gut microbiota has a significant impact on lung health through what is known as the gut-lung axis. Our invention can help maintain a healthy gut microbiota, which in turn can influence lung health. A balanced gut microbiome can reduce systemic inflammation and improve overall immune function, potentially leading to fewer respiratory infections and less severe symptoms.

[0040] The Invention provides a composition comprising: a) a microbial consortium comprising plurality of Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9-4-C11 strains isolated from high vaginal swab samples, said strain characterized to express genes encoding one or more bacteriocins, one or more anti-inflammatory peptides, acid tolerance proteins, and glycogen degrading enzymes; b) a pharmaceutically acceptable carrier; c) buffering agents and stabilizers, wherein the consortium produces lactic acid from glycogen metabolism to establish a local pH ranging from about 3.8 to about 4.5.

[0041] In an embodiment, the microbial consortium comprises Lactobacillus crispatus strains that genomically express plurality of CRISPR-Cas type II A systems.

[0042] In an embodiment, the method of preparing the composition as described above, comprises the steps of:

[0043] (a) collecting high vaginal swab samples from reproductive-age women under sterile conditions;

[0044] (b) isolating a plurality of Lactobacillus crispatus strains from the collected samples under anaerobic conditions High vaginal swab (HVS) samples were collected from pregnant Indian women at the time of enrolment in GARBHIni cohort if they fulfilled the inclusion criteria: (i) age > 18 but < 40 years; (ii) period of gestation < 20 weeks; (iii) natural conception; (iv) no local (vaginal) antibiotics usages in last 3 months; (iv) absence of hypertension, diabetes, hypo- and hyperthyroidism, and any chronic health disorders and (vi) no evidence of sexually transmitted diseases. The participants were positioned in the lithotomy position. The midpoint of the vagina was accessed using a Cusco’s speculum and HVS samples were collected by holding the Sterile Catch-All™ Sample Collection swabs gently against the mid vaginal wall on the right side for 20 s ensuring that there is no contamination by the cervical mucus.

[0045] (c) culturing the isolated strains in growth media formulated to simulate an acidic environment; HVS samples collected in ~ 750pl of phosphate buffer saline (PBS) was transferred to laboratory7at the Translational Health Science and Technology Institute (THSTI) on ice within 2 h. HVS samples were streaked and spread with up to dilutions I CT4onto de Man, Rogosa, and Sharpe (MRS) .Agar (Sigma, USA) plates for screening and isolation of Lactobacillus species. The plates were then incubated at 37 °C in anaerobic conditions for 48-72 h. Colonies with distinct morphologies were selected randomly and inoculated in AIRS Broth, for isolation and downstream microbiological analysis. Initial identification was done with the 16S rRNA gene sequencing using Sanger Sequencer and further whole genome sequenced (WGS).

[0046] (d) performing genomic sequencing and biochemical assays on the isolated strains to identify genes encoding bacteriocins, anti-inflammatory peptides, acid tolerance proteins, and glycogen degrading enzymes; said step identifies gene clusters encoding for ribosomally and non-ribosomally synthesized bacteriocins;

[0047] (e) assessing their in vitro antibacterial efficacy of the two strains includedin the consortium using both well diffusion assay and time kill assays. The strains demonstrated broad spectrum inhibitory activity against clinically relevant bacterial pathogens including Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus and Gardnerella vaginalis, all of which associated with various human infections.

[0048] (f) assessing the anti-steatosis functions of the Lactobacillus crispatus consortium as evidenced by pre-clinical diet induced cardiometabolic disease mice model. Lactobacillus crispatus supplementation effectively mitigated the systemic metabolic, hepatic, and cardiac pathologies induced by a choline- deficient high-fat (CDHF) diet in mice. Lactobacillus crispatus supplementation significantly attenuated hepatic lipid accumulation, cellular ballooning, and collagen deposition, as confirmed by histology and non-invasive ultrasound echogenicity.

[0049] (g) assembling the selected strains into a consortium in ratios sufficient to maintain a metabolic activity resulting in lactic acid production that lowers pH to a range of about 3.8 to about 4.5; The consortium comprises two well characterized Lactobacillus crispatus strains combined in a 1 : 1 ratio with 1X109each CFU / mL and formulated within a pharmaceutically appropriate carrier matrix.

[0050] (h) incorporating the consortium into a formulation comprising a pharmaceutically acceptable carrier, buffering agents, and stabilizers. The formulation comprise of 10% sucrose v / v which acts as the cryoprotectant and stabilizer.

[0051] The present invention is defined by non-limiting examples such as:

[0052] Example 1:

[0053] Example supporting Genomic insights of the L. crispatus strains used for developing the consortium:

[0054] These Lactobacillus crispatus strains were obtained from high vaginal swabs (HVS) of women delivering at term and cultured under anaerobic conditions. High vaginal swab (HVS) samples were collected from pregnant Indian women at the time of enrolment in GARBHIni cohort if they fulfilled the inclusion criteria: (i) age > 18 but < 40 years; (ii) period of gestation < 20 weeks; (iii) natural conception; (iv) no local (vaginal) antibiotics usages in last 3 months; (iv) absence of hypertension, diabetes, hypo- and hyperthyroidism, and any chronic health disorders and (vi) no evidence of sexually transmitted diseases. The participants were positioned in the lithotomy position. The midpoint of the vagina was accessed using a Cusco’s speculum and HVS samples were collected by holding the Sterile Catch-All™ Sample Collection swabs gently against the mid vaginal wall on the right side for 20 s ensuring that there is no contamination by the cervical mucus. HVS samples collected in ~ 750pl of phosphate buffer saline (PBS) was transferred to laboratory at the Translational Health Science and Technology Institute (THSTI) on ice within 2 h. HVS samples were streaked and spread with up to dilutions 10”4onto de Man, Rogosa, and Sharpe (MRS) Agar (Sigma, USA) plates for screening and isolation of Lactobacillus species. The plates were then incubated at 37 °C in anaerobic conditions for 48-72 h. Colonies with distinct morphologies were selected randomly and inoculated in MRS Broth, for isolation and downstream microbiological analysis. Initial identification was done with the 16S rRNA gene sequencing using Sanger Sequencer and further whole genome sequenced (WGS). Subsequent genomic analysis revealed significant insights: the Lactobacillus crispatus strains harbour numerous secretory transcriptional regulators associated with antiinflammatory responses. According to gene annotation, the inventors found gene encoding to pullulanase which were abundant in Lactobacillus crispatus isolates. With the help of this enzyme, Lactobacillus crispatus breaks down glycogen and produces lactic acid, which maintains the low pH and inhibits pathogenic bacteria in the vagina. In addition, there were genes responsible for redox reactions and acid tolerance in the vagina, such as Fe-S cluster assembly protein SufB, and manganese transport protein.

[0055] The antimicrobial resistance gene analysis (ARGs) was performed for the Lactobacillus crispatus strains through CARD and Resfinder databases. The strains were found to have putative genes, which are annotated as ARGs, but they seem to be only false partial hits, which may be responsible for other cellular functions. The strains of Lactobacillus crispatus (Genome ID : 47770.951 and Genome ID: 47770.957) were found to possess three signal peptides having anti-inflammatory properties encoded within their genome. The findings show that these secretory transcriptional regulators localize in the cytoplasm, nucleus or mitochondria in the host cell thereby influencing the growth of microorganisms and decreasing the invasion of pathogenic bacteria.

[0056] Table 1: Number of secretory signal peptide identified in the genome of Lactobacillus crispatus strains used in the consortium

[0057] Moreover, these strains of Lactobacillus crispatus harbours ribosomally and non- ribosomally synthesized antimicrobial peptides in their genome. The highest number of antimicrobial peptides was identified in Lactobacillus crispatus as compared to other Lactobacillus species. All the draft genomes of Lactobacillus species isolated from the HVS were screened for the presence of a secondary' metabolite bacterial gene cluster (BGC) using the latest antiSMASH (version 7.0.0). Upon analysis of the bacterial gene clusters (BGC) in the 133 Lactobacillus genomes. Secondary metabolite gene clusters were identified in 124 genomes.

[0058] Table 2: Number of Lactobacillus crispatus strains possessing antimicrobial peptides and the different types of antimicrobial peptides identified in different species of Lactobacillus.

[0059] Bacteriocin-IIc or other unspecified ribosomally synthesized and post-translationally modified peptide product (RiPP) clusters were identified only in the Lactobacillus crispatus isolates. Besides bacteriocin class II, additional proteins (streptolysin S and YcaO) from class I bacteriocin group (LAP, Linear azol(in)e family) were also seen. We also found several phage lysins, autolysin, endolysin, streptolysin and plantaricin (Pin) genes in abundance in the strains of Lactobacillus crispatus.

[0060] Table 3: Number of antimicrobial peptide (AMP) identified in the genome of Lactobacillus crispatus strains used in the consortium

[0061] The presence of CRISPR-Cas type II A system in the strains of Lactobacillus crispatus genome provides adaptive immune protection against foreign genetic elements. The array harbours Cas9 endonuclease gene along with CRISPR associated Casl, Cas2 and Csnl protein. Notably, the genome of Lactobacillus crispatus contains a substantial number of genes encoding anti-inflammatory and antimicrobial peptides.

[0062] Table 4: Lactobacillus isolates with number of CRISPR array, spacers and length of repeat sequence

[0063] Since Lactobacillus crispatus genome harbours a maximum number of antiinflammatory and antimicrobial peptides our next focus was to check for the probability of pathogenicity in the isolates. In silico safety of the isolates were conducted using the ProbioMinServer. The ProbioMinServer integrates robust bioinformatic pipelines such as Mash v2.3 to confirm the bacterial isolate, and EggNOG-mapper v2 for genome annotation with >90% identity and >80% coverage as parameters. To assess antimicrobial resistance (AMR) the Comprehensive Antibiotic Resistance Database (CARD) Variants v4.0.0 and ResFinder v4.0 was used. Further, pathogenic potential is assessed using VirulenceFinder v2.0.3. Combining the results of each assessment, the platform computes the probiotic potential risk score (PPRS) which categorizes the isolate’s safety. A score of >6 denotes high risk while a score between 4-6 denotes medium risk and <4 denotes low risk. Safety assessment via the ProbioMinServer yielded a PPRS score of ‘0’ for the Lactobacillus crispatus strains used in the consortium. Resistome analysis using CARD and ResFinder identified no strict matches for AMR genes. Similarly, evaluation of virulence factors revealed no pathogenic traits in the strains. Inquiry on pathogenicity of the strain using Pathogen Finder tool revealed that these strains are non-human pathogen. Analysis of the proteome from cell-free culture supernatants of Lactobacillus crispatus identified the expression of lysin and bacteriocin across all the strains. Lysins are highly evolved enzymes known for potent antibacterial activity, suggesting their potential as alternatives to antibiotics for treating bacterial vaginosis caused by pathogens such as Gardnerella vaginalis. Bacteriocin immunity protein encoding plantaricin (pin) gene was also identified in one of the strains of Lactobacillus crispatus. Plantaricin has been proven to bind to the lipopolysaccharide of E. coli interfering with the integrity of the bacterial outer membrane. Other than antimicrobial peptides, Inventors also found Gram-positive signal peptides, e.g.,YSIRK family peptides, in the strains corroborating with our findings from in-silico analysis. The functionality of these identified proteins was validated through in vitro experiments using cell-free culture supernatants against bacterial pathogens including Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus and Gardnerella vaginalis. The Lactobctcilhis crispatus strains demonstrated inhibitory activity against the pathogens as evidenced by well diffusion assay showing zone of inhibition larger than that of the control which was MRS medium pH adjusted with lactic acid. This suggests the involvement of antimicrobial peptides. Furthermore, time-kill assay conducted on Escherichia coli demonstrated that the cell free supernatants of the Lactobacillus crispatus strains achieved more than 4 logw reduction in viable cells within 3 hours. The effect was particularly pronounced against .S', aureus with the Lactobacillus crispatus cell free supernatant inducing a 4 logw reduction in CFU / mL within just 1 hour of co-incubation. The outcomes will be relevant for the development of potential therapeutics for combating different human infectious diseases.

[0064] In an embodiment, the present invention, an indigenous consortium of Lactobacillus crispatus with reproductive tract, respiratory tract and gastrointestinal tract associated bacterial pathogens resistance and anti-steatosis functions for nutraceutical and therapeutic uses. The consortium contains two well characterized Lactobacillus crispatus strains formulated at 1 : 1 ratio within a pharmaceutically suitable carrier.

[0065] The consortium of Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9- 4-C11 has better pathogen resistance functions compared to the individual isolates, as mentioned above. This consortium can inhibit the growth of Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus aureus and Gardnerella vaginalis in their optimal growth condition.- Is their nay amount in which the bacterias will be used in the composition

Claims

CLAIMSWe Claims:

1. A composition comprising: a) a microbial consortium comprising plurality of Lactobacillus crispatus S10-5-C2-2 and Lactobacillus crispatus S9-4-C11 strains isolated from high vaginal swab samples, said strain is characterized to express genes encoding one or more bacteriocins, one or more anti-inflammatory peptides, acid tolerance proteins, and glycogen degrading enzymes; b) a pharmaceutically acceptable carrier; wherein the consortium produces lactic acid from glycogen metabolism to establish a local pH ranging from about 3.8 to about 4.5.

2. The composition as claimed in claim 1, wherein the microbial consortium comprises Lactobacillus crispatus strains that genetically express plurality of CRISPR-Cas type II A systems.

2. A method of preparing the composition as claimed in claim 1, comprising the steps of:(a) collecting high vaginal swab samples from reproductive-age women under sterile conditions;(b) isolating a plurality of Lactobacillus crispatus strains from the collected samples under anaerobic conditions(c) culturing the isolated strains in growth media formulated to simulate an acidic environment;(d) performing genomic sequencing and biochemical assays on the isolated strains to identify genes encoding bacteriocins, anti-inflammatory peptides, acid tolerance proteins, and glycogen degrading enzymes; said step identifies gene clusters encoding for ribosomally and non-ribosomally synthesized bacteriocins;(e) assessing their in vitro antibacterial efficacy of the two strains included in the consortium using both well diffusion assay and time kill assays, said strainsdemonstrated broad spectrum inhibitory activity against clinically relevant bacterial pathogens comprising Escherichia coli, Klebsiella pneumoniae, Enterobacter honnaechei, Staphylococcus aureus and Gardnerella vaginalis, all of which associated with various human infections; (g) assembling the selected strains into a consortium in ratios sufficient to maintain a metabolic activity resulting in lactic acid production that lowers pH to a range of about 3.8 to about 4.5 wherein the consortium comprises two well characterized Lactobacillus crispatus strains combined in a 1 : 1 ratio with 1X109each CFU / mL and formulated within a pharmaceutically appropriate carrier matrix.(h) incorporating the consortium into a formulation comprising a pharmaceutically acceptable carrier, buffering agents, and stabilizers, whereinthe formulation comprise of 10% sucrose v / v which acts as the cryoprotectant and stabilizer.

6. The method as claimed in claim 5, wherein the HVS samples were streaked and spread with up to dilutions 10~4for screening and isolation of Lactobacillus species.

7. The method as claimed in claim 5, wherein the plates were then incubated at 37 °C in anaerobic conditions for 48-72 h.

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