DESC:Technical field:
This invention relates to compositions comprising a heat stable component of lactic acid bacteria which helps in enhancement of immunity and reduction of inflammation which is associated with pathological diseases such as diabetes, cancer, rheumatoid arthritis, celiac disease (CD), inflammatory bowel disease (IBD), atherosclerosis etc.

More particularly, the invention relates to compositions and methods of administration of heat killed purified culture supernatant of Bacillus coagulans Unique IS2 for immunomodulation and reducing inflammation through inhibition of COX-2 and inflammatory cytokines via inhibition of NF-kB activity.

Background and prior art:
Inflammation has long been associated with development of cancer by researchers. Inflammatory responses co-ordinate host defence mechanisms against microbial invasions and mediate tissue repair and regeneration, which may occur due to infectious or non-infectious tissue damage. Epidemiological evidence points to a link between inflammation and a predisposition for the development of cancer. Surveys and studies have estimated that nearly 15 to 20% percent of the worldwide cancer incidences are associated with microbial infection. Inflammation has been generally considered as a defensive mechanism the body initiates towards a tissue injury, wound or infection. However, chronic inflammation leads to many pathological diseases other than cancer such as diabetes, rheumatoid arthritis, celiac disease (CD), inflammatory bowel disease (IBD), atherosclerosis etc.

Biochemical mediator molecules released during inflammation builds up and promotes the inflammatory response. These mediator molecules when activated can act locally or systemically. The important cell-derived mediators of inflammation include prostaglandins produced by the action of cyclooxygenase (COX) enzyme and the cytokines produced by macrophages and lymphocytes (Liles and Van Voorhis, 1995; Williams et al., 1999). Treatment measures involve the use of non-steroidal anti-inflammatory drugs (NSAID’s) which inhibit two isoforms of the COX enzyme – COX-1 and COX-2. To overcome gastric side effects of NSAID’s caused due to inhibition of COX-1 enzyme, selective COX-2 inhibitors, coxibs, have been developed (Laine, 2003). Due to the excessive use of these drugs, the gut microflora is adversely affected leading to upper gastrointestinal complications thereby exacerbating intestinal inflammatory disorders such as inflammatory bowel disease (IBD), celiac disease(CD) etc. and therefore, nutritional supplements are normally recommended to counter such conditions. Other untoward effects include increased mucosal permeability, mucosal inflammation, anaemia, malabsorption, protein loss, ileal dysfunction, diarrhoea, mucosal ulceration, as well as active bleeding and perforation. Hence, there arises an urgent need to solve this problem of chronic inflammation with an agent having no adverse effects on the gastrointestinal system and the microflora inhabiting therein.

The beneficial effect of probiotics has revealed that live bacteria colonize the gut and replenish gut microflora (Tuohy et al., 2003). Probiotics such as Bifidobacterium and Lactobacillus sps are shown to be beneficial in many pathological conditions including IBD (Hedin et al., 2007), CD (Rahimi et al., 2008), cancer (De Moreno de LeBlanc et al., 2007), bacterial and viral infections (Rolfe, 2000), etc. Studies have reported that these bacteria have anti-inflammatory and immunomodulatory effects (Isolauri et al., 2001; Kekkonen, 2009; Ng et al., 2009). The cell free culture supernatant of Bifidobacterium was shown to possess strong anti-inflammatory activity (Imaoka et al., 2008).

US Patent No. 8568744 discloses a purified cell wall fraction of Bacillus coagulans bacterium to be administered in an immune enhancing amount which aids in the induction of certain interleukins, the Tumour Necrosis Factor-a (TNF-a) and interferon ?. However, US’744 fails to provide any direct results relating to the use of the cell wall fraction or fragment or the culture supernatant for inhibition of NF-?B also termed as p65, or other protein complexes having a vital role in controlling the transcription, cytokine production and overall cell survival as well as fails to teach reduction in levels of cyclooxygenase using gut microflora or components thereof.

There is ample literature available on the benefits of a few species of Bifidobacterium, Bacillus and Lactobacillus. However, there has been no anti-inflammatory composition comprising a bacterial component having inhibitory activity against the p65 factor, which is a cancer biomarker. Furthermore, it is necessary to explore alternative probiotics that are more beneficial, but with negligible side-effects.
Furthermore, for most probiotic strains to be viable, refrigeration is necessary during transport and storage of therapeutic compositions comprising whole cell micro-organisms, thus increasing the cost and handling requirements of the probiotics. In order to reduce the stability issues with regard to the storage of probiotics and to find an effective, shelf stable therapeutic composition, an alternate probiotic solution for diseases associated with inflammation and cancer needs to be devised.

In the light of the above, it is an objective of the present invention to provide a heat stable composition comprising Bacillus coagulans Unique IS2, for use in enhancing the immunity and in the reduction of inflammatory markers associated with pathological diseases such as diabetes, cancer, rheumatoid arthritis, celiac disease (CD), inflammatory bowel disease (IBD), and atherosclerosis.

Summary of the invention:
In line with the above objective, the present invention provides a composition comprising heat killed sterilized culture supernatant (HSUP) of Bacillus coagulans Unique IS2 which aids in the treatment of cancer, boosting immunity, reducing inflammation associated with pathological diseases.

In an aspect, the present invention provides a composition comprising a heat killed, purified extracellular component from Bacillus coagulans Unique IS-2 along with one or more pharmaceutically acceptable excipients, wherein the said composition comprises anti-inflammatory compounds having size ranging from 0.1 to 0.5 kD.

Accordingly, the present composition comprises anti-inflammatory compounds selected from the group consisting of fatty acids, styrene metabolites, butanoate, pyruvate and phenylalanine.

Further, the present composition may contain other active ingredients selected from the group consisting of vitamins, minerals, antioxidants, prebiotics, antibacterial, antiviral and antifungal agents.

In a further aspect, the invention provides a method for enhancing immunity and for reducing inflammation in a subject which method comprises administering an appropriate amount of heat-killed sterilized Bacillus coagulans Unique IS2 culture supernatant (HSUP) in association with one or more excipients to the subject in need thereof.

In another aspect, the present invention provides a method for inhibiting the p65 factor in a subject, which method comprises administration of effective concentrations of heat killed sterilized supernatant fraction of Bacillus coagulans Unique IS2 in association with one or more excipients to the subject in need thereof.

In yet another aspect, the present invention provides a method of treatment of pathological diseases like diabetes, cancer, rheumatoid arthritis, celiac disease (CD), inflammatory bowel disease (IBD), atherosclerosis associated with inflammation, which method comprises administering at sufficient doses, the anti-inflammatory composition comprising a heat killed purified fraction of Bacillus coagulans Unique IS2.

Further, the heat killed supernatant of Bacillus coagulans Unique IS2 may be formulated alone or with other active ingredients and/or excipients in the form of lyophilized or spray dried powder, tablets, capsules, powders, gels, suspensions, and ointments.

Description of the drawings:
Figure 1 depicts the effect of BC IS2 HSUP on cell viability of RAW 264.7 cells after 24 h and 48 h. LPS (1 µg/ml) is taken as control. Values are taken as mean + SE of duplicates from 2 independent experiments;
Figure 2 shows the effect of BC IS2, HSUP on COX-2 and NF-kB. A. Top - RT-PCR analysis of COX-2 gene. Bottom- Densitometric graph showing fold-difference of bands in gel normalized to b-actin. * p<0.001 B. Top – Western blot of COX-2 protein. Bottom – Densitometric graph of the bands in immunoblot. * p<0.001. C. p65 NF-kB levels determined by ELISA in cellular cytoplasmic and nuclear extracts treated with or without LPS and HSUP. * p<0.05;
Figure 3(A) shows pro-inflammatory and Figure 3(B) shows anti-inflammatory cytokine levels in the culture supernatants of the RAW 264.7 cells treated with or without LPS and/or HSUP.* p<0.001;
Figure 4(A) shows reduction in the Th2 Cytokine level and Figure 4B (Th1) in the culture supernatants of PBMCs treated with or without HSUP indicating the immunomodulatory effect. * p<0.001;
Figure 5 depicts the GC/MS chromatogram of metabolites obtained from the heat killed Bacillus coagulans Unique IS 2 supernatant (subjected to heat treatment and rota evaporation);
Figure 6 depicts the GC/MS chromatogram of metabolites obtained from the lyophilized supernatant of Bacillus coagulans Unique IS 2 wherein there was no heat treatment or rota evaporation (no heat treatment and no rota evaporation);
Figure 7 depicts the GC/MS chromatogram of the control, i.e. the filter sterilized supernatant from Bacillus coagulans Unique IS 2 not subjected to heat treatment but evaporated by rota evaporation;
Figure 8 depicts the anti-inflammatory activity of the composition comprising the heat killed culture supernatant of Bacillus Coagulans Unique IS2 in an in-vivo rat pouch model, Figure 8 (A) shows carrageenan induced inflammation of the rat air-pouch, Figure 8(B) shows the air pouch region of an affected rat post treatment with celecoxib, Figure 8(C) shows the air pouch region of the affected rat post treatment with control supernatant of Bacillus coagulans Unique IS 2 i.e. supernatant not subjected to heat, and Figure 8(D) shows the air pouch region of the rat post treatment with composition of the present invention; and
Figure 9 depicts the GC/MS chromatogram collectively of the heat killed culture supernatant of Bacillus coagulans Unique IS2 (depicted in blue), control sample depicted in green and lyophilized sample depicted in yellow.

Detailed description of the invention
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

In the present invention, the phrase heat killed, purified extracellular component of Bacillus coagulans Unique IS2 is meant to refer to the heat activated extracellular component(s) produced by B. coagulans Unique IS2, or Heat killed Sterilized Bacillus coagulans IS2 culture supernatant (HSUP). The aforesaid terms may be used interchangeably.

In a preferred embodiment, the present invention provides a composition comprising a heat killed, purified extracellular component from Bacillus coagulans Unique IS2, along with one or more pharmaceutically acceptable excipients, wherein said composition comprises anti-inflammatory compounds having size ranging from 0.1 to 0.5 kD.

Accordingly, the invention provides a composition comprising heat activated metabolites obtained from the supernatant of Bacillus coagulans Unique IS2, for use in enhancing cell mediated immunity and reducing levels of inflammation in pathological diseases selected from diabetes, cancer, rheumatoid arthritis, celiac disease (CD), inflammatory bowel disease (IBD), and atherosclerosis. The Bacillus coagulans Unique IS2 used in preparation of the heat killed supernatant ranges from 1x10 6 to 1x10 10 cfu.

Accordingly, the heat killed activated, purified extracellular components of Bacillus coagulans IS2 was analysed for its metabolomic profile. The corresponding peaks of the compounds are provided in chromatograms depicted in Figures 5 to 7 and Figure 9. This composition comprises anti-inflammatory compounds selected from the group consisting of fatty acids, styrene, butanoate, pyruvate and phenylalanine.

As referred above, the Bacillus coagulans Unique IS2, is isolated and characterized by Unique Biotech Limited and deposited at the ATCC and MTCC with assigned numbers ATCC PTA-11748 and MTCC 5260, respectively. Bacillus coagulans Unique IS2 is a highly stable probiotic strain with proven efficacy in reducing diarrhoea, along with cholesterol lowering effects and in reduction of symptoms of bacterial vaginosis (Ratna Sudha et al., 2012). Bacillus coagulans Unique IS2 is reported to have efficacy in reducing diarrhoea, bacterial vaginosis and reducing cholesterol levels (Ratna Sudha et al, 2011, 2012a, 2012b).

In an embodiment, the invention provides a composition comprising a heat killed, purified extracellular component from Bacillus coagulans Unique IS2, optionally comprising one or more active ingredients and pharmaceutically acceptable excipients
The present composition optionally comprises a pharmaceutically acceptable carrier and/or excipient. Examples of suitable excipients include, but are not limited to surfactants and other solvents, polymers, preservatives, stabilizers, flavourings, colouring agents, buffering agents.

The disintegrant employed in the present composition is selected from the group consisting of sodium croscarmellose, crospovidone, gellan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate.

The glidant is selected from the group consisting of silicon dioxide, colloidal silicon dioxide, and talc.

The lubricant is selected from the group consisting of calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumerate, and vegetable based fatty acids.

Suitable surfactants include tyloxapol, polysorbate 20, polysorbate 60, and polysorbate 80 surfactants. Polymers are selected from the group consisting of dextrose, lactose, maltodextrin methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropylethyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, poly(methyl methacrylate), polycarbophil, gelatin, alginate, poly(acrylic acid), polyethylene oxide and chitosan or a derivative thereof.

Suitable stabilizers include chelating agents, such as edetate disodium, and antioxidants, such as ascorbic acid and citric acid, sucrose, sorbitol, glycerol, trehalose, and mannitol.

Solvent(s) are selected from the group consisting of monohydric alcohol or polyhydric alcohols such as polyethylene glycol, and polypropylene glycol.

In addition, the present composition may contain other active ingredients selected from the group of vitamins, minerals, antioxidants, prebiotics, antibacterial, antiviral and antifungal agents.
Prebiotics include but are not limited to the group consisting of inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose and lafinose.

In a further embodiment, the composition may be in the form of a tablet, capsule, granule, liquid or a powdered form.

In another preferred embodiment, the present invention provides a composition comprising effective amount of the heat killed purified fraction of Bacillus coagulans Unique IS2 ranging between 0.01 gm to 1gm.

The effective concentration of the extracellular component of Bacillus coagulans Unique IS2 employed in the present invention does not show any inhibitory/cytotoxic effect on macrophages, it however inhibits and modulates the levels of inflammatory markers selected from p65 NF-kB and cyclooxygenase 2. Cell viability MTT assay clearly demonstrated that BCIS2 HSUP was not cytotoxic to mouse macrophage Abelson murine leukemia virus transformed macrophage (RAW 264.7) cells up to 48 h. Therefore, to evaluate the efficacy of the HSUP fraction of BC IS2, cell toxicity studies, anti-inflammatory and immunomodulatory effects were studied.

Cytotoxicity studies with different probiotic fractions of Bacillus coagulans Unique IS2 were carried out (live BC IS2 (WLO); cell-free culture supernatant (SUP); heat-killed BC IS2 (HKO) and heat-killed sterilized BC IS2 culture supernatant (HSUP) on different cancer cell lines.

The anti-proliferative effects of the present composition were evaluated on human colon cancer (COLO 205), cervical cancer (Hela), and chronic myeloid leukemia (K562) cell lines. The heat killed supernatant of Bacillus coagulans Unique IS2 was thus found to have anti-proliferative effects in cancerous cell inhibition ranging from 20% to 35%.

The cytotoxic MTT assay performed using the heat killed supernatant of Bacillus coagulans Unique IS2 demonstrated a % cell inhibition of 22%, 31.7% and 19.5% of COLO, HeLa and K562 cell line, respectively without effecting the growth of normal cells (Human embryonic kidney cells, HEK).
In yet another preferred embodiment, the present invention provides a heat killed purified, activated extracellular component obtained from Bacillus coagulans Unique IS2 comprising anti-inflammatory compounds inhibiting the expression of p65 NF-kß and COX-2 levels.

According to Figures 2 and 3, HSUP fraction (BCIS2) exhibited anti-inflammatory activity on LPS-induced RAW 264.7 macrophages by inhibiting COX-2 levels, inducing anti-inflammatory cytokines selected from the group consisting of IL-2 and IL-10 and inhibiting pro-inflammatory cytokines selected from the group consisting of IL-6, IL-12, TNF-a and IFN-? via the inhibition of the inflammation regulatory transcription factor, NF-kB.

The anti-inflammatory activity of BC IS2 HSUP was evaluated against important inflammatory proteins, COX-2 and NF-kB by Western blot analysis and ELISA respectively. RT-PCR and Western blot analysis of COX-2 showed a significant decrease in the expression levels of COX-2 mRNA and protein respectively (Figure 2A and 2B). NFkB-p65 a subunit of NF-kappa-B transcription complex plays a crucial role in inflammatory and immune responses. Therefore, in order to evaluate the levels of p65 NF-kB in cytoplasmic and nuclear extracts of cells; the cells were treated with and without BC IS2 HSUP. The lower levels of p65 (p<0.05) in nuclear as well as cytoplasmic extracts of cells treated with the present composition of BC IS2 indicates inhibition of NF-kB (Figure 2C) and thus COX-2.

Further, in-vivo animal studies performed indicate that the heat-killed culture supernatant (HSUP) of BC IS2 is effective in reducing inflammation and is at par with celecoxib, a known anti-inflammatory drug (Figure 8).

In one preferred embodiment, the present invention provides a heat killed purified, activated extracellular component from Bacillus coagulans Unique IS-2 modulating cell-mediated immunity by increasing the levels of Th1 cytokines selected from the group consisting of IL-2, TNF-a and IFN-?.

The inventors have evaluated immunomodulatory effects of BC IS2 on PBMCs, levels of cytokines released into the culture supernatant by measuring the same using ELISA. In accordance with the same BC IS2 HSUP induced Th1 cytokines, TNF-a, IFN-? and IL2. The other Th2 cytokines, IL-6 and IL-12 are not modulated by HSUP significantly. However, BC IS2 induced the release of IL-10 cytokine (Figure 4 A & 4B).

In one embodiment, the present invention provides a composition comprising heat-killed sterilized Bacillus coagulans Unique IS2 culture supernatant (HSUP), optionally with one or more excipients or active ingredients or both for use:
(a) in enhancement of immunity and in the reduction of inflammation associated with pathological diseases such as diabetes, cancer, rheumatoid arthritis, celiac disease (CD), inflammatory bowel disease (IBD), and atherosclerosis, and
(b) as an adjuvant in the treatment of cancers selected from the group consisting of colon cancer, cervical cancer and chronic myeloid leukemia.

In one more preferred embodiment, the invention provides a method for enhancing immunity and reducing inflammation in a subject which method comprises administering an appropriate amount of heat-killed sterilized Bacillus coagulans Unique IS2 culture supernatant (HSUP), in association with one or more excipients to the subject in need thereof.
In another preferred embodiment, the present invention provides a method of inducing the expression of interleukins IL-10 and IL-2 and inhibition of interleukin IL-6 and Tumour Necrosis Factor-a (TNF-a) by administration of effective concentrations of the heat killed sterilized supernatant fraction of Bacillus coagulans IS2 in association with one or more excipients to the subject in need thereof.

In yet another embodiment, the invention provides a method of reducing p65 levels in nuclear extracts of a cancerous cell in a subject which method comprises administering an appropriate amount of heat-killed sterilized Bacillus coagulans Unique IS2 culture supernatant (HSUP), in association with one or more excipients to the subject in need thereof.

In one embodiment, the present invention provides a liquid formulation comprising the heat killed, purified extracellular component synthesized from Bacillus coagulans Unique IS-2, wherein the said component in an amount of 0.1 million to 10 billion cfu /ml is combined with other excipients selected from sodium phosphate buffer, pH 7; an organic co-solvent, selected from the group consisting of polysorbate, polyethylene glycol (PEG), propylene glycol, and a combination thereof and a stabilizing agent selected from the group consisting of sucrose, sorbitol, glycerol, trehalose, and mannitol.

Additionally, the composition in the form of granules, powder or liquid may be used as a food ingredient in different food categories which include but are not limited to baked goods and baking mixes; beverages and its bases; breakfast cereals; chewing gum; coffee and tea; condiments and relishes; confections and frostings; dairy product analogs; fruit juices; frozen dairy desserts and mixes; gelatins, puddings, and fillings; grain products and pastas; hard candy; herbs, seeds, spices, seasonings, blends, and extracts, jams and jellies; milk; milk products; nuts and nut products; plant products; processed fruits and vegetables, vegetable juices; snacks; soft candy; soups and soup mixes; sweet sauces, toppings, and syrups. The composition in the form of granules, powder or liquid and may be sprinkled or poured directly onto different foods.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Examples
Example 1:
Cultivation of BC Unique IS2 culture and preparation of heat-killed sterilized culture supernatant
Overnight culture of BC IS2 cells which were grown in Luria Bertani (LB) medium were heat killed for 1 hr at 95°C in a boiling water bath. The cells were separated from the culture medium by centrifugation at 10000 rpm for 10 min at room temperature. The heat-killed supernatant (HSUP) was then filter sterilized using 0.22µm syringe filter. The filter sterilized supernatant from cells without heat killing was used as control.
Example 2:
Materials and Methods:
(i) Cell culture to determine cytotoxicity of HSUP
RAW 264.7 cells (ATCC® TIB71™) were grown in a complete medium comprising DMEM medium, 10% heat inactivated fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin and 2mM L-glutamine) at 37°C and 5% CO2. Cell viability was determined by trypan blue dye and cells were sub-cultured twice a week, with seeding at a density of 2×103 cells/ml.
(ii) MTT assay
Cytotoxicity of the heat killed sterilized supernatant of Bacillus coagulans IS2 was evaluated by the MTT assay. The concentration of HSUP used was 25, 50 and 100 µl in a final volume of 200 µl.
(iii) Cytokines analysis
The expression of cytokines including interleukins IL-2, IL-6, IL-10, IL-12; TNF-a and interferon IFN-? were analyzed from the culture supernatants of Bacillus coagulans IS 2 using Opti-EIA kits from BD Biosciences. RAW 264.7 cells were pre-treated with HSUP for 1 h and then stimulated with Lipopolysaccharide (LPS) for 18 h. The media was collected and stored at -20°C for the estimation of cytokines and PGE2 release.
(iv) PBMCs culture
Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient method according to manufacturer’s protocol (Sigma). The cells were washed with PBS and resuspended in RPMI medium supplemented with 10% FBS. 2 X 105 cells/well were added into 24-well plate and stimulated with HSUP (10-1000 µl) for 2- 72 hrs. The cell culture supernatants were collected and stored at -20 °C for cytokine estimation.
(v) Immunoblot and RT-PCR analysis
Immunoblot and RT-PCR analysis of COX-2 and b-actin was carried out to according to the protocol described by Arunasree et al., 2008.
(vi) NF-kB ELISA assay
The activity of NF-kB was measured by quantifying the p65 subunit in cytoplasm and nuclear extracts of cells treated with or without LPS and or HSUP using NF-kB p65 measuring kit from Cell Signaling Technologies, USA according to the manufacturer’s protocol.

Example 3
Evaluation of cytotoxicity of BC IS2 HSUP on LPS-induced RAW 264.7 cells
To evaluate the anti-inflammatory and immunomodulatory effects of BC IS2 HSUP, cytotoxic effects were studied using mouse macrophage cells. The RAW 264.7 cells were treated with different concentrations of BC IS2 HSUP (ranging from 10-500 µl) and incubated for different time periods of 4 h to 72 h. The results from MTT assay suggested that BC IS2 HSUP did not induce cell death of macrophages as depicted in Figure 1 up to 24 h, but induced 10% of cell death at 48 h. The macrophage cell growth inhibition was significantly lesser in BC IS2 HSUP treated cells when compared to LPS-treated cells.

Example 4
Evaluation of inhibitory effect of BC IS2 HSUP on inflammatory proteins - COX-2 and NF-kB
Anti-inflammatory activity of BC IS2 HSUP was evaluated on important inflammatory proteins, including COX-2 and NF-kB by Western blot analysis and ELISA respectively. RT-PCR and Western blot analysis of COX-2 showed a significant (p<0.001) decrease in the expression levels of COX-2 mRNA and protein respectively (Figures 2A and 2B). Further evaluation of levels of p65 NF-kB in cytoplasmic and nuclear extracts of cells treated with or without BC IS2 HSUP showed lower levels of p65 (p<0.05) in nuclear extracts of cells treated with BC IS2 suggesting inhibition of NF-kB (Figure 2C) and thus COX-2. The ELISA data measuring the p65 levels of NF-kB in the cytoplasmic and nuclear extracts of LPS-stimulated RAW cells treated with BCIS2 HSUP clearly suggested that it inhibited NF-kB translocation in nucleus thereby inhibiting its transcriptional activity.

Example 5
Evaluation of inhibitory effect of BC IS2 HSUP on pro-inflammatory cytokines
Effect of BC IS2 HSUP on LPS-induced inflammation of RAW 264.7 was further evaluated by measuring the pro- and anti-inflammatory cytokines. LPS stimulation of RAW 264.7cells induced expression of all pro-inflammatory cytokines (IL-6, IL-12, TNF-a and IFN-g) with inhibition of anti-inflammatory cytokines (IL-2 and IL-10) as determined by ELISA. BC IS2 HSUP decreased IL-6 and TNF-a cytokines significantly (Figure 3).The anti-inflammatory cytokines, IL-10 and IL-2 are significantly induced by HSUP confirming the anti-inflammatory effects of BC IS2.

Example 6
Evaluation of immunomodulatory effects of BC IS2 HSUP on PBMCs, levels of cytokines modulated Th1 immune response
To further evaluate the immunomodulatory effects of BC IS2 on Peripheral Blood Mononuclear cells (PBMC’s), levels of cytokines released in to the culture supernatant were measured by ELISA. BC IS2 HSUP induced Th1 cytokines, TNF-a, IFN-g and IL2. The other Th2 cytokines, IL-6 and IL-12 are not modulated by HSUP significantly. However, BC IS2 induced the release of IL-10 cytokine (Figure 4). Thus, the invention clearly demonstrates that heat-killed cell-free culture supernatant of Bacillus coagulans Unique IS2 has immunity enhancing activity and potent anti-inflammatory activity by inhibiting COX-2, NF-kB and pro-inflammatory cytokines. BCIS2 HSUP also has immunomodulatory effect by activating the cell-mediated immunity, specifically the Th1 response through phagocytosis. Further, the heat killed supernatant of the probiotic Bacillus coagulans Unique IS2 was found to elicit an immune response.

Example 7:
Metabolomic analysis of control and heat killed cell culture supernatant of Bacillus coagulans IS2
Metabolomic analysis was performed to depict the role of the secondary metabolites in conferring anti-inflammation properties to the present composition.
(i) Protocol
Bacillus coagulans Unique IS2 was grown in 3000 ml LB broth at 37oC, 200 rpm overnight. The overnight grown culture was centrifuged at 8000 rpm for 20 min at 4oC to pellet cells. The supernatant was separated and filtered through 0.22 µm membrane filter and the filtrate was subjected to further process steps. The three test samples were designated as Control (Cont), Heat-killed (HK) and lyophilized (LY) and processed separately for metabolite extraction and GC-MS analysis as below.
Control (CONT: The water in the filtrate was removed by rotary evaporation at a temperature of 45-50oC and at reduced pressure. To the concentrated filtrate, diethyl ether was added and metabolites were extracted by stirring for 24h at room temperature. Ether was removed by rotary evaporation under reduced pressure at temperature of 25oC.
Heat-Killed (HK): The water in the filtrate was removed by rotary evaporation at a temperature of 45-50oC and at reduced pressure. To the concentrated filtrate, diethyl ether, organic solvent, was added and metabolites were extracted by stirring for 24h at room temperature. Ether was removed by rotary evaporation under reduced pressure and a temperature of 25oC.
Lyophilized (LY): The water in the filtrate was removed by lyophilization. To the concentrated filtrate, diethyl ether, organic solvent, was added and metabolites were extracted by stirring for 24h at room temperature. Ether was removed by rotary evaporation under reduced pressure and a temperature of 25oC.
The aforementioned samples were dried after ether removal, CONT, HK and LY, were dissolved in hexane (100% MS grade SIGMA) and injected into GC-MS (Agilent 7850) for further analysis.

(ii) Data analysis
The control sample without heat killing and the heat killed culture supernatants did not show any difference using GC-MS analysis. This result was reproducible several times. Then it was realized that the metabolites in control supernatant when subjected to rotary evaporation at 45-50oC were being modified and therefore chromatograms of CONT and HK appeared alike. To overcome this, the control supernatant was lyophilized (LY) to remove water content and the metabolites were extracted. During the complete extraction process, care was taken to not expose the LY sample to temperatures higher than room temperature. Chromatograms of three samples (CONT, HK, LY) clearly indicated the effect of temperature on metabolite extraction. The LY sample did not show any metabolites at lower retention time (RT). CONT sample showed same intensity of peaks as the HK sample. However, HK sample showed more metabolites especially at lower RT points and this was attributed to complete lysis of bacteria by heating at 95oC, releasing all secondary metabolites and stable metabolite extraction (Fig 1).
To identify the pathways of the metabolites extracted, the top 200 compounds (Data not shown) obtained in GC-MS were subjected to data analysis using MetaboAnalyst 3.0 software. Data from all the three samples, Control (CONT), Heat killed (HK) and lyophilized (LY) were analysed. (Figures 5, 6, 7 and 9) Till date there is no comprehensive database for microbial metabolites specific for Bacillus coagulans. Few databases were available for E. coli. Therefore, data analysis has been carried out using E. coli database to map the metabolites of Bacillus coagulans Unique IS2.
Lyophilized sample (LY)
The pathway analysis of LY sample indicated that metabolites extracted belong to naphthalene and anthracene degradation pathway and purine metabolism pathway. Naphthalene and anthracene belong to family of Polycyclic Aromatic Hydrocarbons (PAH). Many bacterial species including Bacillus have been identified which degrade these PAH and use their metabolites as carbon source [Annweiler, E., et al., ppl Environ Microbiol, 2000. 66(2): p. 518-23]. However, role of this pathway in BC IS2 is not known. In humans, high intake of purine-rich diet results in uric acid accumulation in blood leading to hyperuricemia. Administration of the present composition, helped in dietary purine degradation and to overcome hyperuricemia.
Control and Heat-killed samples (CONT & HK)
In control sample (CONT) and heat-killed (HK) samples, metabolites of fatty acid metabolism and biosynthesis of unsaturated fatty acid were extracted. Apart from the metabolites of these two pathways, HK sample also showed production of metabolites belonging to styrene degradation, butanoate metabolism, pyruvate metabolism and phenylalanine metabolism.

(iii) Analysis of metabolites
Crucial Metabolites observed to be present in the heat killed supernatant of Bacillus coagulans IS2 and their role in the treatment of diseases are provided below:
Fatty acid metabolism and Biosynthesis of unsaturated fatty acids
Fatty acids form an important source of energy in organisms. Breakdown of fatty acids results in short-chain fatty acids (SCFAs) (Rautiola et al 2013). SCFAs produced by gut microbes are important for intestinal health maintenance (Flint, H.J., et al 2012). Butyrate SCFA produced by gut microbiota forms an important source of energy for colon cells of the intestine and also stimulates their barrier function (Usami, M., et al World J Gastroenterol, 2015. 21(41): p. 11597-608). It is also known to possess anti-inflammatory and anti-cancer activities (van Zanten, G.C., et al.,PLoS One, 2012. 7(10): p. e47212).
Unsaturated fatty acids (UFA), especially monounsaturated, poly unsaturated fatty acids (PUFAs) and conjugated fatty acids (CFAs) are beneficial to human health. Lactic acid bacteria in gut produce high amount of CFA’s such as conjugated linoleic acid (CLA) (Kishino, S.,et al.,2009) which possesses anticancer and anti-inflammatory activities.
The metabolomics results and data analysis suggested the beneficial effects of Bacillus coagulans Unique IS2 were attributed to SCFAs and biosynthesis of unsaturated fatty acids as evident from the pathway analysis.
Styrene metabolites
Styrene is both industrial and environmentally important molecule. Styrene is toxic to humans and some bacteria are known to degrade styrene. (J.A. de Bont, et al Appl Environ Microbiol, 1990. 56(5): p. 1347-51.) Metabolites of styrene degradation pathway are intermediates of pyruvate metabolism pathway which ultimately result in energy production.
Butanoate metabolism
Butyrate (butanoate) is a short chain fatty acid and is an important source of energy for colon cells. Without butyrate, colon cells undergo autophagy. (Donohoe, D.R., et al Cell Metab, 2011. 13(5): p. 517-26.)
Pyruvate metabolism
Pyruvate is the key intermediate formed through many metabolic pathways. This enters in Krebs cycle to yield ATP. (Velagapudi, V.R., et al. 2010)
Phenylalanine metabolism
Phenylalanine is an important essential amino acid involved in the synthesis of tyrosine which is a precursor for many neurotransmitters.

The above data suggested that heat induced changes in metabolic profile of the present composition thereby rendering presence of several metabolites therein having beneficial effects on human health. GC-MS data and pathway analysis indicated that the culture supernatant of heat killed BC IS2 harboured many metabolites, from different pathways.

Example 8:
Anti-inflammatory activity of heat killed culture supernatant of Bacillus Coagulans IS2 in an in-vivo rat pouch model
The efficacy of the heat-killed sterilized culture supernatant (HSUP) of Bacillus coagulans Unique IS2 in inhibiting cancer cell proliferation along with its anti-inflammatory activity was determined by evaluation of its in vivo anti-inflammatory efficacy in a carrageenan-induced rat air pouch model of inflammation.
Accordingly, 150g male Wistar rats were obtained from the National Institute of Nutrition (NIN), Hyderabad and housed in an animal house for 7 days. The air pouch formation in rats was carried out subcutaneously by injecting 20 ml of sterile air via a 0.22 µm syringe filter three times each at a 2-day interval. 0.1% carrageenan in PBS was injected into the air pouch to induce inflammation. The filter sterilized supernatant from cells without heat killing was employed as a control. After 24h, the control, i.e. culture supernatant of BC IS2 (250 and 500 µl) and HSUP fraction (250 and 500 µl) of BC IS2 was injected into the air pouch. Celecoxib, a known anti-inflammatory drug, was used as positive control at 20 mg/kg body weight concentration.

The control group of Wistar rats were administered with Phosphate Buffer saline (PBS). After 24h of drug treatment, blood was collected and animals were euthanized in a CO2 chamber and the air pouch were cut open. The air pouch was rinsed with PBS and the fluid as well as tissue were collected for cytokine analysis and were stored at -80°C until use. The carrageenan induced inflammation in air pouch was characterized by redness. The air pouch injected with celecoxib showed significant reduction in inflammation. The air pouch injected with control supernatant (CONT) showed yellow pus accumulation indicating secondary infection. However, the air pouch injected with heat killed culture supernatant (HSUP) showed complete inhibition of inflammation (Figure 5). The heat-killed culture supernatant (HSUP) of Bacillus coagulans Unique IS2 was effective in reducing inflammation in vivo and was at par with celecoxib.

Example 9:
Preparation of capsules comprising the present composition
The optimized preparation of the freeze-dried powder comprising the heat killed, purified extracellular component synthesized from Bacillus coagulans Unique IS2 was as follows: 5% trehalose and glucose in a 1:1 ratio was used as cryoprotector, the heat killed fraction of BC IS2 was freezed for 24 h in vacuum frozen-dryer after being pre-freezed for 24 h. This heat killed, purified extracellular component synthesized in the form of a freeze-dried powder.100mg of lyophilized heat killed supernatant, (lactose and/or maltodextrin were thoroughly blended. The blended mixture was filled in gelatin capsules.

Example 10:
Preparation of lyophilized liquid formulation
The liquid lyophilized formulation comprises 20mg/ml of heat killed, purified extracellular component synthesized from Bacillus coagulans Unique IS2, 5 mM of sodium phosphate buffer, pH 7.0; 0.01 of polyethylene glycol (PEG), and, 1% of sucrose.

Example 11
Capsules containing the following active ingredients
100 mg of the lyophilised powder of heat killed supernatant of Bacillus coagulans Unique IS2, 100mg prebiotic, 100mg fructooligosaccharide- and 1000 mg Vitamin C is blended together and filled in gelatin capsules.

Advantages of the invention:
- For most probiotic strains to be viable, refrigeration at 4°C is necessary during transport and storage thus increasing the cost and handling requirements. The present composition comprising the heat killed purified, activated extracellular component obtained from Bacillus coagulans Unique IS2 addresses the disadvantages posed by transportation, and
- The present composition is effective against inflammation, enhances immunity and is an alternate probiotic solution with negligible side-effects.
,CLAIMS:We claim,

1. A composition comprising a heat killed purified, activated extracellular component obtained from Bacillus coagulans Unique IS2 along with one or more pharmaceutically acceptable excipients, wherein the said composition comprises anti-inflammatory compounds having size in the range of 0.1 to 0.5 kD.

2. The composition according to claim 1, wherein the heat killed purified extracellular component synthesized from Bacillus coagulans Unique IS-2 comprises anti-inflammatory and immune enhancing compounds inhibiting the expression of p65 NF-kß and COX-2 levels, inducing anti-inflammatory cytokines selected from the group consisting of IL-2 and IL-10 and inhibiting pro-inflammatory cytokines selected from the group consisting of IL-6, IL-12, TNF-a and IFN-?.

3. The composition according to claim 1, wherein an effective amount of the heat killed purified fraction of Bacillus coagulans Unique IS2 comprises between 0.01 gms to 1gm.

4. The composition according to claim 1, wherein the heat killed purified, activated extracellular component comprises anti-inflammatory compounds selected from the group consisting of fatty acids, styrene metabolites, butanoate, pyruvate and phenylalanine.

5. The composition according to claim 1, further comprising active ingredients selected from the group of vitamins, minerals, antioxidants, prebiotics, antibacterial, antiviral and antifungal agents.

6. The composition according to claim 1, wherein the pharmaceutically acceptable carriers and or excipients are selected from the group consisting of disintegrants, glidants, lubricants, surfactants, polymers, co-solvents, diluents, stabilizing agents and preservatives.

7. The composition according to claim 7, wherein (a) the disintegrant is selected from the group consisting of sodium croscarmellose, crospovidone, gellan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate;
(b) glidant is selected from the group consisting of silicon dioxide, colloidal silicon dioxide, and talc,
(c) lubricant is selected from the group consisting of calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumerate, and vegetable based fatty acids,
(d) surfactant(s) is selected from the group consisting of polysorbate 80 and 20, polyethylene glycol esters, polyethylene glycols, glycerol ethers or mixtures of those compounds,
(e) polymer is selected from the group consisting of dextrose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxylpropylethyl cellulose, carboxymethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, poly(methyl methacrylate), polycarbophil, gelatin, alginate, poly(acrylic acid), polyethylene oxide and chitosan or a derivative thereof,
(f) co-solvent(s) is selected from the group consisting of monohydric alcohol or polyhydric alcohols such as polyethylene glycol, and polypropylene glycol, and (g) the stabilizing agent is selected from the group consisting of sucrose, sorbitol, glycerol, trehalose, and mannitol.

8. The composition according to claim 1, wherein the said composition is in the form of a powder, tablet, capsule, granule, suspension, gel or cream.

9. The composition according to claim 1, wherein the said composition is lyophilized or spray dried.