Modulation of immune function by <i>Bacillus coagulans</i>

Abstract

The present invention discloses a composition comprising heat inactivated spores and/or comprising heat inactivated vegetative cells of probiotic bacteria Bacillus coagulans, and a process for preparing the same. The invention also discloses a method of modulating immune function in mammals by activating macrophages, using a composition comprising Bacillus coagulans in the form of live or heat inactivated spore and/or vegetative cells.

Claims

1. A method of modulating immune function by macrophage polarization in mammals, said method comprising step of bringing into contact mammalian macrophages with an effective concentration of Bacillus coagulans MTCC 5856 in the form of spore or vegetative cells, to bring about the effect of immune modulation by polarizing macrophages to M1 type.

2. The method as in claim 1, wherein the spores include heat inactivated or dead spores of Bacillus coagulans.

3. The method as in claim 1, wherein the vegetative cells include heat inactivated or dead or lysed vegetative cells of Bacillus coagulans.

4. The method as in claim 1, wherein the polarisation of macrophages to M1 type is brought about by inducing the expression of pro-inflammatory genes and cells surface receptors.

5. The method as in claim 1, wherein the pro-inflammatory genes are selected from the group comprising IL-1β, IL-6, IL-12p40, IL23, TNF-α, TNOS and iNOS.

6. The method as in claim 1, wherein the cell surface receptors are selected from the group comprising CD80, CD83, CD86, MHC-II, F4/80 and CD16/32.

7. The method as in claim 1, wherein the mammal is human.

8. The method as in claim 1, wherein the composition comprising heat inactivated spores and/or vegetative cells of Bacillus coagulans is formulated with pharmaceutically/nutraceutically accepted excipients, adjuvants in the form of powder, infant formulation, suspension, syrup, emulsion, tablets, capsules, eatable or chewable.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

(2) FIG. 1 shows the flow cytomeric results the detection if live and dead cells of heat inactivated spores of Bacillus coagulans MTCC 5856 A—Unstained and B—Stained. Q1 denotes dead cells, Q2—Viable but non culturable cells, Q3 denotes damaged cells, and Q4 denotes Live cells.

(3) FIG. 2 A shows microscopic picture of wet mount of live spores of Bacillus coagulans MTCC 5856.

(4) FIG. 2 B shows microscopic picture of Gram staining of live spores of Bacillus coagulans MTCC 5856.

(5) FIG. 2 C shows microscopic picture of spore staining of live spores of Bacillus coagulans MTCC 5856.

(6) FIG. 2D shows microscopic picture of wet mount of heat inactivated spores of Bacillus coagulans MTCC 5856.

(7) FIG. 2E shows the microscopic picture of Gram stain of heat inactivated spores of Bacillus coagulans MTCC 5856.

(8) FIG. 2F shows the spores staining of heat inactivated spores of Bacillus coagulans MTCC 5856.

(9) FIG. 3A is the graphical representation showing the effect of heat inactivation on the viability of spores and vegetative cells of Bacillus coagulans MTCC 5856 determined by following Flow Cytometry (FCM) technique.

(10) FIG. 3B is the graphical representation showing the effect of heat inactivation on the viability of spores and vegetative cells of Bacillus coagulans MTCC 5856 determined by following plate count method.

(11) FIG. 4 is the graphical representation showing Cell viability of RAW264.7 cells following treatment with different probiotic concentration. Treatment was done at concentration of 1.5×10.sup.6, 1.5×10.sup.7, 1.5×10.sup.8, 1.5×10.sup.9 cfu/ml at 6 h points.

(12) FIG. 5A is the graphical representation showing the expression of IL-1β gene in RAW 264.7 co-culture with 1.5×10.sup.8 cfu/ml live cells, heat inactivated cells and heat inactivated spores for a duration of 6 h

(13) FIG. 5B is the graphical representation showing the expression of TNF-α gene in RAW 264.7 co-culture with 1.5×10.sup.8 cfu/ml live cells, heat inactivated cells and heat inactivated spores for a duration of 6 h.

(14) FIG. 5C is the graphical representation showing the expression of IL-6 gene in RAW 264.7 co-culture with 1.5×10.sup.8 cfu/ml live cells, heat inactivated cells and heat inactivated spores for a duration of 6 h.

(15) FIG. 5D is the graphical representation showing the expression of IL-12p40 gene in RAW 264.7 co-culture with 1.5×10.sup.8 cfu/ml live cells, heat inactivated cells and heat inactivated spores for a duration of 6 h.

(16) FIG. 6 is the graphical representation showing the expression of i-NOS gene in RAW 264.7 co-culture with 1.5×10.sup.8 cfu/ml live cells, heat inactivated cells and heat inactivated spores for a duration of 6 h.

(17) FIG. 7 is the graphical representation showing the expression of M1 related genes activated by live cells of Bacillus coagulans MTCC 5856.

(18) FIG. 8A is the graphical representation showing the levels of NO in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856.

(19) FIG. 8B is the graphical representation showing the levels of i-NOS in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856

(20) FIG. 8C is the graphical representation showing the levels of TNOS in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856

(21) FIG. 8D is the graphical representation showing the levels of IL-6 in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856

(22) FIG. 8E is the graphical representation showing the levels of IL-1β in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856

(23) FIG. 8F is the graphical representation showing the levels of TNF-α in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856.

(24) FIG. 8G is the graphical representation showing the levels of TGF-β in RAW 264.7 cells cultured with live cells, heat inactivated cells and heat inactivated spores of Bacillus coagulans MTCC 5856.

(25) FIG. 9A is a flow cytometric graphical representation showing the effect of Bacillus coagulans on M1 surface receptors CD80, CD83, CD86 and MHC-II of RAW264.7 macrophages

(26) FIG. 9B is a flow cytometric graphical representation showing the effect of Bacillus coagulans on M1 surface receptors CD 16/32 and F 4/80 of RAW264.7 macrophages

DESCRIPTION OF PREFERRED EMBODIMENTS

(27) In a most preferred embodiment, the invention discloses a composition comprising heat inactivated spores of probiotic bacteria Bacillus coagulans, prepared by the process comprising steps of: a) Preparing pure culture of Bacillus coagulans by inoculating the bacteria in a sterile seed medium and incubating at 37-40° C. for 22-24 hours with constant shaking and confirming the purity through microscopic techniques; b) Preparing the seed inoculum by mixing the pure culture of step a) in a suitable media and adjusting the pH to 6.5±0.2 with ortho-phosphoric acid; c) Inoculating the seed medium of step b) to a suitably sterilized fermentation medium (broth) and incubated at 37-39° C. for 35-37 hours with agitation and suitable aeration; d) Identifying sporulated cells using microscopic techniques and harvesting the spores by centrifuging the broth containing 80-100% sporulated cells, at 7000-15000 rpm; e) Adding 10% w/v maltodextrin or suitable protective agent to the biomass of sporulated cells in the ratio of 1:1 and filtering the slurry through sterile mesh; f) Inactivating the slurry of step e) by heat treatment at 110±2° C. with 0.8±0.2 bars of pressure for 5 to 8 hours; g) Spray drying the heat inactivated spores at 115 to 150° C. inlet temperature and 55 to 70° C. outlet temperature; h) Subjecting the spray dried powder containing heat inactivated spores to further heat treatment at 121±2° C. with 1.5±0.2 bars of pressure for 15 to 30 minutes to ensure that spore viable count is <10.sup.3 cfu/g; i) Diluting with maltodextrin or suitable protective agent to obtain a composition comprising heat inactivated spores of Bacillus coagulans; j) Enumerating viable, dead and viable but not culturable cells by flow cytometry.

(28) In a related embodiment, the Bacillus coagulans strain is specifically Bacillus coagulans MTCC 5856. In another related embodiment, the media of step a) and step b) is selected from the group comprising MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media. In another related embodiment, the fermentation media of step c) is selected from the group comprising MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media. In another related embodiment, the fermentation media of step c) preferably comprises dextrose, corn steep powder, calcium carbonate, Manganese (II) sulfate and ammonium sulphate.

(29) In another preferred embodiment, the composition is used as a supplement/additive for increasing the immune function in mammals. In a related aspect, the mammal is preferably human. In another related embodiment, the composition comprising heat inactivated spores of Bacillus coagulans is formulated with pharmaceutically/nutraceutically accepted excipients, adjuvants and administered in the form of powder, infant formulation, suspension, syrup, emulsion, tablets, capsules, eatable or chewable.

(30) In another most preferred embodiment, the invention discloses a composition comprising heat inactivated vegetative cells of probiotic bacteria Bacillus coagulans, prepared by the process comprising steps of: a) Preparing pure culture of Bacillus coagulans by inoculating the bacteria in a sterile seed medium and incubating at 37-40° C. for 22-24 hours with constant shaking and confirming purity through microscopic techniques; b) Preparing the seed inoculum by mixing the pure culture of step a) in a suitable media and adjusting the pH to 6.5±0.2 with ortho-phosphoric acid; c) Inoculating the seed medium of step b) to a suitably sterilized fermentation medium (broth) and incubated at 37-39° C. for 35-37 hours with agitation and suitable aeration; d) Identifying vegetative cells using microscopic techniques and harvesting the cells by centrifuging the broth at 7000-15000 rpm; e) Adding 10% w/v maltodextrin or suitable protective agent to the biomass of vegetative cells in the ratio of 1:1 and filtering the slurry through sterile mesh; f) Inactivating the slurry of step e) by heat treatment 100±2° C. with 0.2±0.1 bars of pressure for 5 to 8 hours; g) Spray drying the heat inactivated vegetative cells at 115 to 150° C. inlet temperature and 55 to 70° C. outlet temperature; h) Diluting with maltodextrin or suitable protective agent to obtain a composition comprising heat inactivated vegetative cells of Bacillus coagulans; i) Enumerating viable, dead and viable but not culturable cells by flow cytometry.

(31) In a related embodiment, the Bacillus coagulans strain is specifically Bacillus coagulans MTCC 5856. In another related embodiment, the media of step a) and step b) is selected from the group comprising MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media. In another related embodiment, the fermentation media of step c) is selected from the group comprising MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media. In another related embodiment, the fermentation media of step c) preferably comprises dextrose, corn steep powder, calcium carbonate, Manganese (II) sulfate and ammonium sulphate.

(32) In another preferred embodiment, the composition is used as a supplement/additive for increasing the immune function in mammals. In a related aspect, the mammal is preferably human. In another related embodiment, the composition comprising heat inactivated vegetative cells of Bacillus coagulans is formulated with pharmaceutically/nutraceutically accepted excipients, adjuvants and administered in the form of powder, infant formulation, suspension, syrup, emulsion, tablets, capsules, eatable or chewable.

(33) In yet another most preferred embodiment the invention discloses a method of modulating immune function in mammals, said method comprising step of administering effective concentration of Bacillus coagulans in the form of spore and/or bacterium to said mammals to bring about the effect of immune modulation by polarizing macrophages. In a related embodiment, the spores include viable or heat inactivated or dead spores of Bacillus coagulans. In another related embodiment, the bacterium includes viable or heat inactivated or dead or lysed vegetative cells of Bacillus coagulans. In another related embodiment, the Bacillus coagulans strain is preferably Bacillus coagulans MTCC 5856. In a related aspect, the modulation of immune function is brought about by polarizing the macrophages to M1 type. In another related aspect, the polarisation of macrophages to M1 type is brought about by inducing the expression of pro-inflammatory genes and cells surface receptors. In yet another related aspect, the pro-inflammatory genes are selected from the group comprising IL-1β, IL-6, IL-12p40, IL23, TNF-α, and iNOS. In a further related aspect, the cell surface receptors are selected from the group comprising CD80, CD83, CD86, MHC-II, F4/80 and CD16/32. In yet another related embodiment, the mammal is human. In another related embodiment, the composition comprising heat inactivated spores and/or vegetative cells of Bacillus coagulans is formulated with pharmaceutically/nutraceutically accepted excipients, adjuvants and administered in the form of powder, infant formulation, suspension, syrup, emulsion, tablets, capsules, eatable or chewable.

(34) The following illustrative examples further describe in detail the preferred embodiments of the invention:

EXAMPLES

Example 1: Process of Heat Inactivation of Spores and Vegetative Cells of Bacillus coagulans

(35) Heat inactivation of spores of Bacillus coagulans is carried out by the following steps: a) Preparing pure culture of Bacillus coagulans by inoculating the bacteria in a sterile seed medium (MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media) and incubating at 37-40° C. for 22-24 hours with constant shaking and confirming the purity through microscopic techniques; b) Preparing the seed inoculum by mixing the pure culture of step a) in a suitable media (MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media) and adjusting the pH to 6.5±0.2 with ortho-phosphoric acid c) Inoculating the seed medium of step b) to a suitably sterilized fermentation medium (broth—comprising dextrose, corn steep powder, calcium carbonate, Manganese (II) sulfate and ammonium sulphate) and incubated at 37-39° C. for 35-37 hours with agitation and suitable aeration; d) Identifying sporulated cells using microscopic techniques and harvesting the spores by centrifuging the broth containing 80-100% sporulated cells, at 7000-15000 rpm; e) Adding 10% w/v maltodextrin or suitable protective agent to the biomass of sporulated cells in the ratio of 1:1 and filtering the slurry through sterile mesh; f) Inactivating the slurry of step e) by heat treatment at 110±2° C. with 0.8±0.2 bars of pressure for 5 to 8 hours; g) Spray drying the heat inactivated spores at 115 to 150° C. inlet temperature and 55 to 70° C. outlet temperature; h) Subjecting the Spray dried powder containing heat inactivated spores to further heat treatment at 121±2° C. with 1.5±0.2 bars of pressure for 15 to 30 minutes to ensure that spore viable count is 10.sup.3 cfu/g; i) Diluting with maltodextrin or suitable protective agent to obtain a composition comprising heat inactivated spores of Bacillus coagulans; j) Enumerating viable, dead and viable but not culturable cells by flow cytometry.

(36) Similarly, the heat inactivated vegetative cells are prepared by the following process: a) Preparing pure culture of Bacillus coagulans by inoculating the bacteria in a sterile seed medium (MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media) and incubating at 37-40° C. for 22-24 hours with constant shaking and confirming the purity through microscopic techniques; b) Preparing the seed inoculum by mixing the pure culture of step a) in a suitable media (MRS, dextrose media, tryptic soya media, nutrient media, yeast peptone media, corn steep media) and adjusting the pH to 6.5±0.2 with ortho-phosphoric acid c) Inoculating the seed medium of step b) to a suitably sterilized fermentation medium (broth—comprising dextrose, corn steep powder, calcium carbonate, Manganese (II) sulfate and ammonium sulphate) and incubated at 37-39° C. for 35-37 hours with agitation and suitable aeration; d) Identifying vegetative cells using microscopic techniques and harvesting the cells by centrifuging the broth at 7000-15000 rpm; e) Adding 10% w/v maltodextrin or suitable protective agent to the biomass of vegetative cells in the ratio of 1:1 and filtering the slurry through sterile mesh; f) Inactivating the slurry of step e) by heat treatment 100±2° C. with 0.2±0.1 bars of pressure for 5 to 8 hours; g) Spray drying the heat inactivated vegetative cells at 115 to 150° C. inlet temperature and 55 to 70° C. outlet temperature; h) Diluting with maltodextrin or suitable protective agent to obtain a composition comprising heat inactivated vegetative cells of Bacillus coagulans; i) Enumerating viable, dead and viable but not culturable cells by flow cytometry.

(37) The flow cytometric results differentiate viable but non-culturable cells, from dead and live cells (FIG. 1A and FIG. 1B). The heat inactivation step is vital for preparing a stable composition responsible for the biological function of a probiotic strain. If the heat provided is inadequate, it leads to partial inactivation and if the heat is more, the spores die and cannot be revived. Hence, the right temperature as mentioned in the above steps was decided through rigorous experimentation which shows that the cell integrity is maintained (FIG. 2A-2F) after heat inactivation which resulted in retaining the biological function of such composition containing heat inactivated spores and/or vegetative cells of probiotic strain. FIGS. 2A, 2B and 2C shows the morphology live spores of Bacillus coagulans whereas FIGS. 2 D, 2E and 2F show the morphology of heat inactivated spores of Bacillus coagulans. It is evident that the heat inactivation step has not significantly changed the cell morphology/structure, thus, found to be suitable for preparing a stable composition which exhibits biological function i.e. modulating immune function. The viability of the cells was determined by flow cytometric method (FIG. 3A) and plate count (FIG. 3B). For determining viable cells, the flow cytometic method is much more efficient than plate count method. It is very clear from the flow cytometric data (FIG. 1A, FIG. 1B and FIG. 3A) that the heat inactivated spores and vegetative cells obtained by following said process had viable vegetative cells and spores of Bacillus coagulans but they were not culturable as indicated by the plate method of vegetative cells and spores of Bacillus coagulans enumeration (FIG. 3B)

Example 2: Modulation of Immune Function by Macrophage Polarisation

Experimental Section/Materials and Methods

(38) Reagents

(39) Dulbecco's modified eagle's medium (DMEM), LPS (Escherichia coli 055:B5), and FITC-dextran (40,000 Da) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Kits for Cell Counting (Kit-8), nitric oxide (NO), BCA protein, and nitric oxide synthase (iNOS) were purchased from Beyotime Biotechnology (Haimen, China). Antimouse antibodies FITC-CD80, FITC-CD83, APC-CD86, APC-MHCII, FITC-F4/80 pro-inflammatory markers were purchased from Beijing 4A Biotech Co., Ltd (4A Biotech, china. Eosin-methylene blue medium (EMB) agar were obtained from solarbio (solarbio, china). Phospho-ERK1/2, ERK1/2, phospho-JNK, INK, phospho-p38, p-38, β-actin and HRP-conjugated anti-mouse IgG were obtained from Cell Signaling Technology (Massachusetts, USA).

(40) Cell Culture and Probiotics

(41) The mouse monocyte/macrophage cell line, Raw264.7, was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 100 μg/mL streptomycin, and 100 U/mL, penicillin (Sigma-Aldrich, USA). Cells were maintained at 37° C. in a humidified atmosphere of 5% CO2. B. coagulans MTCC 5856 commercially known as LactoSpore®, Registered trademark of Sabinsa Corporation, USA) and heat inactivated vegetative cells and heat inactivated spores were used for the experimentation.

(42) Cell Viability Assay

(43) Cells were seeded at 2×10.sup.4 cells/well in 96-well culture plates and incubated for 6 h. then RAW264.7 cells were further cultured with PBS, Lipopolysaccharide (LPS, 200 ng/mL) for 24 h or live cells (1.5×10.sup.8 cfu/mL) and heat inactivated spores (1.5×10.sup.8 cfu/mL) respectively for 6 h. Stimulation with LPS (200 ng/ml) was included in each experiment to ensure functional differentiation into M1 subtypes. Cell counting kit-8 (Beyotime) was used to determine the cell cytotoxicity according to the manufacturer's instruction. Briefly, 10 μl CCK-8 was added into each well and incubated for 1-4 h at 37° C. The optical absorbance at OD450 was measured by SpectraMax M5 (Molecular Devices, Sunnyvale, Calif.).

(44) Relative Transcription of iNOS, IL-1β, IL-6, IL-12p40 and TNF-α

(45) After treatment of RAW264.7 cells (1.0×10.sup.6 cells in six-well plates) with either Lipopolysaccharide (LPS, 200 ng/mL) for 24 h or live cells (1.5×10.sup.8 cfu/mL), heat inactivated cells or heat inactivated spores (1.5×10.sup.8 cfu/mL alone for 6 h at 37° C. under 5% CO2, Macrophages were lysed and total RNA was extracted using Trizol (Sangon Biotech). The concentration, purity, and quality of isolated RNA were measured with a NanoDrop One spectrophotometer (ThermoFisher Scientific) and 1,000 ng of total RNA was immediately reverse transcribed into cDNA using HiScript® II Q RT SuperMix (Vazyme, R223-01). Relative expression levels of iNOS, IL-1β, IL-6, IL-12p40 and TNF-α were evaluated by quantitative real-time reverse transcription PCR (RT-qPCR), using ChamQTM SYBR® qPCR Master Mix (Vazyme, Q341-02) and CFX96 Real Time PCR System (Bio-Rad). The RT-qPCR comprised an initial step of 95° C. for 10 min, thereafter 95° C. for 15 s followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. All data were normalized to the level of actin transcripts amplified from the same sample, and then to untreated control mRNA. The data were analyzed with 2-ΔΔT method. The gene-specific primer sequences are given below:

(46) TABLE-US-00001 IL-1β (SEQ ID NO: 1) F: GCAACTGTTCCTGAACTCAACT (SEQ ID NO: 2) R: ATCTTTTGGGGTCCGTCAACT IL-6 (SEQ ID NO: 3) F: TAGTCCTTCCTACCCCAATTTCC (SEQ ID NO: 4) R: TTGGTCCTTAGCCACTCCTTC IL-12p40 (SEQ ID NO: 5) F: CCCATTCCTACTTCTCCCTCAA (SEQ ID NO: 6) R: CCTCCTCTGTCTCCTTCATCTT TNF-α (SEQ ID NO: 7) F: CCCTCACACTCAGATCATCTTCT (SEQ ID NO: 8) R: GCTACGACGTGGGCTACAG iNOS (SEQ ID NO: 9) F: CTCACCTACTTCCTGGACATTAC (SEQ ID NO: 10) R: CAATCTCTGCCTATCCGTCTC β-actin (SEQ ID NO: 11) F: CGTTGACATCCGTAAAGACC (SEQ ID NO: 12) R: AACAGTCCGCCTAGAAGCAC

(47) Evaluation of Nitric Oxide Synthesis

(48) Monolayers of RAW 264.7 macrophages in 12-well microplate were cultured in DMEM supplemented with 10% FBS at 37° C. in 5% CO2 under optimal humidity. Cells were incubated with PBS, Lipopolysaccharide (LPS, 200 ng/mL) for 24 h, live cells (1.5×10.sup.8 cfu/mL) and heat inactived spores (1.5×10.sup.8 cfu/mL) respectively. Nitric Oxide and Nitric Oxide Synthase (iNOS, tNOS) in supernatant was determined using Nitric Oxide and Nitric Oxide Synthase typed assay kit (Nj jiancheng, China).

(49) Cytokine Profile

(50) The concentrations of iNOS, NO, IL-1β, IL-6 TNF-α and TGF-β secreted by macrophages after treatment of LPS or 1.5×10.sup.8 cfu/ml Bacillus coagulans (live cells, heat inactivated spores) were determined in macrophage cells supernatants by ELISA (4A Biotech, china) following the manufacturer's recommendation. The cytokines levels were determined by comparison with a standard calibration curve.

(51) Dextran Phagocytosis Assay

(52) RAW264.7 cells were seeded at 1.0×10.sup.5 cells in 12-well plates followed by treatment with either Lipopolysaccharide (LPS, 200 ng/mL) for 24 h or live cells (1.5×10.sup.8 cfu/mL) or heat inactivated spores (1.5×10.sup.8 cfu/mL) for 6 h at 37° C. under 5% CO.sub.2, following 1 h starvation in serum-free medium. Then, cells were washed with PBS for two times repeat and incubated with FITC-dextran (1 mg/mL; Sigma, FD40S) for 1 h at 37° C. under 5% CO.sub.2. Thereafter, cells were washed with PBS and harvested followed by centrifugation (500×g, 5 min, 4° C.). Data were processed using flow cytometry analysis (FACS) at least 10,000 events to determine Mean fluorescence intensity (MFI) of intracellular FITC-dextran.

(53) Flow Cytometric Analysis

(54) RAW264.7 cells were seeded at 1.0×10.sup.5 cells in 12-well plates followed by treatment with either Lipopolysaccharide (LPS, 200 ng/mL) for 24 h or live cells (1.5×10.sup.8 cfu/mL) alone or heat inactivated spores (1.5×10.sup.8 cfu/mL) for 6 h at 37° C. under 5% CO2. After the final incubation, macrophages were washed with PBS and treated with 0.04% ethylenediamine tetra acetic acid (EDTA, Sinopharm), Cells were incubated with Fc Block TM (BD Biosciences), and stained with either an FITC anti-mouse CD80, FITC anti-mouse CD83, APC anti-mouse CD86, APC anti-mouse MHCII, FITC anti-mouse F4/80 and FITC anti-mouse CD16/32 antibody (BioLegend) or an isotype control, 30 min, 4° C. in the dark. After washing with PBS for two times repeat, stained cells were analyzed by fluorescence-activated cell sorting (FACS) for at least 10,000 events to determine Mean fluorescence intensity (MFI).

(55) Statistical Analysis

(56) Data are presented as means±SEM at least three independent experiments. Statistical analysis was performed using SPSS20.0 and OriginPro Software. Statistical significance was assessed using a one-way analysis of variance followed by Dunnett's or Tukey's test for multiple comparisons. The value of P<0.05 was considered as statistical significant.

(57) Results

(58) Viability Analysis of Bacillus coagulans on RAW264.7 Macrophage

(59) To evaluate the cytotoxicity of probiotic strain Bacillus coagulans (Live cells and heat inactivated cells) on murine macrophage cell line, RAW264.7 cells were treated with probiotic Bacillus coagulans for 6 h and cell viability was determined using the CCK-8 assay. In this assay, no significant decrease (p>0.05) of viability was observed when RAW264.7 cells were treated with Live cells or Heated inactivated spores at a range of concentrations (from 1.5×10.sup.6 to 1.5×10.sup.8 cfu/ml) (FIG. 4). However, viability was decreased when cells were exposure to 1.5×10.sup.9 cfu/ml Bacillus coagulans (P<0.05). At 1.5×10.sup.8 cfu/ml Live cells were found to increase cell viability (P<0.01) compared with the control. Therefore, 1.5×10.sup.8 cfu/ml were used in subsequent experiments.

(60) Bacillus coagulans Upregulates the Gene Expression Level of Markers for M1 Macrophage In Vitro

(61) The expression of pro-inflammatory genes (IL-1 β, IL-6, IL-12p40, TNF-α) and iNOS was evaluated following treatment of RAW 264.7 with 1.5×10.sup.8 cfu/ml Bacillus coagulans (Live cells, heat inactivated cells and heat inactivated spores) for a duration of 6 h. mRNA level revealed proinflammatory genes except for IL-12p40 were dramatically increased (P<0.01) in RAW 264.7 treated with live cells and LPS treatment relative to untreated time-matched control cells (FIG. 5A-5D). Furthermore, heat inactivated spores up-regulate expression of IL-6 (P<0.01) and TNF-α (P<0.05) (FIG. 5C,5B). The probiotic Live cells significantly upregulated (P<0.01) the expression of iNOS, compared to untreated samples, showing a higher potency compared to LPS. (FIG. 6). Nevertheless, it is noteworthy that Bacillus coagulans tended to induce up-regulation of iNOS which indicated live cells has a complex role in promoting macrophage Raw264.7 polarization.

(62) Alternatively, human monocytes THP1 cells were treated with PMA to differentiate them to macrophages. These macrophages were cultured in low serum containing media to induce a M0 phase. M1 macrophages were differentiated using bacterial LPS and IFN-γ to induce an M1 polarization (Micontrol) and IL4 to induce M2 polarization (M2 control). M0 cells were incubated with live cells (1000 cells) for 6 hours. Cells were washed and processed for RNA isolation and RT PCR. Supernatants stored for cytokine estimation

(63) The results indicated that Bacillus coagulans MTCC 5856 (live cells) was found to induce a M1 phenotype by increasing the expression of genes related to M1 phenotype (TNF-α, MCP-1, IL6, IL 1β) (FIG. 7).

(64) Bacillus coagulans Induced the Immune Response of RAW264.7 Macrophages

(65) Cytokine profiling may indicate whether the RAW264.7 macrophages have acquired a pro-inflammatory phenotype. Hence, soluble mediators of total secreted TNOS, iNOS, NO, IL-1β, IL-6, TNF-α and TGF-β were quantified by ELISA following treatment with 1.5×10.sup.8 cfu/ml Bacillus coagulans (Live cells, heat inactivated spores) NO levels were significantly increased (P<0.01), compared to control samples, (FIG. 8A-8C), In particular, compared with LPS, the secretion of NO was higher with Live cells treatment. LPS was most potent in inducing cytokine IL-6 (P<0.01) and IL-1β (P<0.05) secretion (FIGS. 8D and 8E), compared to, probiotic Live cells or heat inactivated spores. Live cells were highly potent (P<0.01) in inducing cytokine TNF-α (FIG. 8F). Meanwhile, the amounts of secreted TGF-β were dramatically decreased (P<0.01) relative to control macrophages (FIG. 8G). The effects were associated with an increase in the expression of polarized-gene in macrophages co-culture with Bacillus coagulans.

(66) Bacillus coagulans Promotes the Activation and Maturation of RAW264.7 Macrophages

(67) Gene analysis of M1 related and specific cytokine expressed by RAW 264.7 macrophage treated by Bacillus coagulans, indicating an activation of M1-like polarization of RAW264.7 macrophages. The expression of receptor on macrophage surfaces, including CD80, CD83, CD86, MHC-II, F4/80 and CD16/32, induced by Bacillus coagulans was confirmed by flow cytometry. The expression of the CD80 and MHC-II molecule on the surface of RAW 264.7 macrophage were lower (FIG. 9A) in cells co-cultured with 1.5×10.sup.8 cfu/ml Bacillus coagulans (Live cells, heat inactivated spores) for 6 h, relative to the PBS control group. Live bacteria not only significantly reduced CD86 expression, but also increased the CD83 cell surface marker expression, while heat inactivated spores increased CD83 and CD86 expression (FIG. 9A). The expression of Mature Mouse Macrophage Markers F4/80 and representative Receptor for M1 Polarization CD16/32 showed an higher expression in the presence of B. coagulans. Flow cytometry results demonstrated that live and heat inactivated spores could significantly polarize Raw264.7 macrophage to become M1-like macrophages, and promote cell surface antigen F4/80 and CD 16/32 expression in Raw264.7 macrophage (FIG. 9B). Above all, compared with positive LPS treatment, the activation and maturation of Raw264.7 is complicated and diversified surfaces receptor for M1 macrophage polarization.

(68) The results indicated that both live and heat inactivated cells and spores of Bacillus coagulans induced M1 type macrophage polarisation. Since M1 polarization is important for increasing immunity against bacterial and viral infections, both live and heat inactivated spores/cells of Bacillus coagulans MTCC 5856 can be use for increasing the immunity of subjects in such need, especially in children and infants. The heat inactivated spores/cells of Bacillus coagulans MTCC 5856 can be formulated into finished products such as beverage and infant formulations and can be administered as a dietary supplement for increasing the immune function of the individual.

(69) Other modifications and variations to the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention. The scope of the invention is to be interpreted only in conjunction with the appended claims.