Description:FIELD OF INVENTION
The invention relates to field of biotechnology particularly to a probiotic composition and a method of obtaining the same.
BACKGROUND OF THE INVENTION
Composition of the Cow’s milk has major constituent as water i.e. 85.5 % to 88.7%, Lactose (sugar) range from 3.8 % to 5.3%. The fat content ranges from 2.4 % to 5.5% and the proteins, ranges between 2.3 % and 4.4%. The major protein of milk, casein ranges from 1.7% to 3.5. The milk is therefore a complete medium that provides ideal environment for the microbial proliferation. Cow milk has a huge bacterial population which can be classified into four phylum, Firmicutes (76%), Actinobacteria (4.9%), Proteobacteria (17.8%) and Bacteroides (1.3%). In addition, milk also has thermoduric populations which resists pasteurisation such as sulphate-reducing clostridia, Listeria monocytogenes, Salmonella, coagulase-positive staphylococci, Escherichia coli, Enterobacteriaceae, coliforms and Bacillus cereus among others. Probiotics are live microbes, which on administration to a subject confer health benefits. Bacillus species have been used as probiotics for long. Bacillus are known to form spores. Spore-forming bacteria have distinct advantage over the non-spore forming bacteria as spore-forming bacteria are heat stable and survive low pH of the gastric environment. Of the species that have been most extensively examined these are Bacillus subtilis, Bacillus clausii, Bacillus cereus, Bacillus coagulans and Bacillus licheniformis. The spore forming bacteria belonging to the Firmicutes are Bacilli, Clostridium, Negativicutes, Erysipelotrichia and Thermolithobacteria. Among these, Bacillus (aerobic spore forming bacteria) is the dominant classes. Bacterial Bacillus spores are able to survive post-pasteurization also as they have potential to subsequently germinate when they get favorable conditions.
Bacillus spores are tough, metabolically insert structures which are made by vegetative bacterial cells under extreme conditions as their survival strategy in response to environmental conditions. Bacillus spores produce endospores that have the ability to resist heat, radiation and chemical treatment.
The milk also in addition is a source of Ca2+ ions that has ability to make a bacterial cell competent. Combining Ca2+ ions with cold/heat shock (ice/heat) is known to make a bacterial cell competent. In this method divalent (Ca2+) on ice is followed by treatment with elevated temperature as a heat-shock, which produces a temperature imbalance. Molecules with increased Brownian motion outside the cell are likely to push the DNA molecule inside the cell. It is also inferred that lowering of temperature actually contributes to protein loss, while heating contributes to lipid loss, and thus together these cycles increase transformation efficiency as it enlarges the pore size on the cell surface. Moreover, due to loss of lipids and proteins, the membrane is depolarized, further reducing the repulsion between the DNA molecule and the membrane. Moreover, cell density can also affect the efficiency of transformation and it has been reported that maximum competency is observed at cell density ranging from 107 to 108 cells ml in the log phase. The temperature imbalances and treatment with Ca2+ help deal with the barriers to DNA uptake, such as charge repulsions and pore sizes.
Divalent calcium cations (Ca2+) interact to stabilize the negatively charged phosphates of DNA surface and the negatively charged LPSs in the outer membrane. Calcium chloride solution is added at ice-cold temperature to enhance brining DNA and LPS molecules in proximity, mediated through Ca2+, by reducing the degree of disorder in the system. LPSs in the outer membrane shield membrane proteins that are required for DNA binding, so Ca2+ divalent cation abundant in solution reposition of the LPSs to facilitate DNA insertion into the cell at heat shock. By using these Ca2+ ions of the milk makes the Bacteria competent by creating the pores in the membrane of the Bacteria.
Ghee is analogous to drawn butter fat, which is produced by heating butter to separate out milk solids and water. It is prepared from cow’s milk, buffalo’s milk and the mixed milk’s as well.
Ghee contains fats, cholesterol, vitamins, tocopherols, lanosterol, free fatty acids, carotenes,
ubiquinone’s, etc. Ghee is prepared from butter but the impurities and the milk solids are removed, so people having casein and lactose intolerance do not suffer the symptoms on consumption. Ghee is a rich source of omega-3 fatty acids and omega-6 fatty acids. The omega fatty acids have been reported to alleviate cardiovascular diseases and other chronic diseases.
Ghee also has anti-inflammatory properties. Butyrate is a kind of fatty acid with known anti-inflammatory properties present in ghee. Butyrate decreases TNF production and pro-inflammatory cytokines mRNA expression. Butyrate downregulates proinflammatory cytokines expression via inhibition of NF-kB activation and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) degradation. Β-carotene is present in high amount in cow’s milk when as compared to Buffalo’s milk. Β-carotene is also known as pro vitamin-A, as human body easily converts them into retinol (vitamin-A). The range of β-carotene concentration in cow’s milk is 0.05-0.20 mg/L.

Tulsi (Osmium sanctum) is an aromatic plant and has been employed in the treatment of various diseases such as antimicrobial infection, antifungal, anticancer, arthritis, chronic fever, antifertility, eye disease, hepatoprotective, antispasmodic, and analgesic, antiemetic, cardio protective. Tulsi is known to reduce blood glucose levels, making it an effective treatment of diabetes. Osmium sanctum is considered to have diuretic, stimulant property. It also serves in multidirectional ways by providing therapeutic and medicinal values which is significantly beneficial as it has phytochemicals which provide some antioxidant and anti-inflammatory values. Anti-inflammatory activity of phytochemicals from Osmium sanctum against cyclooxygenase 2 (COX-2) is already known. COX-2 is expressed by inflammatory cells, such as macrophages and can be induced by TNF and EGF. It is the inducible form of cyclo-oxygenase and catalyses the conversion of arachidonic acid to prostaglandins. It initiates the formation of prostaglandins and thromboxane which cause the pain and swelling of associated with inflammation. COX-2 is be inhibited by many phytochemicals present in the Osmium sanctum plant. Various types of the phytochemical of Osmium sanctum (tulsi) acts as a COX-2 inhibitor, like many modern painkillers, due to its high concentration of eugenol. Eugenol (4-allyl 2-methoxyphenol) is a naturally occurring phenolic compound. It possesses antiseptic, analgesic, antibacterial, anti-inflammatory and antiphylactic properties. In lipopolysaccharide (LPS)- induced acute lung injury, pre-treatment with eugenol inhibits the inflammatory response and leukocyte recruitment into the lung tissue by the downregulation of proinflammatory cytokines (IL-6 and TNF-α) expression and NF-kB signaling. Eugenol also inhibits N-methyl-d-aspartate (NMDA) receptors which are involved in pain sensitivity.
Honey and jaggery have been used as sweetening and medicinal agents. Honey is a homogenous mixture of sugar and water. The sugar in honey are simple sugars namely fructose and glucose, accounts for 95%–99% of honey dry matter. Honey has shown to have an antimicrobial action against a broad spectrum of bacteria and fungi and also used as an agent for preventing autolysis and putrefaction. However, It has been observed that if honey is diluted with water, it supports the growth of non-pathogenic bacterial strains and killing of dangerous strains. Jaggery is obtained from sugarcane, and contains high content of reducing sugars. Jaggery is rich in important minerals (Calcium-40-100 mg, Magnesium-70-90 mg, Potassium-1056 mg, Phosphorus-20-90 mg, Sodium-19-30 mg, Iron-10-13 mg, Manganese-0.2-0.5 mg, Zinc-0.2- 0.4 mg, Copper-0.1-0.9 mg, and Chloride-5.3 mg per 100 g of jaggery), vitamins (Vitamin A-3.8 mg, Vitamin B1-0.01 mg, Vitamin B2-0.06 mg, Vitamin B5-0.01 mg, Vitamin B6-0.01 mg, Vitamin C-7.00 mg, Vitamin D2-6.50 mg, Vitamin E-111.30 mg, Vitamin PP-7.00 mg), and protein-280 mg per 100 g of jaggery The micronutrients present in jaggery have antitoxic, antioxidant, and anticarcinogenic effects. Jaggery has been used as carbon source in bacterial medium.
Curd is obtained from milk by sour¬ing, by a harmless lactic acid or other bac-terial culture. Curd contains 80-85% water, 5-8% protein, 3.2 -3.4% proteins, 4.6-5.2% lactose, 0.5-0.11% Latic. Curd is an excellent source calcium, phosphorus, potassium, magnesium and sodium, and is a significant source of vitamin B12 as well as a certain amount of folic acid. The natural microflora of curd is Lactococcus lactis, Streptococcus diacetylactis, Streptococcus cremoris, Lactobacillus. bulgaricus and Streptococcus thermophilus.
In 1915, Bacteriophage was discovered by Felix Twort and Felix d’Hérelle. It is well-known that bacteriophages are very specific; they attack only host bacterial cells without any effect on normal microflora. Bacteriophages of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group member and S. typhi. from different sources of water samples have been identified in ganga water. These bacteriophages are therefore known to kill a number of pathogenic bacteria in the ganga water. It is one of the reason behind the self-cleansing property of the ganga water. Apart from this the ganga water might be containing high amounts of genetic material of the millions of microorganism or natural flora of the ganga water. This might get transforming into the naturally occurring bacteria found in ganga water and cause genetic changes in the bacteria.
Exosomes are small vesicles produced endogenously by cells for intercellular communication. Extracellular vesicles (EVs) are nano‐sized membranous structures which are lipid-bound vesicles secreted by cells into the extracellular space. The content, or the cargo, of EVs may be lipids, nucleic acids, and protein or other proteins associated with the plasma membrane, cytosol, or proteins involved in lipid metabolism. EV cargo, specifically nucleic acids, i.e., mRNA and miRNA to the recipient cells EV has compositional properties such as surface lipid/protein content and nano-mechanical properties including size and colloidal ability, which are important in EV-cell interaction. EVs can also serve as messengers by binding to signalling receptors on recipient cells or via transfer of functional cargo such as microRNAs (miRNAs), mRNAs, proteins and lipids. EVs can also infiltrate biological barriers through transcytotic processes or, for the smaller EVs, through small vessel fenestrations. EVs generally show minimal native immunogenicity when compared with artificial nanoparticles, and the immune response can be avoided using autologous. They can be modified by loading specific cargo or by changing EV surface molecules to target the EV to, or away from, specific cell types. EVs are known to secrete by all kind of cells including mammalian and bacterial cell. However due to the thick peptidoglycan layer, thick cell wall and lack of an outer membrane, the mechanism of secretion of EVs remains unclear in gram positive bacteria. Due to the versatility of the EVs, it has been long recognized as potential drug delivery system.
A few of the prior art discloses use of bacillus in probiotic composition. US20030003107A1 published on 2nd January 2003, discloses compositions derived from an isolated Bacillus species, spores, or an extracellular product of Bacillus coagulans comprising a supernatant or filtrate of a culture of said Bacillus coagulans strain, suitable for topical application to the skin or mucosal membranes of a mammal, which are utilized to inhibit the growth of bacterium, yeast, fungi, virus, and combinations thereof. The present invention also discloses methods of treatment and therapeutic systems for inhibiting the growth of bacterium, yeast, fungi, virus, and combinations thereof, by topical application of therapeutic compositions which are comprised, in part, of isolated Bacillus species, spores, or an extracellular product of Bacillus coagulans comprising a supernatant or filtrate of a culture of said Bacillus coagulans strain. In addition, the present invention also discloses compositions, methods of treatment, and therapeutic systems for inhibiting the growth of bacterium, yeast, fungi, virus, and combinations thereof, comprising an extracellular product of Pseudomonas lindbergii comprising a supernatant or filtrate of a culture of said Pseudomonas lindbergii strain. However the composition is directed towards a topical composition.
A probiotic drink that encompasses milk, curd, ghee, tulsi extract, honey and jaggery along with ganga water containing extra vesicle encapsulating ex-RNA can be used to cure chronic inflammatory conditions which are otherwise difficult to cure.

OBJECT OF THE INVENTION
The object of the present invention is to develop probiotic composition that can aid in curing pro-inflammatory conditions.
Yet another object of the present invention is to extract vesicular compartment (extracellular vesicles) containing extracellular ribonucleic acid (exRNA) from biofilm- associated extra polymeric substances (EPS) and use the same for its therapeutic potential in a pro-biotic drink as a cure.
Yet another object of the present invention is to combine the anti-inflammatory, anti-oxidant properties of the natural ingredients such as curd, ghee, milk, honey, jaggery, tulsi etc. with that of the therapeutic potential of extracellular vesicles encapsulated microRNA (miRNA).

SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that is further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.

The present application discloses a probiotic composition comprising a probiotic composition comprising; milk 75-80%; sweetener 5-10%; clarified butter, comprising glycerides (majorly triglycerides), free fatty acids, phospholipids, sterols, sterol esters, fat soluble vitamins, carbonyls, hydrocarbons, carotenoids 1-2%; curd 6-10%; osmium extract in 0.1-0.5% water 2-10%; and probiotic factor 8-10%, wherein the probiotic factor comprises ex-RNA encapsulated within extracellular vesicle, wherein the water is ganga water.
The present invention also discloses a method of obtaining probiotic factor comprising; mixing milk 75-80%; sweetener 2-10%; clarified butter 1-2%; curd 6-10%; osmium extract 0.1-0.5% and water 2-10% to obtain the pro-biotic composition-1; homogenizing the pro-biotic composition-1 at 1000 rpm for 5 mins at room temperature; and adding probiotic factor in a ratio of 1:10 to the above mixture to obtain the pro-biotic composition.

BRIEF DESCRIPTION OF FIGURES:
The above mentioned objectives and descriptions, features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:

Figure 1 depicts the using microRNA quantity as estimated in qubit fluorophore analysis in probiotic drink;
Figure 2 depicts effect of probiotic drink on IL-17. IL-1B, IL-6, NFKB and TNFα as compared to PBS in the mammalian A549 cell line; and
Figure 3 depicts effect of probiotic drink on IL-17. IL-1B, IL-6, NFKB and TNFα as compared to PBS in the mammalian A549 cell line in presence of RNase.
Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of the aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures 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. 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.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.

Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

Extracellular vesicles (EVs) are derived from extra polymeric substances (EPS) of the non-pathogenic, gram-positive bacterial species. EVs are the lipid bound vesicles that consists of lipids (phospholipids), nucleic acids, lipopeptides, lipoteichoic acid, aspartate, alanine, aspartic acid, tryptophan, tyrosine, histidine, glutathione.
Extracellular RNA (exRNA) are RNA molecules which are encapsulated in extracellular vesicles derived from gram positive bacteria more specifically non-pathogenic bacteria.
exRNA may be selected from group of micro-RNA, siRNA, circular RNA, piwi RNA, gRNA.
Extracellular vesicles (EVs) are the lipid bound vesicles of the extracellular matrix (ECM) secreted out of the cell. The content or cargo of EV consists of lipids (mainly phospholipids), nucleic acids, lipopeptides, lipoteichoic acid, aspartate, alanine, arginine, aspartic acid, polysaccharides, propanoic acid, glutathione proteins, specifically proteins associated with the plasma membrane, cytosol and those involved in the lipid metabolism.
Total RNA is defined as total cellular RNA present in a cell including ribosomal RNA (rRNA), precursor messenger RNA (pre-mRNA), mRNA, and various classes of noncoding RNA (ncRNA).
MicroRNA- MicroRNAs (miRNAs) are a class of small noncoding RNAs having approximately 22 nucleotide sequences in length which are involved in the regulation of gene expression at the posttranscriptional level by degrading their target mRNAs and/or inhibiting their translation.
Ganga water has a natural microflora bacteria Staphylococcus spp., E. coli, Vibrio spp., Bacillus subtilis, Campylobacter spp., Helicobacter spp., Brucella spp., Micrococcus spp., Corynebacterium spp., Pseudomonas spp., Hemophilus spp., and Clostridium spp. A number of multi drug resistant bacteria are also found in the ganga water, however still ganga water is found to be self-cleansing probably due to the presence of bacteriophages in ganga water. A recent report by National Environmental Engineering Research Institute (NEERI 2017) also demonstrated that Ganga contained around 1100 types of bacteriophage. In addition, bacteriophages against Escherichia coli (B), Escherichia coli (K12), Vibrio cholerae, Enterococcus faecalis, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa.
The inventor of the present invention has thus utilized antimicrobial properties of ganga water along with other ingredients such as cow’s milk, curd and ghee obtained from cow’s milk, jaggery, honey. These ingredients are natural probiotic or have high antioxidant and anti-inflammatory properties. The ingredients are then combined with probiotic factor which is obtained from the extra polymeric substances of the cellular matrix of a non-pathogenic bacteria. The probiotic factor comprises of microRNA encapsulated in a extracellular vesicles. This micro RNA is therapeutic in nature and inhibits the inflammatory cytokines such as APOE (Apolipoprotein E), CTSB(Cathepsin-B), IL-1β (Interleukin -1 beta), IL-6(Interleukin-6), Presenilin 1 (PSEN-1), TNF-α (Tumour necrosis factor -α),), IL-4 (Interleukin-4), IL-5 (Interleukin-5), IL-6 (Interleukim-6), IL-9 (Interleukim-9), IL-12(Interleukin-12), IL-13(Interleukin-13), IL-15(Interleukin-15), IL-17(Interleukin-17), IL-18 (Interleukin-18), IL-23 (Interleukim-23), IL-33(Interleukim-33) (Interleukim-6), TGF-β (Transforming growth factor-β), TNF-α (Tumour necrosis factor -α), IL-17, IL-23, IL-6, NfκB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), TLR-9 (Toll like receptor-9), TNF-α, TGF-β, IFN-ϒ(Interferon-Ƴ), IRS-1(Insulin receptor substrate 1), CXCL-10(C-X-C motif chemokine ligand 10), ESR-1 (Estrogen Receptor 1), KISS-1, KLK-7(Kallikrein-1), CCL-11(C-C motif chemokine ligand-11), CCL-2(C-C motif chemokine ligand-2) by antisense mechanism or ribosomal cleavage.
The present application discloses a probiotic composition comprising a probiotic composition comprising; milk 75-80%; sweetener 5-10%; clarified butter, comprising glycerides (majorly triglycerides), free fatty acids, phospholipids, sterols, sterol esters, fat soluble vitamins, carbonyls, hydrocarbons, carotenoids 1-2%; curd 6-10%; osmium extract in 0.1-0.5% water 2-10%; and probiotic factor 8-10%, wherein the probiotic factor comprises ex-RNA encapsulated within extracellular vesicle, wherein the water is ganga water. The osmium extract here is used as an anti-inflammatory agent. Ghee is used here for its anti-inflammatory property, however in minimal amount.
In one embodiment of the invention sweetener is mixture of jaggery and honey and is 2-10% and 2-5% respectively. Jaggery and honey again here are used as antibacterial, antitoxic, antioxidant, and anticarcinogenic agents. In a preferred embodiment of the present invention the milk is cow’s milk. In another embodiment of the invention the ganga water is filtered through filter having pore size of 0.22µm.
In a further embodiment the clarified butter may be preferably obtained from cow’s milk. In still another embodiment the extract of Osmium sanctum (Tulsi) is obtained by a method comprising collection of fresh leaves of Osmium sanctum (Tulsi) and through washing under running water and drying at room temperature; grinding of dried leaves into fine powder; carrying out the extraction by suspending 1g of powdered leaves of osmium sanctum (tulsi) in 20 ml of distilled water for 22 hours; and filtration of the extract.
In in particular embodiment of the invention the probiotic factor is derived from extra polymeric substances (EPS) of a gram positive non-pathogenic bacteria.
In one embodiment of the present invention discloses a method of producing pro-biotic composition comprising; mixing milk 75-80%; sweetener 2-10%; clarified butter 1-2%; curd 6-10%; osmium extract 0.1-0.5% and water 2-10% to obtain the pro-biotic composition-1; homogenizing the pro-biotic composition-1 at 1000 rpm for 5 mins at room temperature; and adding probiotic factor in a ratio of 1:10 to the above mixture to obtain the pro-biotic composition.
In a further embodiment of the invention the gram positive non-pathogenic bacteria is a bio-film-producing-bacteria produces extracellular vesicle encapsulating ex-RNA.
In one embodiment extracellular vesicle encapsulating ex-RNA in the composition has anti-inflammatory properties which is effective in downregulating the pro-inflammatory cytokines.
In another embodiment of the invention the probiotic composition downregulates the pro-inflammatory gene selected from a group comprising APOE (Apolipoprotein E), CTSB(Cathepsin-B), IL-1β (Interleukin -1 beta), IL-6(Interleukin-6), Presenilin 1 (PSEN-1), TNF-α (Tumour necrosis factor -α),), IL-4 (Interleukin-4), IL-5 (Interleukin-5), IL-6 (Interleukim-6), IL-9 (Interleukim-9), IL-12(Interleukin-12), IL-13(Interleukin-13), IL-15(Interleukin-15), IL-17(Interleukin-17), IL-18 (Interleukin-18), IL-23 (Interleukim-23), IL-33(Interleukim-33) (Interleukim-6), TGF-β (Transforming growth factor-β), TNF-α (Tumour necrosis factor -α), IL-17, IL-23, IL-6, NfκB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), TLR-9 (Toll like receptor-9), TNF-α, TGF-β, IFN-ϒ(Interferon-Ƴ), IRS-1(Insulin receptor substrate 1), CXCL-10(C-X-C motif chemokine ligand 10), ESR-1 (Estrogen Receptor 1), KISS-1, KLK-7(Kallikrein-1), CCL-11(C-C motif chemokine ligand-11), CCL-2(C-C motif chemokine ligand-2).
In another embodiment the present application discloses a method of obtaining the pro-biotic composition comprising; mixing milk in ratio of 1:8 and double distilled water to obtain -pro-composition-1; incubation of pro-composition-1 at 370C overnight followed by centrifugation at 5000 rpm for 3 minutes and discarding supernatant to obtain a pellet; mixing 1:8 curd solution to the pellet and mixing well incubating followed by centrifugation at 5000 rpm for 3 minutes and discarding supernatant to obtain a pellet; mixing 10 ml of ganga water to the pellet, mixing well to obtain a homogenous solution and placing the tube on ice for 30 mins, followed by incubation of at-40-450C for 60-90 secs, followed by incubation on ice for 2 mins; incubation of the homogenous solution at 370C for 30-60 mins; and adding clarified butter in a ratio of 1:48; curd in a ratio of 1:8; osmium extract in a ratio of 1:48; milk in a ratio of 1:8; sweetener comprising a mixture of jaggery and honey and is in a ratio of 1:8 and 1:2; curd in a ratio of 1:8 to obtain the pro-biotic composition.

EXAMPLE
The following examples are for illustration purposes and are not to be construed as limiting the invention disclosed in this document to only the embodiments disclosed in these examples.
Example 1
Gram-positive non-pathogenic bacteria was mutated in lab through homologous recombination method using a suitable bacteriophage strain, mutated bacteria was grown on a suitable culture media for 22-24 hrs. at 37oC. After formation of the full grown biofilm on the medium. The biofilm was scrapped from the surface and suspended in the conversion buffer to extract extra polymeric substances (EPS) from the biofilm. The conversion buffer used for suspension of extra polymeric substances (EPS) were carried out in 6 different solution- double distilled water; 10% ethanol (10 ml ethanol in double distilled water); 5% Dimethyl sulfoxide (DMSO) (5ml DMSO in double distilled water); 0.9% Sodium chloride (NaCl) 0.9gms NaCl dissolved in 90ml double distilled water, making up the volume to 100ml; Phosphate buffer saline (PBS), 0.819gms PBS dissolved in 90ml double distilled water, making up the volume to 100ml, adjusting the pH to 7.4.
The homogenization of the mixture was carried out at room temperature (250C) for 1 minute in a homogenizer (NAMCO, Asia 1936). The suspension obtained after the homogenization is then centrifuged, (REMI- R8C) at 4500rpm for 5 mins. The supernatant obtained in then filtered twice with membrane filter pore size, 0.45 µm followed by membrane filter pore size, 0.22µm.
microRNA isolation from Extracellular matrix:
For isolation of microRNA, 200µl of 1:20 ratio of filtered sample (obtained above) was taken and 200µl of isolation buffer was added, the solution was kept for 15 minutes at 56ºC and followed by addition of 300µl of ethanol. The sample was loaded on to binding column and centrifuged at 6000rpm for 1min. The column was washed with 700µl of wash buffer followed by centrifugation for 1 min, this was followed by a second wash with 500µl of wash buffer followed by centrifugation for 1 minute. The unwanted material in the column gets washed and miRNA was then eluted and dried. Centrifugation done again for 3 mins at 6000rpm followed by adding 50µl of RNase free water for elution and solution was kept at 56ºC for 2 mins. Centrifugation was repeated at 12000rpm for 1 minute to obtain miRNA.
Estimation of microRNA in the drug sample
Qubit™ RNA Assay Kits (Invitrogen) for RNA estimation in the drug sample obtained above. 190µl of Qubit microRNA buffer and 1µl of microRNA reagent was added and mixed properly. 20µl of drug sample (1:20 ratio) was added. The sample kept for incubation at room temperature for 2 minutes. Finally, the reading of sample taken by using Qubit fluorophotometer. The OD was taken duplicates and the experiment was repeated thrice. The microRNA was estimated to be 4-12µg/ml.
Size distribution of the extracellular- vesicular
The exosome is an anionic molecule with a zeta potential (-1) to (-30) mV and 20-500nm in size as elucidated by the size distribution by intensity reports.
Estimation of total RNA in the drug sample by 260/280 ratio
Drug sample was diluted in double distilled water in 1:20 ratio and homogenized. The homogenized sample was filtered by 0.22µm Millipore membrane filter using a filter syringe. Samples were read at 240-300nm using a BioTek Epoch 2 microplate spectrophotometer. The estimation of the 260/280 ratio was carried out. Nucleic acids have absorbance is maxima at 260 nm. The ratio of this absorbance maximum to the absorbance at 280 nm has been used as a measure of purity in both DNA and RNA extractions. A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. Samples showed 260/280 ratio in duplicate samples (sample 1 and 2) to be 1.926 and 1.899 respectively. The Total RNA was estimated to be 90.942 µg/ml and 92.856 µg/ml
Extracellular- vesicular layer
Exosomal layer was lysed with the help of a lysis buffer to dissolve the exosomal layer in the M9 and M9-1 (composition of the present invention (Sample A), and in a separate control no lysis buffer was added (Sample B). The sample A & B were passed by binding column. and reading (260/280 ratio) was taken in microtiter spectrophotometer. Nucleic acids have absorbance is maxima at 260 nm. The ratio of this absorbance maximum to the absorbance at 280 nm has been used as a measure of purity in both DNA and RNA extractions. A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. In Sample A, the (260/280) ratio of the reading obtained was 2.122 and the concentration of RNA was detected to be of 12.669ng/µl. Whereas, in Sample B the (260/280), the ratio of the reading obtained was 1.417 (same as distilled water) and its concentration was 2.695ng/µl.
Example 2
Production of the probiotic composition
Mixing cow’s milk (750ml) 75-80%, jaggery 100gms (2-10%), honey 50ml (2-5%); cow ghee 20ml (1-2%), curd 100 ml (6-10%), osmium extract 5 ml (0.1-0.5%) to obtain the pro-biotic composition-1. The mixture obtained was homogenized using a homogenizer at 1000 rpm for 5 mins at room temperature to obtain the pro-biotic composition-1. To the pro-biotic composition-1, 100 mL of probiotic component was added to the obtain the final pro-biotic composition and volume was made up to 1000ml with filtered and processed ganga water.
Example 3
Extraction of RNA from Probiotic Drink
50ml of sample was taken in a autoclaved falcon tube and centrifuged for 10 min at 2000g in a cooling centrifuge. Pellet was discarded and supernatant collected in another tube. Supernatant was centrifuged for 20 min at 14000g in a cooling centrifuge. Pellet was discarded and supernatant collected in another tube. Supernatant was centrifuged for 30 min at 20000g in a cooling centrifuge. Pellet was discarded and supernatant collected in another tube. The supernatant lyophilized in 10ml aliquots in a lyophilizer machine/ freeze dryer for 12 hours. After 12 hours the concentrated sample was further used for RNA isolation.
The lyophilized sample was reconstituted in 5ml in nuclease free water (NFW) and out of which 200µl taken in an empty 1.5ml lysis tube. 220µl of lysis solution supplemented with 5µl carrier RNA was added to the sample and mix thoroughly by vortexing. The sample was incubated for 15 min at 56°C in a water bath. The sample was then centrifuged for 3-5 sec at full speed to collect drops from the inside of the lid. 300 µl of ethanol was added to the sample and mixed by vortexing. The sample was incubated at room temperature for 3 min and centrifuged the sample for 3-5sec at full speed to collect drops from the inside of the lid. The lysate was transferred to a prepared spin column preassembled within the wash tube. The column was centrifuged for 1min at 6000g. The wash tube was discarded containing flow-through and the spin column was placed into a new 2ml wash tube. 700µl of wash buffer VWB supplemented with ethanol was added to the spin column. The column was then centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column was placed into 2ml wash tube. 500 µl of wash buffer VWB3 supplemented with ethanol was added to the spin column. The column was centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column placed into 2ml wash tube. 500 µl of was buffer VWB3 supplemented with ethanol was added to the spin column. The column was centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column was placed into 2ml wash tube and centrifuged for 3 min at 12000g. The wash tube containing flow through was discarded. The spin column was placed into a new 1.5 ml elution tube. 50 µl of RNase free water was added and preheated to 56°C to the centre of spin column membrane. The spin column membrane was incubated for 2 min at room temperature and centrifuged for 1 min at 12000g. The spin column was discarded and the purified nucleic acids was stored at -20°C for epoch2 and fluorophore reading. The isolated RNA in a tube were further examined using fluorometry analysis. It was done to measure the purity and quantity of RNAs isolated from probiotic sample. The instrument for the above mentioned process was of Biotek,Model- Epoch Microplate Spectrophotometer. The obtained concentration of RNAs is presented below
The lyophilized sample was reconstituted in 5ml in nuclease free water (NFW) and out of which 200µl taken in an empty 1.5ml lysis tube. 220µl of lysis solution supplemented with 5µl carrier RNA was added to the sample and mix thoroughly by vortexing. The sample was incubated for 15 min at 56°C in a water bath. The sample was then centrifuged for 3-5 sec at full speed to collect drops from the inside of the lid. 300 µl of ethanol was added to the sample and mixed by vortexing. The sample was incubated at room temperature for 3 min and centrifuged the sample for 3-5sec at full speed to collect drops from the inside of the lid. The lysate was transferred to a prepared spin column preassembled within the wash tube. The column was centrifuged for 1min at 6000g. The wash tube was discarded containing flow-through and the spin column was placed into a new 2ml wash tube. 700µl of wash buffer VWB supplemented with ethanol was added to the spin column. The column was then centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column was placed into 2ml wash tube. 500 µl of wash buffer VWB3 supplemented with ethanol was added to the spin column. The column was centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column placed into 2ml wash tube. 500 µl of was buffer VWB3 supplemented with ethanol was added to the spin column. The column was centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column was placed into 2ml wash tube and centrifuged for 3 min at 12000g. The wash tube containing flow through was discarded. The spin column was placed into a new 1.5 ml elution tube. 50 µl of RNase free water was added and preheated to 56°C to the centre of spin column membrane. The spin column membrane was incubated for 2 min at room temperature and centrifuged for 1 min at 12000g. The spin column was discarded and the purified nucleic acids was stored at -20°C for epoch2 and fluorophore reading. The isolated RNA in a tube were further examined using fluorometry analysis. It was done to measure the purity and quantity of RNAs isolated from paramtatva and gangajal sample. The instrument for the above mentioned process was of Biotek,Model- Epoch Microplate Spectrophotometer. microRNAs (miRNAs) are a22
nucleotide) non-coding RNAs that are essential for
controlling the regulation of gene expression. They act
via binding to specific sequences in the 3′-untranslated
region (3′-UTR) of mRNAs, leading to either
translational repression or message decay. miRNAs have
been linked to distinct biological processes in normal de-
velopment and their altered expression is implicated in
many diseases including neurological disorders, immune
diseases and cancers
Table 1
S. No. Probiotic Drink – 260/280 ratio Probiotic Drink – Total RNA quantity in ng/mL
1. 1.796 41.288 µg/mL

microRNA Quantification using qubit fluorophore
The isolated RNA were further examined using qubit fluorophore analysis. It was done to measure the quantity of microRNAs from probiotic drink. The microRNA was estimated to be 2280 ng/mL (Figure 1).
Example 4
Effect of probiotic drink on IL-17. IL-1B, IL-6, NFKB and TNFα in the mammalian A549 cell line
Culture of A549 cell line
Equal amount of cell was seeded in a six well plate and incubated for 24 hours at 30°C in a 5% CO2 incubator for allow to reach 70-80% confluency. After 24 hours 100 µl probiotic drink composition, 100 µl probiotic drink composition plus RNAse was added into wells. 100 µl PBS, 100 µl PBS plus RNAse were kept as control. After 24 hours RNA isolation process was done. The six-well plate was scrapped and homogenized with the help of a pipette The homogenized mixture was pipetted into a lysis tube and centrifuged for 10minutes at 2000g in a cooling centrifuge. The supernatant was discarded and take pellets. 500µl of lysis buffer (Biorad Aurum Total RNA Mini Kit) was added in each sample, mixed well and vortexed. The lysate was transferred to the prepared spin column preassembled within the wash tube and 500 µl of DEPC plus ethanol solution was added in each sample, mixed well and vortexed and centrifuged for 1 minute at 13000g. The supernatant was discarded. 700 µl total RNA low stringency wash solution (Biorad Aurum Total RNA Mini Kit) was added to each sample and centrifuged for 1 minute at 13000g. The supernatant was discarded. 80µl solution containing 75µl DNAse buffer + 5µl DNAse (Biorad Aurum Total RNA Mini Kit) was added to each sample and incubated for 20 minutes in room temperature. 700µl total RNA high stringency wash solution (Biorad Aurum Total RNA Mini Kit) was added each sample and centrifuged for 1 minute at 13000g in a cooling centrifuge. 700 µl total RNA low stringency wash solution (Biorad Aurum Total RNA Mini Kit) was added to each sample and centrifuged for 1 minute at 13000g in a cooling centrifuge, the supernatant was discarded and tubes were air dried centrifuged for 2minutes at 13000g. The spin column was placed into a new 1.5 ml elution tube and 40µl of elution buffer was added in each tube and incubated for 2 min at room temperature. The column was centrifuged for 1min at 12000g. The spin column was discarded and the purified nucleic acid was stored at -20°C for epoch2 and fluorophore reading.
Table 2
S. No. Sample Characterization
1. Probiotic Drink Total RNA quantity in Probiotic Drink – 27.065 µg/mL
2. Probiotic Drink 260/280 ratio -1.878

Example 5
The isolated RNAs were used to prepare cDNA. The cDNA stands for complementary DNA, which the complementary RNA sequences. it is prepared using rtPCR in which RNA serves as the template in cDNA synthesis. Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling. All the eluted RNA sample were normalised (volume equalization). Pipetting was done for proper mixing and subsequently PCR tube was run on the PCR machine for 37 minutes. cDNA was prepared and further used for RT qPCR.
RT-PCR Analysis
The expression of different pro-inflammatory cytokines associated with different autoimmune disorder i.e. IL-17, IL-1β, IL-6, NF-kb, TNF-α. c-DNA 2 µl, primers 5 µl (2.5 µl forward and 2.5 µl reverse) and SYBR green 20 µl was added in a PCR tube and volume makeup was done with NFW. Pipetting was done for proper mixing and then each sample duplet was loaded in 96 well plates. Plate sealer was applied and subsequently, the plate was put on the rotor for proper mixing. The plate was transferred to the PCR machine and according to desired primers temperatures and PCR was run for 1 hour 52 minutes. Cq value of desired genes was analysed at 80-93 °C, multi curved were checked. The gene expression was analysed for genes. The result are presented in Figure 2 and 3. Figure 2 shows downregulation of the inflammatory cytokines IL-17, IL-1β, IL-6, NF-kb, TNF-α. Figure 3 shows that then activity of the probiotic composition is lost when the probiotic composition is treated with RNase (20µg/ml) and incubated at room temperature for 30 mins. The anti-inflammatory activity of the probiotic composition is significantly lost showing that the active ingredient is microRNA present in the probiotic composition.
, Claims:We Claim,
1. A probiotic composition comprising;
a. milk 75-80%;
b. sweetener 5-10%;
c. clarified butter, comprising glycerides (majorly triglycerides), free fatty acids, phospholipids, sterols, sterol esters, fat soluble vitamins, carbonyls, hydrocarbons, carotenoids 1-2%;
d. curd 6-10%;
e. osmium extract in a ratio of 0.1-0.5%
f. water 2-10%; and
g. probiotic factor 8-10%
wherein the probiotic factor comprises ex-RNA encapsulated within extracellular vesicle.
wherein the water is ganga water.
2. The composition as claimed in claim 1, wherein the composition downregulates the pro-inflammatory gene selected from a group comprising APOE (Apolipoprotein E), CTSB(Cathepsin-B), IL-1β (Interleukin -1 beta), IL-6(Interleukin-6), Presenilin 1 (PSEN-1), TNF-α (Tumour necrosis factor -α),), IL-4 (Interleukin-4), IL-5 (Interleukin-5), IL-6 (Interleukim-6), IL-9 (Interleukim-9), IL-12(Interleukin-12), IL-13(Interleukin-13), IL-15(Interleukin-15), IL-17(Interleukin-17), IL-18 (Interleukin-18), IL-23 (Interleukim-23), IL-33(Interleukim-33) (Interleukim-6), TGF-β (Transforming growth factor-β), TNF-α (Tumour necrosis factor -α), IL-17, IL-23, IL-6, NfκB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), TLR-9 (Toll like receptor-9), TNF-α, TGF-β, IFN-ϒ(Interferon-Ƴ), IRS-1(Insulin receptor substrate 1), CXCL-10(C-X-C motif chemokine ligand 10), ESR-1 (Estrogen Receptor 1), KISS-1, KLK-7(Kallikrein-1), CCL-11(C-C motif chemokine ligand-11), CCL-2(C-C motif chemokine ligand-2)

3. A method of producing pro-biotic composition comprising;
a. mixing milk 75-80%; sweetener 5-10%; clarified butter 1-2%; curd 6-10%;
osmium extract in a ratio of 0.1-0.5% and water 2-10% to obtain the pro-biotic composition-1;
b. homogenizing the pro-biotic composition-1 at 1000 rpm for 5 mins at room temperature; and
c. adding probiotic factor 8-10% to the pro-biotic composition-1 to obtain the pro-biotic composition.

4. The method as claimed in claim 9, wherein the probiotic factor comprises ex-RNA encapsulated within extracellular vesicle and is derived from extra polymeric substances (EPS) of a gram positive non-pathogenic bacteria.